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DuPont-2548
DuPont-2548
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MORsE LABORATORJES INC SOP Meth-93
Revision __tM_ Date OSn096
DETERMJNATON OF HEXAZINONE AND ITS METABOLITES IN WATER
Reason for Revision To incorpoiate the addition of a sonication step for the final sample extract in mixed solvent to ensure consistent recovery for Hexazinone metabolite 03170 in soil pore (lysimeter) Water This change will be used for all water samples
10 INTRODUCTION
This standard op=ring procedure (SOP) is intended for the determination of residues of helta7inone and he=inone metabolites A (lN-13937) B (lN-AJ928) c (lNshyD935) Ai (IN-G3453) I (IN-JS472) and G3 li0 (IN-G3170) in water
20 EQUIVALENCE STATEMENT
During the conduc of this analysisbullbullequivalen~ appararus solvents glassware or technlciues (such as sample con=tration) may be substiruted for those specified in this metho~ except where otherwise noted rnmiddot the event an equivalent piece of equipment or an equivalent technique is used its use will be documented in the study records when appropriate middot middot middot middot middot middot
30 MATERJALS
31 Apoiritus ind Eauipment
Assorted laboratory glassware including graduated cylinders short stem glas~ funnels pipets volumetric flasks evaporating flasks microliter syrin~es
Analytical Balances Analytical balance cipable of weighing to 01 mg for weighing analytical standards
Centrifuge Bottles 250 mL Nalgenc$ polyethylene bottles with screbullv cap closures
Evaporators NmiddotEvap Model 115 attached to a nitrogen source (Orgmomation Associates South Berlin tvlr)
Filter Pipe VVhaunan 54 l
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Gas Chromatograph
GCColumn
Milter
EDP Electronic Pipets with suitable tips
Reservoirs (75 mL 25 mL or 15 mL) and adapters for use with SPE cartridges
Solid Phase E-araction Apparatus
Solid Phase Exiraction Columns
Test Tubes
Ultrasonic Bath
SOP Merh-93 Page 2
Hewlett-Packard 5890E gas chromatograph equipped with an NIP detecor an HP6890 Autosampler and an HP 3365IIChemStation
15 M x 053 = id fused silica column crossbonded with 05 microm filni thickness Rtlt-35
Vortex Genie 2 (VWR Scientific Bridgeport NJ)
Rainin Ridgefield NJ
Varian Analytical Instruments Sunnyvale CA
Vac Elut Mode SPS z4middot (Varian Analytical Instruments Sunnyvale CA) or
Visipreptrade Solid-Phase Extraction Vacuum Manifollti (Supelco Inc Bellefonte PA)
Supelcleantrade Envftrade-Carb SPE Tubes Custom 12 mL Polypropylene Tubes with Teflon Frits paclted with 15 gram ofENVI-CARB (Supleco Inc Bellefonte PA)
Cl8 Mega Bond Elut 6 cc gram (Varian Part 1225- middot 6001) (Varian Inc Harbor City CA)
16 x JOO mm borcsiliette silylated with Teflon-lined screv caps
Branson Model 3200 Ultrasonic Bath (VWR Scientific)
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32 Reaents
Acotone Pesticide residue quality
Dimethyldichlorosilane Supeco Catalog No 3-3009 (Supelco Inc Bellefonte PA)
Ethyl Acotate Pesticide residue quality
Glacial Acotic Acid Reagent grade
Hexane Pesticide =iduc quality
Methanol Pesticide residue qualicy
Toluene Pesticide residue quality
Water HPLC grade
middot 33 Primirv Standards
Analytical grade available from DuPont Agricultural Products Global Technology Division E I duPont de Nemours and Company Wilmington DE
middot Analvte Standard Identification
Hexazinone DPX-A3674 Metabolite A IN-T3937 Metabolite B IN-A3928 Metabolite C IN-T3935 Metabolite Al IN-03453 Metabolite l IN-JS472 Metabolite G3170 IN-03170
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bull
34 ReifJent Prepirition
SOP Meth-93 Page 4 J
34 3mM Acetic Acid
Add 173 microL glacial actic acid pelt one liter ofHPLC grade water Record the pH of this solution middot This solution is stable for appcoximatey two weeks Discard the soltition when it starts showing signs ofbacteria contamination
342 9 I Acetone3mM Acetic Acid
MiX 90 mL acetonewith 10 mL 3mM acetic acid soluticn Prepare fresh daily
343 91 Methanol3mM Acetic Acid
MiX 90 mL methanol with 10 mL 3lllYl acetic acid solutions Prepare fresh daily
3 44 Mixed solvent solution (20 acetone50 ethvl acetate30 toluene GC Diluting ~olutionl middot
In a 50 mL mixing cylinder add 10 mL acetone md 25 mL ethyl acetate Dilute to volume with toluene Mix well Prepare fresh as needed
35 Analvticil Standard Preoarition
35l Stock Standard Solutions
125 mg (corrected for purity) of each anaytictl standard is accurately weighed quantitatively transferred to a 25 mL volumetric flask and brought to_volume with methanol to make individual stock standard solutions having a concentration of 500 microglmL These stock standard solutions are to be stored at 1-8 C when not in use These solutions are stable for approximately six months when stored under these conditions
352 Fortificition CSoikingl Solutions
Typicilly mfaed standard solutions containing all seven anaytes per standard solution ue prepued by adding the appropriate amounts of standud solutions into a volumetric flask and diluting to volume with methaool
For preparation of various standard concentrations see form ML 471 in Appendix I
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Store all soikina solutions in a refrige~cor whei not in use bull 0 shy
353 lntennediate Standard Solutions
Typicilly intermediate standard solutions containing all anaytes are used to prepare the GC standards The intermediate standard solutions and the GC standards are prepared in volumetric flaskS and diluted with mixed solvent solution (20 acetone 50 ethyl acetate30 toluene)
Forpreparatio_n ofvarious standard concentrations see form ML 472 in Appendix L
Store these standard solutions in a refrigerator when not in use
354 GC Calibration) Standard SolutiQfil middot
Typically GC standards containing all seven analytes are prepared For preparation ofthe various standard concentrations see form ML 472 in Appendix I
Store GC standards in a refrigerator when not in use
40 ANALYTICAL PROCEDURE middot
41 Princiole
A suitable aliquot of the sample is acidified and concentrated by solid phase extraction (SPE) using disposable graphitized cirbon cartridges Tue analytes are eluted from the SPE column with acid-acetone solution The eluate is evaporated to dryness and brought to the water phase for a second column clemup iising C 13 SPE colwnn The anaytes are eluted from the column with acid-methanol The eluate is evaporated to dryness and the residue dissolved in a suitable mLxed solvent solution for gas chromatographic analysis using NP detection
42 Simnle Prep1rition
Note Use disposable laboratory equipment whenever possible All glassware used for the analysis must be meticulously cleaned prior to use
l Measure the amount ofsample for analysis in a graduated cylinder Record the volume of sample used for analysis
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2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
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FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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Gas Chromatograph
GCColumn
Milter
EDP Electronic Pipets with suitable tips
Reservoirs (75 mL 25 mL or 15 mL) and adapters for use with SPE cartridges
Solid Phase E-araction Apparatus
Solid Phase Exiraction Columns
Test Tubes
Ultrasonic Bath
SOP Merh-93 Page 2
Hewlett-Packard 5890E gas chromatograph equipped with an NIP detecor an HP6890 Autosampler and an HP 3365IIChemStation
15 M x 053 = id fused silica column crossbonded with 05 microm filni thickness Rtlt-35
Vortex Genie 2 (VWR Scientific Bridgeport NJ)
Rainin Ridgefield NJ
Varian Analytical Instruments Sunnyvale CA
Vac Elut Mode SPS z4middot (Varian Analytical Instruments Sunnyvale CA) or
Visipreptrade Solid-Phase Extraction Vacuum Manifollti (Supelco Inc Bellefonte PA)
Supelcleantrade Envftrade-Carb SPE Tubes Custom 12 mL Polypropylene Tubes with Teflon Frits paclted with 15 gram ofENVI-CARB (Supleco Inc Bellefonte PA)
Cl8 Mega Bond Elut 6 cc gram (Varian Part 1225- middot 6001) (Varian Inc Harbor City CA)
16 x JOO mm borcsiliette silylated with Teflon-lined screv caps
Branson Model 3200 Ultrasonic Bath (VWR Scientific)
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32 Reaents
Acotone Pesticide residue quality
Dimethyldichlorosilane Supeco Catalog No 3-3009 (Supelco Inc Bellefonte PA)
Ethyl Acotate Pesticide residue quality
Glacial Acotic Acid Reagent grade
Hexane Pesticide =iduc quality
Methanol Pesticide residue qualicy
Toluene Pesticide residue quality
Water HPLC grade
middot 33 Primirv Standards
Analytical grade available from DuPont Agricultural Products Global Technology Division E I duPont de Nemours and Company Wilmington DE
middot Analvte Standard Identification
Hexazinone DPX-A3674 Metabolite A IN-T3937 Metabolite B IN-A3928 Metabolite C IN-T3935 Metabolite Al IN-03453 Metabolite l IN-JS472 Metabolite G3170 IN-03170
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~middot
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34 ReifJent Prepirition
SOP Meth-93 Page 4 J
34 3mM Acetic Acid
Add 173 microL glacial actic acid pelt one liter ofHPLC grade water Record the pH of this solution middot This solution is stable for appcoximatey two weeks Discard the soltition when it starts showing signs ofbacteria contamination
342 9 I Acetone3mM Acetic Acid
MiX 90 mL acetonewith 10 mL 3mM acetic acid soluticn Prepare fresh daily
343 91 Methanol3mM Acetic Acid
MiX 90 mL methanol with 10 mL 3lllYl acetic acid solutions Prepare fresh daily
3 44 Mixed solvent solution (20 acetone50 ethvl acetate30 toluene GC Diluting ~olutionl middot
In a 50 mL mixing cylinder add 10 mL acetone md 25 mL ethyl acetate Dilute to volume with toluene Mix well Prepare fresh as needed
35 Analvticil Standard Preoarition
35l Stock Standard Solutions
125 mg (corrected for purity) of each anaytictl standard is accurately weighed quantitatively transferred to a 25 mL volumetric flask and brought to_volume with methanol to make individual stock standard solutions having a concentration of 500 microglmL These stock standard solutions are to be stored at 1-8 C when not in use These solutions are stable for approximately six months when stored under these conditions
352 Fortificition CSoikingl Solutions
Typicilly mfaed standard solutions containing all seven anaytes per standard solution ue prepued by adding the appropriate amounts of standud solutions into a volumetric flask and diluting to volume with methaool
For preparation of various standard concentrations see form ML 471 in Appendix I
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SOP Meth-93 Page 5
Store all soikina solutions in a refrige~cor whei not in use bull 0 shy
353 lntennediate Standard Solutions
Typicilly intermediate standard solutions containing all anaytes are used to prepare the GC standards The intermediate standard solutions and the GC standards are prepared in volumetric flaskS and diluted with mixed solvent solution (20 acetone 50 ethyl acetate30 toluene)
Forpreparatio_n ofvarious standard concentrations see form ML 472 in Appendix L
Store these standard solutions in a refrigerator when not in use
354 GC Calibration) Standard SolutiQfil middot
Typically GC standards containing all seven analytes are prepared For preparation ofthe various standard concentrations see form ML 472 in Appendix I
Store GC standards in a refrigerator when not in use
40 ANALYTICAL PROCEDURE middot
41 Princiole
A suitable aliquot of the sample is acidified and concentrated by solid phase extraction (SPE) using disposable graphitized cirbon cartridges Tue analytes are eluted from the SPE column with acid-acetone solution The eluate is evaporated to dryness and brought to the water phase for a second column clemup iising C 13 SPE colwnn The anaytes are eluted from the column with acid-methanol The eluate is evaporated to dryness and the residue dissolved in a suitable mLxed solvent solution for gas chromatographic analysis using NP detection
42 Simnle Prep1rition
Note Use disposable laboratory equipment whenever possible All glassware used for the analysis must be meticulously cleaned prior to use
l Measure the amount ofsample for analysis in a graduated cylinder Record the volume of sample used for analysis
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SOP Meth-93 Page 6
2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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SOP Meth-93 Page 9
46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
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4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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32 Reaents
Acotone Pesticide residue quality
Dimethyldichlorosilane Supeco Catalog No 3-3009 (Supelco Inc Bellefonte PA)
Ethyl Acotate Pesticide residue quality
Glacial Acotic Acid Reagent grade
Hexane Pesticide =iduc quality
Methanol Pesticide residue qualicy
Toluene Pesticide residue quality
Water HPLC grade
middot 33 Primirv Standards
Analytical grade available from DuPont Agricultural Products Global Technology Division E I duPont de Nemours and Company Wilmington DE
middot Analvte Standard Identification
Hexazinone DPX-A3674 Metabolite A IN-T3937 Metabolite B IN-A3928 Metabolite C IN-T3935 Metabolite Al IN-03453 Metabolite l IN-JS472 Metabolite G3170 IN-03170
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34 ReifJent Prepirition
SOP Meth-93 Page 4 J
34 3mM Acetic Acid
Add 173 microL glacial actic acid pelt one liter ofHPLC grade water Record the pH of this solution middot This solution is stable for appcoximatey two weeks Discard the soltition when it starts showing signs ofbacteria contamination
342 9 I Acetone3mM Acetic Acid
MiX 90 mL acetonewith 10 mL 3mM acetic acid soluticn Prepare fresh daily
343 91 Methanol3mM Acetic Acid
MiX 90 mL methanol with 10 mL 3lllYl acetic acid solutions Prepare fresh daily
3 44 Mixed solvent solution (20 acetone50 ethvl acetate30 toluene GC Diluting ~olutionl middot
In a 50 mL mixing cylinder add 10 mL acetone md 25 mL ethyl acetate Dilute to volume with toluene Mix well Prepare fresh as needed
35 Analvticil Standard Preoarition
35l Stock Standard Solutions
125 mg (corrected for purity) of each anaytictl standard is accurately weighed quantitatively transferred to a 25 mL volumetric flask and brought to_volume with methanol to make individual stock standard solutions having a concentration of 500 microglmL These stock standard solutions are to be stored at 1-8 C when not in use These solutions are stable for approximately six months when stored under these conditions
352 Fortificition CSoikingl Solutions
Typicilly mfaed standard solutions containing all seven anaytes per standard solution ue prepued by adding the appropriate amounts of standud solutions into a volumetric flask and diluting to volume with methaool
For preparation of various standard concentrations see form ML 471 in Appendix I
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Store all soikina solutions in a refrige~cor whei not in use bull 0 shy
353 lntennediate Standard Solutions
Typicilly intermediate standard solutions containing all anaytes are used to prepare the GC standards The intermediate standard solutions and the GC standards are prepared in volumetric flaskS and diluted with mixed solvent solution (20 acetone 50 ethyl acetate30 toluene)
Forpreparatio_n ofvarious standard concentrations see form ML 472 in Appendix L
Store these standard solutions in a refrigerator when not in use
354 GC Calibration) Standard SolutiQfil middot
Typically GC standards containing all seven analytes are prepared For preparation ofthe various standard concentrations see form ML 472 in Appendix I
Store GC standards in a refrigerator when not in use
40 ANALYTICAL PROCEDURE middot
41 Princiole
A suitable aliquot of the sample is acidified and concentrated by solid phase extraction (SPE) using disposable graphitized cirbon cartridges Tue analytes are eluted from the SPE column with acid-acetone solution The eluate is evaporated to dryness and brought to the water phase for a second column clemup iising C 13 SPE colwnn The anaytes are eluted from the column with acid-methanol The eluate is evaporated to dryness and the residue dissolved in a suitable mLxed solvent solution for gas chromatographic analysis using NP detection
42 Simnle Prep1rition
Note Use disposable laboratory equipment whenever possible All glassware used for the analysis must be meticulously cleaned prior to use
l Measure the amount ofsample for analysis in a graduated cylinder Record the volume of sample used for analysis
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2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
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46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
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4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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DuPont-2548
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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~middot
bull
34 ReifJent Prepirition
SOP Meth-93 Page 4 J
34 3mM Acetic Acid
Add 173 microL glacial actic acid pelt one liter ofHPLC grade water Record the pH of this solution middot This solution is stable for appcoximatey two weeks Discard the soltition when it starts showing signs ofbacteria contamination
342 9 I Acetone3mM Acetic Acid
MiX 90 mL acetonewith 10 mL 3mM acetic acid soluticn Prepare fresh daily
343 91 Methanol3mM Acetic Acid
MiX 90 mL methanol with 10 mL 3lllYl acetic acid solutions Prepare fresh daily
3 44 Mixed solvent solution (20 acetone50 ethvl acetate30 toluene GC Diluting ~olutionl middot
In a 50 mL mixing cylinder add 10 mL acetone md 25 mL ethyl acetate Dilute to volume with toluene Mix well Prepare fresh as needed
35 Analvticil Standard Preoarition
35l Stock Standard Solutions
125 mg (corrected for purity) of each anaytictl standard is accurately weighed quantitatively transferred to a 25 mL volumetric flask and brought to_volume with methanol to make individual stock standard solutions having a concentration of 500 microglmL These stock standard solutions are to be stored at 1-8 C when not in use These solutions are stable for approximately six months when stored under these conditions
352 Fortificition CSoikingl Solutions
Typicilly mfaed standard solutions containing all seven anaytes per standard solution ue prepued by adding the appropriate amounts of standud solutions into a volumetric flask and diluting to volume with methaool
For preparation of various standard concentrations see form ML 471 in Appendix I
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SOP Meth-93 Page 5
Store all soikina solutions in a refrige~cor whei not in use bull 0 shy
353 lntennediate Standard Solutions
Typicilly intermediate standard solutions containing all anaytes are used to prepare the GC standards The intermediate standard solutions and the GC standards are prepared in volumetric flaskS and diluted with mixed solvent solution (20 acetone 50 ethyl acetate30 toluene)
Forpreparatio_n ofvarious standard concentrations see form ML 472 in Appendix L
Store these standard solutions in a refrigerator when not in use
354 GC Calibration) Standard SolutiQfil middot
Typically GC standards containing all seven analytes are prepared For preparation ofthe various standard concentrations see form ML 472 in Appendix I
Store GC standards in a refrigerator when not in use
40 ANALYTICAL PROCEDURE middot
41 Princiole
A suitable aliquot of the sample is acidified and concentrated by solid phase extraction (SPE) using disposable graphitized cirbon cartridges Tue analytes are eluted from the SPE column with acid-acetone solution The eluate is evaporated to dryness and brought to the water phase for a second column clemup iising C 13 SPE colwnn The anaytes are eluted from the column with acid-methanol The eluate is evaporated to dryness and the residue dissolved in a suitable mLxed solvent solution for gas chromatographic analysis using NP detection
42 Simnle Prep1rition
Note Use disposable laboratory equipment whenever possible All glassware used for the analysis must be meticulously cleaned prior to use
l Measure the amount ofsample for analysis in a graduated cylinder Record the volume of sample used for analysis
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SOP Meth-93 Page 6
2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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SOP Meth-93 Page 7
6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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middot
SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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SOP Meth-93 Page 5
Store all soikina solutions in a refrige~cor whei not in use bull 0 shy
353 lntennediate Standard Solutions
Typicilly intermediate standard solutions containing all anaytes are used to prepare the GC standards The intermediate standard solutions and the GC standards are prepared in volumetric flaskS and diluted with mixed solvent solution (20 acetone 50 ethyl acetate30 toluene)
Forpreparatio_n ofvarious standard concentrations see form ML 472 in Appendix L
Store these standard solutions in a refrigerator when not in use
354 GC Calibration) Standard SolutiQfil middot
Typically GC standards containing all seven analytes are prepared For preparation ofthe various standard concentrations see form ML 472 in Appendix I
Store GC standards in a refrigerator when not in use
40 ANALYTICAL PROCEDURE middot
41 Princiole
A suitable aliquot of the sample is acidified and concentrated by solid phase extraction (SPE) using disposable graphitized cirbon cartridges Tue analytes are eluted from the SPE column with acid-acetone solution The eluate is evaporated to dryness and brought to the water phase for a second column clemup iising C 13 SPE colwnn The anaytes are eluted from the column with acid-methanol The eluate is evaporated to dryness and the residue dissolved in a suitable mLxed solvent solution for gas chromatographic analysis using NP detection
42 Simnle Prep1rition
Note Use disposable laboratory equipment whenever possible All glassware used for the analysis must be meticulously cleaned prior to use
l Measure the amount ofsample for analysis in a graduated cylinder Record the volume of sample used for analysis
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2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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2 Transfer the sampie Oquot for analysis to a 250 mL polyethylene bottle Fortify ar this poinr ACd water to the sample so that the total volume prior to addition of acid is l 00 ni Swirl to mix
3 Acidify the sampie with 20 microL glacial acetic acid Swirl to mix
4 Filter the acidified smple into a second 250 mL polyethylene bottle The sample is now r=iy for SPE column processing
43 Solid Phase Extraction SPEVConcentrltion ond Purificotion
I Pack the carbon column by gently pushing the column fut onto the cirboii packing Condition the column with one column volume of9 l acetone 3mM acetic acid Elute to dry and dry the column for approximately 30 seconds more with vacuum adjusting the vacuum gauge to minimize loss of small amounts of carbon Continue conditioning the column with two column volumes of water withcut allowing the column to go to dry Discird all elutions middotmiddot
2 Attach a reservoir to the cartridge and pass the acidified sample through the cartridge Use va= to facilitae sample elutionmiddot Do net allow the cartridge to go to dry Dis=d this sample load Remove the reservoir
3 Rinse the sample container with one column volume (10-11 mL) of water and add rinsing to the coiumn Elute to dryness and dry for approximately 30 seconds more with vacucm Adjust the vacuum gauge to minimize the loss of smill amowits ofcarbon Discird this wash
4 Rinse the column with coe column volume ofhexane Allow the hexane wash to go to dryness and drf for approximately 30 seconds more with vacuum Adjust the vacuum gauge to minimize the loss of small amounts of cirbon Discird the hexane wash
5 Elute the analytes into l 16 x 100 mm silanized glass test tube (mirked at approximately l mL md approltlmately 5 mL) with IO mL of 91 aceoneJmM ac=ic aci Elute by gravity However vacuum may be used to start the elution
Note See Appendix II or detailed insnuctions for silylating glassware
THIS IS A STOPPING POINT Refrigerate extracts for further processing
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6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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middot
SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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SOP Meth-93 Page 7
6 Evaporate the sampie extract to approximately l rnL at 40 degC - 50 degc using the N-Evap
7 Wash the sides of the sample test rube with approximately 2 mL acetone and evaporate the extract to 02 to 05 mL
8 Repeat washiilg the sides of the test rube with a second 2 mL portion of acetone
9 Take the extract to drynSS
I0 Dissolve the residue in 200 microL acetone Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
11 Add approximately I mL of water and evaporate to the water phase
12 Add approximately 4 mL HPLC water and continue the evaporation step to ensure removal of all traces of the a~etone
13 Adjust to approximately 5 mL with HPLC water
THIS IS A STOPPING POINT Refrigerate extracts for further processing
14 Vortex Sonicate for 5 minutes Vortex Tac extract is now ready for additional SPE column cleanup
THIS IS A STOPPING POINT Refrigerate extracts for further processing
44 Additional Column Clea nun with C18 SPE Cartridae
Note To ensure unifotmity ofthe column packing in the cartridge tap the cartridge on the lab bench then gently push the column frit on to the column packing prior to use
Pass 5 mL 9 (Methanol31111vAcetic Acid) to the column Elute to dryness and dry for 30 seconds more with vacuum Continue conditioning Vith 2 x 5 mL HPLC water without allowing the column to go to dryness middot
2 Load the aqueous ectract from 434 on to the column and elute to approximately I cm above the column packing
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middot
SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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SOP Meth-93 Page 9
46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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SOP Merh-93 Page lO
463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
DRAFT 6
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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DuPont-2548
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
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DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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middot
SOP~ Meh-93 Page 8
3 Rinse the sample tes~ tube with approximately 2 mL liPLC water and add this rinsing to the column Elute iust to drvness ~iscird the sample load and wash
4 Wash the colwnn with 2 x 5 mL hexane Elute the second 5 mL hexane wash to dryness Continue drying the column for 30 seconds more with vacuum Discard the hexane washes
5 Elute the analytes into a 16 x l00 = silanized glass test tube calibrated at 40 mL with IO mL 91 MehanolJmM Acetic Acid A suitable reservoir may be u-d for this step to facilitate elution Elute by gravity However vacuum may ie used to start the elution
TIDS IS A STOPPING POINT Refrigerate extract for futher processing
45 Concentrltion and Processina of Sample Eluates for GC Analvsis
I Evaporate the srunple extract to approximately I mL at approximately 50 bullc using the N-Evap
2 Wash the sides ofthe sample test tube with approximately l mL methanol ard evaporate the extract to 02 to 05 mL
3 Repcot wasbing the sides of the test tube with a second l mL portion of methanol
4 middot Take the extract to dryness Determine the final volume of the extract at this point so that the concntration of the sample in the final extract is 25 mLmL
5 Dissolve the residue in a volume of actone equivalent to 20 of the final volume ofthe extract Briefly vortex the extract and sonicate for 5 minutes Again vortex briefly
6 Add a volume of ethyl acene equivalent to 50 of the final volume of the earict and ensure complete solution of the analytes by vigorously vortexing the mixrure
7 Adjust the final volume of the sarnple extract with toluene Sonicate for 5 minutes lVix thoroughly fer GC analysis The final concentration of the sample in the extract is l mL = 25 mL
19
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SOP Meth-93 Page 9
46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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SOP Merh-93 Page lO
463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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SOP Meth-93 Page 9
46 GC Analvsis
Note The column and conditions stated in the method have been satisfactory for the matrix being analyzed Tne specific column packingcoating carrier gas column temperature ind flow rate listed are typicil conditions for this analysis Specific conditions used will be noted on each chromatographic run and will not otherwise be documented
46l Ooeraring Conditions
Instrument HP5890E gas chromatogrph equipped with an NIP detector electronicP= controUed inlet (packedpurged) an HP6890 autosampler and an HP 3365 II ChemStation
Colwnn 15 M x 053 mm id fused silica column crossbonded with 05 microm film thickness Rtx-35
Inlet Liner 2 mm Ld liner lightly packed with fused silica wool
Injection Volume 2 microL
Carrier Gas Helium
Flov Column 5 mUmin (constant) Makeup 15mUmin
Temperature fnjecor 290 middotc
Detector 235 middotc
Column Initial 150 middotc Rlte 25 degCmin to 275 bullc hold 22
minutes Rlte 2 10 degCmin Final 235 middotc hold for 100 minute
462 Samule Analvsis
Prepare a four-point stJndard curve by injecting constant volumes of combined analyre standard solutions prepared in J mixed solvent solution Use constant volume injections for sample e~cts as Ve Sample responses not bricketed by the standard curve require dilution and reIBjcction
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SOP Merh-93 Page lO
463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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SOP Merh-93 Page lO
463 CaCularions
Calculations for instrumental analysis are conducted using a validated software application to create a standard curve based on linear regression Tnese regression functions are used to cilculate a best fit line (from a set ofstandard concentrations in microglmL versus peak area response) and to detemine conceitrations of the analyte found during sample analysis from the calculated best fit line_
Tne equation used for the least squares fit is
y=nu+b
whee
y =peak area response
x = microglmL found for peak of interest
m=slope
b = y-intercept
Tne calculations for ppb analyte found and percent recovery (for fortified samples) for each of the seve_l analytes are
I ppb analyte =
microgfmL analyti formd x 1000 x ml final volumt x CC dil fact ml sample
vhee
microglmL analyte is calculated by linear regssicn based on peik area response
1000 conversion facor from microg to ng
mL sample amount of sample analyzed
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SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
120
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2543
SOP Meth-93 Page l l
rnL fincl volume volume of poundinaJ exrrzct submicted to GC
GC dil fact the magnitude of dilution required to bracket the response of the sample within the standard curve responses When the sample requires no dilution the GC dilution factor= l
2 The peCnt r~overy for fortified control samples is calculated as follows
pvb found in forrifieri control - p_ob forurd in control OO-Recro1cry = middot x ppb added
50 REFERENCES
l Dupont Report No AivR 3833-9) (Draft) Analysis of Hexazinone and Metabolites in Soil and Groundwater using GCMS received 11122195
2 Dupont Report No AiVfR 2896-94
120
OuPont-2548
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
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4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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SOPii Meth-93 Page 16
Siylation of Gl-SWare
Puroose
Silylation is a process used to chemicilly treat glassware in order to prevent or minimize binding of aoalyte residues to the glass surface
Caution DO NOT ALLOW DIMETHYLDICHLOROSILANE TO COME IN CONTACT WITH WATER CHLORINE GAS AND HYDROGEN CHLORIDE GAS WilL BE PRODUCED
THIS PROCEDURE MUST BE DONE UNDER A FUME HOOD THE TECHNICIAN MUST WEAR BEAVY LATEX GLOVES
Procedure
- Prepare 100 mL of a 5 (vv) solution middotof dim_thyldichlorosilaoe (DMDCS) in hexane
To a glass stoppered glass container (approximately 200 mL volume) add 95 mL hexaoe Slowly add 5 mL DMDCS Stopper aod invert to mix
Larger volumes cin be prepared using the proportions disCJSsed above however attempt to prepare amounts that will be nearly totally used to avoid disposal of excess solution
2 PourasmallamountoftheDMDCSsolution into the glassware to betrearecL Rotate the glassware to thoroughly coat the inside surfaces Pour excess solution into the next piece of glassware to be treated middot
3 Allow the treated glassware to dxyapproximately 20 minutes) Rinse with deionized Vatcr then acetone Again allov to dry
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SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
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DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
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SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
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4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2548-
SOP Mdi-93 Pag 17
4 Glass~are is now reidy for use
Note bull Any glassware thar is clemd with a brush afte~ it has been silylanized must be resilylanized
bull Store pure DMDCS at room temperature
bull 5 solutions ofDMDCS in hexane are stable for 5 days when stored well-stoppered ar room temperature
SOP Prepared by Frances Brookev
Kevin Clark
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DuPont-2548
Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPont-2548
DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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DuPont-2543
DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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DuPont-2548
Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2548
Dcioct ~on So 92
ANALYTICAL METHOD FOR THE DETERMINATION OF HEXAZJNONE AND
METABOLITES OF INTERST IN SOIL AND WATER USING ELECTROSRAYshy
LCMSMS F W BriII Tom Garrin~r
10 SUMMARY A residue method is dcscnbcd for the atrlcion uriScrioa icd q~ticrion ofbecaziiione (DPXshyAJ674) and meibolitcs A (IN-13937) A-1 (IN-A3453 B (IN-AJ92S) C (INTZ935) 1 (IN-JS472) m4 G31 iO (INQJ l iO) in sail and wa~-
Witer salXlples (20 mL) were filtcml conceunted 1lld purified wing solid phase extracioa cnridgcs (Eivi--Drli and Cl8) The instrum~ analysis for deteeion and cpuntit1tion of the ~ytcs wu accomplished by lOMSIMS with ac ekarospriy intcruce in Jlllllriplc roction moltltorizig (MRf)
Soil mnplcs Sg) were cxtncted l-ith OlM 1CH6DQ)05M NaCl solu~on fitaed_ conc~trted and pUrified using solid phtsc cm-Jccioa cmridges Elvicub and C18) The icstrucnetcal anUyns (or detection and quantimioo of the malytcs was 2ccomplished by LCJMSJMS with an clecrospray intcfacc in multiple rcction monitoring (MRpound)
The limit ofquanQation (10Q) for wittf was OJ 00 ppb forhcxuinonc md ill 6 mctaholitcs The LOQ for soil was ZO ppb for b~ncml all 6 mcUgtolites Test samples wen fortified Jt the LOQ and 5 x LOQ for c3ch amlyte in both soil uid watcr to vtlidatc the method
To be compa~
20 INTRODUCTION Hcuzinaae (DPX-A36i4) is the lctivc in~Cll in Velpaf herbicides-registered for postcCcocc control ofmany annual and biennial wc-ds Analytc StructllrC$ chemical names DuPont code umbers and CiDllkalAlnuaas registry awnbcn are provided in Figure 1
In this method wttcr samples arc lddified 5lted purified uid concitrltcd by SPE A combiiutioa of an Envi-CUb md i CtS SPE step mused to remove most matrix md subst1nces ~tmiy Uncicrc with the iilmumental inalysis
The puri5ed extrio were anllyzed by ESImiddottO-fSMS wing positive mode multiple rciction monitoring (MRf) for bcxainone Jnd metibolilCS A AmiddotI B C l Jnd G3170 Qwlritition was based on the Ditegritioa of l single MR umsition respoase ibis malysis qumtictively detects heuzllone lnd ill 6 rnctabolitcs the LOQ for eich detemined to be 01 ppb in water and 20 ppb in soil
To be competamp
3 0 MATERJALS Equivdcnc equiptbent ind mnerills niy be substiruied unlen otherwise specified (sc section 5J) noie iny specificuions in the following descriptions beta~ making substirutions The cquivUenctlsuitibilicy of uiy substicution should be verified with icc~t1bie cono-ol Jnd fortiticition recovery cbtl Tne -nateriiis 3re listed in order of first 3JlpelnllCC in the ncthcd
JJ Equipmtmt
Stindirds ~iririon
DRAFT J
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DuPont-2548
DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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DuPont-2543
DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
DRAFT 6
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DuPont-2543
Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2548
DuPonr R=ort ~o zgz
~cttlcr AE 63 mUyCol bal3nc Mccier Inmumeu Corp Highmown SJ) Ppctsipipeoors lld ~ suicble for p~rioo of Stlndards llld sample forificiciocs Aisoned fbsks bci= lDd voiwcerie iyililcie-s
Sarrnlc ErnCon Mettl PM460 top-oaiilg inayticl haWice )fcnlc- lnstruIlot Corp)
2S~mL high ccsicy oiypropylcne cltrifuge rubes (Nalgce)
RlpidVapD Evaporation Syncm (Ubconco Corp Klnsas Cty ~o)
Twumiz_-r homogei= Model fIDISIO (Tckmar Cocnpiny Cincanari Ohio)
0 $$JlID filtcr lmit P-~- 150-0045 (Nalgc Company Rochester NY)
Sorvall RCSC celtrifle with SS34 romr (DuPont Compmy SorvilllaquoI Products WilmingtOn DeL) or rEC HN-Sil ceitrffilge (DamocIEC Division Needham Mass) Buchner FUIllel Filter Paper Whatman S (CaL No 1005 OiOJ to fit Buchner FUimel
Samele urifieitionmiddot CI8 Mega Bond EarS 20ccIg PN 1215-6001 (Varian Hubor City CA)
Sipcclemno Eivi-Cugt SPE column 60mUl5g OJSTOM (Supelco Inc Bellefonte h)
IS-tnl cp ~cVoir =opry PJ 1213-1010 tVarian) Luer stopcock P~ 1213-1005 (Vuiao) Boud Eur igttcs PN 1213-1005 (VW) middot
bull N-Eoap Analyticil E-ixgtraror Mocicl 11 l Becton Dicliason amp Co Franklin Lakes NJ)
2-microm 13-mm Aero Disc 130 P1FE syringe filter PN 4422 (Geruim SCcccs Ami AWMichJ 45-microm 25-cn AcroDisc 13CR P17E S)linge filter PN 44 9 (Gclm3n Sciences Am AWMich)
ViCt1Um manifold p_i 5-1030 (Supclco Inc Bellcfotitt Pa)
3200R-4 Bramonicraquo Llttisonic Cicmir(Bransonic Ultnsonics Corp Danbury Cocn) LCMS Analvsls The following -oPLCMS systcn 11S used for this method induding cquipmeit o~essary to divert the flow going into the lllllS speeromca o waste 3Dd o(postolumo splitting oCthc HPLC flow
bull MicirMus Quaaro Il riplc sector quadrupole instrumct with API sourcFinterfac configured (or ESI opeirion MusIynx dul xquisieoa so~ running undcr Wiildows NT Waun Model 1100 HC systcm including pumJ) module autosamplir dc3wilig module column comprmcut module Watm CorP Milford Mass) Elcctricilly-aeu2ted ligh-pRSSUre 6-pon switching valve with l16 in fittings tfEC5W usd (or cfnuot divcsioa from MS (4-pon valve would be adequate EC~V) (Valeo lnsmnnent Co Inc Houston TX) High-pressure sttinless steel 116 in fitting tt= ZTI for use in post-cOlumn floV splitting Split ruio 3Cijastid by altering restriction on waste-side oCtec by altering JCI)gtb oC cipillary tubing (Valeo Instrument Co Inc)
bull Zorbaxbull 46 mm id x 50 mm Rx-CS Smicrom paniclc siu PN 880967-901 (MAC-MOD Analyricil Inc Chaamps Ford P3) Substitute columns m3Y give sub-op1imiI pcrlonnioce
jJ Reagrnts and Standards
Rc~llei
OauiSolv 11 gJSs discied icooc hc~c mc~ol
DRAFT 4
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DuPont-2543
DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
DRAFT 6
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont-2548
DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2543
DuPoct Ron ~o 2292
Baker Anilyzcd Reigent pocmium phospil3te dJbuic cryscl PN 3252--0l KHPO (1T Baker Phlllipsburg N)
Baker Anilyzd RC3got sodium chloricie cystll P-~- 325-01 KHPO (T Baker Phillipsburg NJ)
GR F31 actic icid (EM SciClc)
All ~tcr was Milli-Q distilled dciouiz-d W3ter r-fiiliorc Corp Bedforc ~au) Stancbrds All stlndards were syuthesizd by DuPoa Agrii-tlrunl Produi-s Wilmington Del Stancims should be grcter than 95~ purity
DPX-A3674-213 (hallincnc)
IN-TI937-3 mccbolitc A)
IN03453-2 (mccbolite A-1)
IN-A392S--4 (mccbolite B)
IN-T3935-3 (mcabolitc q IN 15472-2 (moaboliie I) IN G3171)2 (metabolite G3li0)
JJ Safety and Heallll
No UlULqWJy hzzardous nucrfals are 11Scd in this method All 1ppropriuc mJteriil safcy data sbcts should be r=d and followed md proper pmonal mitecrive equipment should b used
40 METHODS
~I Princlpl~ ofMaytical tftthoi ~-middot
flI Samof~Earaaion
The 0IM KHPQJOSM NaCl sail canction solution yielded high movcrics uid has bcei dcionstrucd as in effective extr1ction agent for hcxIZinonc uuI its mcWgtoli1es Vatc is nee cunc1ed it is dimtly filtered concntrltcd and purified on he SPE columns
4 1 Erracc Plrificanmiddotan
Cmon piwc (Envi-Clrb) md Reverse phlse (Cl) Bond Elu~ SPE irocedurcs ire wed to provide extensive cxtr1ct purificuion The wucr and he soil cttr1c11 are pwcd oa10 and rcained on Eivi-Cm columns The column is then washed with water uuI hcnne co remove most of the Dl3trix ma1erial Eution is with 3n aectonc3 mM acric acid (9010 vv) solution The acetone in he el1111e is mnoved and the sampJe is brought up in wa1cr md loaded onto a ClScolumn The C18 is the washed with waler and hexanc following which it is eluted with methanol The sample is blown dowu to dryness through a series of procedures designed to minimize loss of malyte on the etinoincr wills and is brought up to a finaI volume in witer for a fm1l filtration md LOMS analysis
41J LCMS Analvru
The method utilizes electrosprly ionizatioo (ESI) md is opm1cd in positive ion mode wing ~fultiplc Reicrioo Monitoring (MRi) on the Miaomass imuummatioo for all ofthe a1Uytcs A single transition wu monicored for each u1dytc pm+t] fngmentiog 10 the (M-cycloballlc moietyr Seleion of these ~itions was based upon he mus spcctrl gcicritcd dwin-g the merbod devclopmenl process with the instrument in sannUig mode The spectri gcim1cd by ESI-LOfS for the lllllytes ue shown in Figure The bue ion [MHr for eicb milyte is scleeed for qiuntiatioo The sensitivities of eicJ milyte vuied the G3 I70 showing the eist se11Sitivity in the positive ESI SR[ rcde the hegttizicone showing the most
DRAFT 5
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DuPont-2548
Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
DRAFT 6
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Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
- 43 shy
140
bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2548
DuPont-2548
Duoct Reon No 92
Analyrjazi Procedure
J] I Glasrwan and Eauimnnrt Oeaning PnJcedura
Disposable labwuc ~ used whenever possible in this method Rcuublc 3~ whicb indudcs the evapol2tiou vessels and vohrmetrics for standard soluriom u dcned by wuhing with a bboruory gridC dctegeu followed by ap witcr rinses (3) and disnlled wate -inses (3) A~ icctoue tnsc InlY be used to remove he residual water llld promote drying Ccuin i1e=s of glusware llil1St be ttcted sifarUzcd to pnvent absorption o( mil~ onto the glass
411 PrmaratiDn and Stabilitv ofReagmtSolutiotq
OIM KHJraquoOJQ5MNaa Solutioti Add 136g KR~ and 292g NaCl into a 1 Utervolumcaic flask DWolvc the CompoUDds with HPLCor Milli-Q wua Bring to voam md mix welt Record chc pH of the solution (shouli be -4) This solnticn is sable for 1mcmb disclrd ~lie Uit show signs of turbidity or bacteria gniwth Sale volume as neccsmy middot
3 mM rcttic Acid Add 173 microL ofglral acetic icid pcr l L ofHPLC or WllimiddotQ wate The solution is sable for 1 month Discrd eirlicr ii it shows signs of mrbiciity or bacefa growth
1110 diluttd Glacial Acetic Acid Prepare l 110 dilution of laciil acti aCd by jgtladng 9 mL ofBPLC or Milli-Q water into a IS mL plaortic ccnttifuge tube or my otic suitable conlJine with a eip Add 1 mL of glacial 3Cetic acid mix cucfully Dp when not in me
Extraction ygtJution for Soilmiddot is pRpUCd by combining acemnc and the 0IM KHP0)05M Naa So1utioa in a 9010 vv ratio This scJntion is ~arcd daily ss needed ho~evcr will be sable for I tllOnth
9010 Acetone-3mM Acetic Acid SolutiOi Mix 90 mL ofic=tanc with 10 ml of3 mM Actic Acid solution This solution is used for SPE column conditioning and elution SciJc volume as needed stible for l month
9010 Mctbano13mM Acetic Acid Solution Mix 90mL ofmetbmol with IO mL of3 middotmM Acetic Acid solution This solution i~ used for SPE column conditioning acd elution Sele volume is ncdcd suble for I month
HPLC Aaueous Mobile Phase A OOIM actic add solution far use u mobile phuc is p~arcd by filling a l L volumcttic flask with distilled deionized wmr Jdding 600 microL of cooeeitrated accic add geitly agiuting the mixtuR and diluting to volume in distiilcd dcionited wirer This solution should be prepared as needed or sooner if turbidity or bactaW growth is observed
J13 SroclcSolutions rnJararion
U possible stmcbrds witb ~purity grcirr thin 95 ue ro be tued lndividuU llJ02-fglmL 12poundk mncbrds solutions for bmrinonc md the 6 liled meoboli1s were prepand by weighing 501 mg of cicb stmdird in a separiie taRd 50-mL voluxneaic fbsk acd diluting 10 volume in acetone Sample weights wen determined to 3 significant 5gurts The malyricJ btlacce mwt provide a weight precision lo 3 significmt figures or the amCunt acd volume bas 10 be ~jus1ed 10 meet tltis crirerfa For example in~ the miount to 10~2 mg acd use 2 100-mL volumaic fluk OciIy fabcl each stock solution with dare prepared maJytc 211d concentruion Srod solutioos ire stored under refrige-ition (41 ~q md mwt be replaced at IC25t every 110 days or when approrimitcy hJlfvolwrc Keep stoclc solutions stoppeed 10 restrict solvent eviporation
DRAFT 6
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DuPont-2548
DuPont-2543
Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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DuPont-2548
DuPont-2548
DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont-2548
DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont-2548
DuPont-2548
DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont-2548
DuPont-2548
DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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138
DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2543
Duicnt l~on No 2292
4-1 ntU111ttiiartSrandard and FomficJrion Soiunon Prnxrr=on
Vne7Soi1 rir~diatc Suncbrd n ln1erneciiatc 10 microgtcl iiii scudlrd soiurioo S =acie from the 7 individuU 100 microgimL stock solurions PJc 05 mL o( e=C of he 7 iodiviciwl 00 microgll nod soutiocs inm a SO mL volumeaic fluk wicg a pipct PUce the voitncric oato the ~-Evap and au iitrogei through the fbU to blow otrall of the acetone rcnovc ~rly Add HPLC or Milli-Q water and fill tD the 50 mL liDc Vo~ sonicte far 5 min
Wner Forrifiettioa uid Standard Solutionmiddot A SO ngmL -nter fortificuioo 3ld scndird soiuion is ~from the mixed I0-microgimL Inimncdbte Stwbtd Rcnove the Inccrmcdiatc Saniiud from the nfrigm1or ind allow it to reich room tcmpencmc Tben UWcc by huid to insure stm6ni consistencymiddot before making dilutions Pbc 25 mL of the LO pgml imemcdiltc swck solution into a O ml volumetric flask Fill up to the Imc with witer and mix Libel the solution with the dl~ re-arcd uialytcs uid conceitration Store the solution in the cfrigmtDr It is Stlblc for 90 days
Soil Fonifiarioo and Standard Solution A 200 llampmL wucr fonification and sandard solution is prpared from the mixed 10microYml Intctnediatt Standard Rcmave de lntcmcdiatc Standard from the refrigrritor and illow it to resch room tempof3ntte Then hake by hand to imwc mndlrd cocsistcicy before making dilutiooi Place 100 mL o(the 10 11Jml intcn=diatc stock solution into a 50 mL volumtric flask Fill up to the line with water and mix Lahcl the solution wirh die dn prc-iarcd malytes and conc~trition Store the sohtion in the rcfrigcntor It is stble (or 90 day
425 LC Calibration Sumtlm-ri Solution Sn Prroaration
Wafr The ealIbruionstandardset is made from the SOD~ Water Fortilkationmiddotand Swuiud solution Reuovc the WJur Forciiicition ind Stmdud solution 6-om the refi1igcrator md 3llow it to rcicb roam tcDptrllUrc Then shake by hand to insure stU1dard c~ before makins
0
diDtions Rf== to the 11blc below Pl3c the specified aliquot of the 500 microgmL WurFortification mdStmdardsolution into a 10 mL volumetric flask Fill to the line with HELC or MilliQbull water Label is ahbmtion mndlrd solutions with date prepared inaiytcs md coDcntration Score these solutions in the rcfrigeicor They are stable for 90da
Tdeitifier Aliauot ofO ngmL Stdfull Final Vol SClndard Concmrion (nel WSI 2000 IOmL 10 WS2 1000 OmL 50 WSJ 400 IOmL 20 WS4 134 IOmL 06i
Cleuly label the clib~tion solutions with the d3rc prcpami lnalyccs mc1middot collcntrition Autosampler vUls should be filled co approxim1ccly 113 cipacity 3nd C3pptd to minimize solvent cvaporuioo The 10 mL vol~ttic fluks oCstloduds will be s11blc for 2 WC~ Use fresh aliquou from then for ciclI imlysis set
~The cihbrition stutdud set is lll3de from lhe 200 ngiri1L Soil Forrificuion and Saadard solutfon Remove the Soil Fortificiticn md Standard solution from the refrigerator and allow it ta rcxh roo01 lenpclturc Then shake by biod to insure scuidard consistocy before making dilutions Rer co the uble below Pbce the specified aliquoc of the 2000 microgmL Soil Fortification and Stmdlrd solution into a lo mL vohune~c tbsk Fill to lhe line with HPLC or MillimiddotQbull wirer Ube is cilforition sun6rd solutions with due pr~ircd lDllytcs llld conc~i1r1ticn Store these solutions in the rfrigcntor The~middot are stable for 90dJys
Alicuor ofOO miltrtl SrdfuL Fin Vol Stiodird Conc~rlrion fnltirnL) SS 2500 lOmL 500 ~
DR-Ff 7
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133
DuPont-2548
DuPont-2548
DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont-2548
DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont-2548
DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont R40n Sc 192
SS2 l~O Oml 250 SSJ ~00 OmL 100 SS-I 167 Oml jJJ
Ccriy laix the calibration solutioos w1cb the dace ~ired ~)1d aad concuntion A~ vials should be iillcd to approxiimtely 213 czpaciry ind cppcd to ciinimi solveuc evtporiCcn Te o mL voumcric flasks o( stmduds will be sable for 2 weeks Use frcl aliquots from thcn for och aoalysis set
426 Souret anti Oaractn-itition ofSamDlu
427 Storagt and Prqroecsi11g ofSamples
423 Samolt Forrificatiorr Procttiurt
Water Ail warir test samplcs ~ fortifiedwitb ampcilnonc llld th 6 listed mctibQlics from~ SO ~middotmt Water Foniticition and Stmdud solutionL Tiu ippropriitt fottificcion volune is dnwn into a pipetpiFltlor and placed into 20 mL owurr The water is then mixed Rcfcr to the table bcow for e volumes oWatcr FortifiQtion md Stmdard solution to use for nllillg the LOQ and S x LCQ onifictions
~All soil test samples are fortified W4h hcxizinanc and the 6 listed mctlbolit= from the 200 ogL Soil Fortificujon ind Sbndard solutions The rpprcpriit onificttiiio volume isdrawn into a bull pipctlpipettnr acdplaced into Sg of soiL he soil is allowed to stand for 10 minmcs Refer to~ table below (or the volumes ofSoilFortifictioo aodSbndard solutiot1 to we for making the LOQ ~ 5 x LOQ fonificitions
Amount to Volume Fortiiiotion Soln Volume Fortifiorioo Sotn Leoter an inlvtes LOO Forrifvl Low Forrificition (LQQl Hie-h Fortificirion fLOOl and 5 x LOO
Witer Soil
20mL Sg
40 microL lG
200 microL 250
01 pb 05 ppb 20 b 10 ppb
29a Cbncmtration arid Pirificatfpn Prrxrduns (ar Water
Solution RcquircmencsSimple (CXaictioo + purificitioo) Milli- Q W3tcr 50 mL metbuiol 10 tnL hcWle J0 mL 90 l 0 acetot1e 3 mM Acetic Acid 10 mL 9010mctuool3mMAceticAcid 10 rnL
SAMPLE CONCENTRATION AND PL1UFICATION
I Remove sampes ampom nligmtor or freezer ind allow to wann to room =pe~ 2 Mosure 20 mL o( tbe wltcr sunplc lDd plice it into a 50 mL ceitrifug Cnlc 3 Fortify test simplcs U rquired 4 Add SO ml o(Milli-Q wllct ind lbm acidify by adding oo microL of the llO Uu-=
glicUI ictic acid solution Clp ind briefly vonci mit
DRAFT 3
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DuPont-2548
DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont-2548
DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont-2548
DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
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DuPont-2548
DuPont-2543
DuPont Reper ~o 292
t Plle the S ~ 21irb Sand Euis~ iDto the yen1Ctim mmifold ind condition hem by placing IO nL of 9( l 0 3ceoneJ mf 1ctic ldd solution into the rubes uid adjusting the ilow ~a fast c- (-10 mUminwc] UsiDg 1 grntc ~cuum to pull through ill of the liquid let he cohi dry for 30 seconds~ the Ls1 of the liquid has aitcd Ioad uid collWiion he c0Ju12 witb rwo JO ml volwnrs of wmr Do nor let the liquid level drop beiow the op of b sorbcu tfter the second witcr load has passed throu~ Do not allow the tnhe to Cry
2 ~the water suiie onto the column in severii portio~ ifnetcssuy due to column reservoir cpaCty~ Tarn on the vac1um md allow the sample to pw through the column u a slow -p Da aoc allow tbe coltmlll 14 go dry Do not eollec the liquid
3 Pl3c 5 mi of~ltn nro the ccipty 50 mL centrifuge mbc vona Place this rinse onto the Bood Eut md iilow to pass through it a slow drip Pumicro ill the 1112tcr througll allow to dry passing air rough for abow 30 Sctends iitcr last drop has come o~
4 Place 5 mL ofbctabc mto the Bolld Elui and allow to passthrough at a slow drip Allow to dry~ air through for UioutJO seecads aitcr Wt drop has come Qt
5 Opc1 the nc-mm miold and place pbstit lS mL t=ttiiuge mbcs into the manifold in ordct to cone-a~ elU3tc
6 Pla~ IO ml of 9010 acel0nc3 mM actic acid soluton in10 the Bond Elut tube md aDow o pass dao~ u a stow drip Pull all ~ttho liquid thOugh into the collc-ttion tlbes Bni1t the vxum md mnove the tubes comaining the eluate
7 Place sample an m NmiddotEvap and ~ratc ID approximately 1 ml at 40SO deg 8 Wash the siks ofesmiple tcst lllbe witb ~Jy 2 mU ofacetone llid
evzporau Qc aru to 02 to O~ mL ~t this vrashing with I second 2 mL volume axeio= EY2pCI3M the sample to deyrdS
9 Add 200microLofaccne10 the tube voncx md ronicte for S ininutcs vortex lgain Add approximm1y 1 cl of water md CV2p0ntc lO the Natef pbsc Add approximately 4 mL ofHPLC trltt md coacinat the evapondoa n to =sun rtmoval ofall tnces of actoDc This is 2 Teri cridoI step the praence or iny icetone will prevent the polar analytes from bdnC rctLined on the C18 column in bter steps
IO Adjust to rpprorplusmnucly S ml with HPLC water Vortex sonicte for S lllinutcs_ vortet
I I Poc~ed dfrttfy to Oc Ct8 SPE cemmiddotup TIW isl napping poirit Samples en be pbced into the rpoundgeltor for stotlge for up to l week
ClS BO-tD ELUT CIZlUPIFINAL PREPARATION L Place the Cl g Bacd flu~ tub= onto lhe vacu~ mmifold and condition them by
placns S mL of~olJmM Arnie Acid (9010 vv) solution into euh tube md adjunizJg de flow EJ a bst drip DrUn lht rube 2Dd illow ro dry for 30 stcoiuh more under~ la~ SJmC mmncr condition next with 10 mL of water in two S mL inc-mems W2iting mal the lim S mL is completely below the 6ii before adding the scolld S mL Stop Qc Oow when the level of the water drops slightly below the top of the sorbcm n the bottom rube Do not let the tubes dry
z_ Place the Eivi-Cati eimre solutioa (step I I ENYJ-CARB dcmup above into the CIS Bond Eu~ rubebullbulliiy VJCUum open lhe scopcock md load the cluite onco the colwnn wilh av~ 5Jow ~-Stop when the level of the liquid is 1 cm lbovc the top oftbe sorbenL Discni aoucous effiuenL Bickrinse Cc ori~ Eivi-Carb eluue cont1iner by pbcing 20 rnL of wutcr into it vorex tniciig for a middot=w seconds Pour this rinsate into the CJS c~dgc Allow this (rinse)wit- lrlSb deg lSS through the C18 ind disclfd Allow lll of the wiur to be dnwn out oj he ~~ Umnedii1cly shut off vicmun discard Joad volumt
DRAFT
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DuPont-2548
DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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DuPont-2548
DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
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DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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DuPont-2548
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FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
DuPont R~on -10 ii92
4 Plac 50 mL oiheucc into the ClS orridge Allow the hcune ro pass through 3t i
moderate dip Afee the heune bu COIIi)icely puscd hrougb the coiumn iCd lllorher 50 mL ofbexine lDd Jilow dlt ro compleceiy pw througD the column as weiL Allow the vacuum to pull air through the Qitridge unc1 there is 110 Clore drippiDg
5 Biuk vacuum md pbc a new IS mL pbstic eeitriiuge tube into the vacuum mmifold under the C13 tubes to coUect the euue
6 Plice 10 mL o(mctunol3 m Acetic Acid (9010 vv) solutioo in10 the cohmll Allow the illate to pw through it i modaue drip 1lld collect Discutl the C18 oolumn
7 When elution is finished pbcc the tube C011tUning the cxt3C1 onto the N-Evap and blow it doll-n to approximately I mL middot
3 Wuh Ibe siclcs o(the tubewith ipproximauly I mL methallol md blow down to approiimately 02 t 05 mL Rcpat with anathcr 1ml wash uid blow down 10 dryness lmmcdiateJy $IDJ lhe nitrogen Dow and mDOVC tbc lllhe from the N-Evllp
9 Add~ 20 mL ofHPLC or Milli-Q ~to the tube conWning the evaporited Cl8 eluate Voncx tna or 5 secends md pbcc iilto the ultruonic balhAllow the samplt r=nain in the bath (srt at JO t0 45 dtgms) or S to 10 minutes
10 Filter ipproxllmrely 10 mL of this finaI voluc~ ~ollioo through a 02 micron AcroDise liltcr into m appropriate wtosamplcr vial Dp
I I This sample is now tfJJi (or LCMSMS malysis Ir eio be stored iorup 10 3 wrb in a refrigerator at 4 bullc
429b Canceiitrttlon attd Purification Procrulurq (or Soil
Solution RcquircnentsSamile (extraction+ purification Millimiddot Q Vllet 50 ml Extr1cioo solution 20 mL methanol middot 10 mL hence 10 mL 9010 acetone 3 mM Acetic Acfd 10 ml 9010methanol3mMAccticAcid 10 mL
SA1JILE CONCENTRATION ~D PURlFICATIOl
SAMPLE PREPUATION AND EXIlACTION
1 Remove samples fnim re6igeruor or frecr and illow to wimi co room tenpenrurc 2 Weigh out a 5 g $3IDplc oCthe soil and place it into a SO mL centrifuge bottle 3 Fonify samples it requlrcd 4 Add 20 mL o(OIM KHP005M NaO cnxtion solution MmwJly shake 10 times
Pbcc onro a wrist-action siakcr and smke for 10 minutes at tnUimum speed 5 Centritiige the simple for 10 minutes at approximately 2500 rpm 6 Atuch a 20 mL syringe 10 a OAS pm Acrodi$c filter Dccant the supcnutent into the 2
syringe Filter into a SO mL centrifuge tube Place tube immediately onto m N-Evap u 40 10 SO bullc md begin cviporition
7 Rcpcit steps$ through S When there is suffitlcic room in the SO mL centrifuge rube (on the N~Evip) filler the slaquoond supcrmccitinto il Rinse syringe with 2 x 10 ml volwncs of icronc ind place icctone into the 50 ml cenaifugc rube concining the fiJtrllC
S Eviponce the combined extractrinse to the witcrphise (3pproxinutcy 15 mt) 9 Add HPLC water witil the volume is ipproUmltcly SO mL I0 Jwt prior 10 procding 10 the SPE dcinup sooici1c cttrac for 5 min ind vonex mi-
DRFf 10
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136
DuPont-2548
DuPont-2548
DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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137
DuPont-2548
DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
- 41
138
DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
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DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
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bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
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------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
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DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
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149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
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150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
DuPont Rort No 92
1 Phcc the J5 g EnviDrb Bond Ems~ into the vacuum wtlfold md condition hett by placing 10 mL of9010 acelOllcJ CL ac1ic icid solution into the tubes and adjusting the flow tO i fast drip (-10 mUmizutc) Using i geule vac-Jum to pull through ill oftbc liquid let the column dry for 30 smmds after the Wt of the liquid bu exited Load and condition the columa wilh two 10 mL volumes ofwate Do not let the liquid level drop below the top of the sorbctt aAcr the second Mter oid has passed through Do not
allow tbc mbc to dry 2 kQ9 the soil attiet onto the colucm m scvcnl portions ifnecessary due to column
rcscvoit apaciry Tum ot1 the VXmD and allow the sample to pass through the column at i stow drip Do t10t allow the eolmm to go drj Do nol collect the liquid
3 Place S mL ofvnterimo tbc empty SO mL CC1aifilge tube vona Place this rinse onto the Bond Elm and allow tD pass through at a slow drip Pull all the water through allow to dry passing air through iorabom30 seconds aftc Wt drop w come of[
4 Place 5 mL ofhccme into the Bond Elut and allow to pus through at a slow drip Allow to dry passing air tbrooth or 3boul 30 seconds tfter last drop hu come oft
S Opo the vatrum manifold andplac plastic 15 mL ceiaiPlgc tubCs into the manifold io order to coOeet thC euaJc
10 Place IOmL oI9010 aotoue3 mM ~acid sclutiun into the Bond EJut rube md allow to pass through ac a slow drip Pull all ofthe liquid through into the collcetion t11bes BrW the nammmiddot md remove the tishcs containing the eluate
11 Place sample oc an N-Enp md cvaporatc to approxiimcly 1 mL at 40-50 deg 12 Wash the sides ofthe sample trstaibc widizpprmimately 2 mal ofacetone and
imapo~ the cx=cc to 02 to 05 mL ~this Wl$lUng yith a seond 2 mL volwe ofacetone Evaporate the sample mdryness ~
13 Add 20o )11 of acetone to die tube vona andsani~ for 5 miiiutes vortex again Add approximately I mL ofwater md cnporaa to the water phlsc Add approximately 4 ruL of HPLC water and comimic the evaporation step to ensure removal of all trtees of acetone Ibis is a very cridal step the presence of any acetone llilJ prevent the polar walytcs from btinc rcbined on the C18 column in later steps)
10 Adjust to ipproximately 5 mL with HPLC water Vortex soniclte for 5 minutes vortet
11 Proceed directly to the Cl8 SPE cc=-up lhis is a stepping point Samples =i be placed into the rcirigmtor for stongc for up to I weei
Cl8 BOND ELTff ClEANUPFNAL PlEPARATION I Place the CIS Bond Eiurl tubes oDtD the 2CUUID manifold md condition them by
placing 5 mL omethaaolJmM Actic Acid (9010 vlv) solution in10 cich tube md adjusting the Dow to a ~drip Drtia W tube uid illow to dry for 30 seconds more under vacuum In the same manner iondirion next with 10 rnL ofwater in two S mL inacmcntS waiting until the first 5 mL is complcrely below the frit before adding the second S mL Slop the flow when the level oftbc witer drops slightly below the top o
the sorbeit in the bortom rube Do oor let the tubes dry 2 Pace the Eivi-Cltb duarc solution (step I I ENVI-CARB dcuiup above) in10 the ClS
Bocd Elurl rube Apply vacuum open the siopcock llld loid the cU3ce onro the column with 3 vcrv slow drip Slop wbcu the level of the liquid is l rm above the top of the scrbcnt Discmi the aqueous cfflucll
3 Backrinsc the original Envi-Cirb chute coccin~r by pbcns O mL ofwltc~ into it vonet mixing for a few seconds Pour this rinsuc into the CI S c1Ctridge Allow this
DRAFT 11
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DuPont-2548
DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
- 41
138
DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
- 42 shy
139
DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
- 43 shy
140
bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
(rinsc)watrr wash 10 pass through the Ct3 ~ discrd Allow ill of the wu- to be dnVU out of the ubc Imntciiltdy shut oITvacuwn disord loid voiwic
4 Place 50 mL ofhcuoc into the Cl8 c~dgc Allow the hetlDc o pass rougi1 lt l mode2te drip fie the hcuoc has compietcly passed through the ccluc Jdd another 50 mL ofhcUllc md allow Wt to compictcly pass through the column u VCU Allow the vacuum to pull air throu~ the cinridgc unril lhac is oo ClOre drippiDg
5 BreU V3Clum and place a -ocw 15 mL pWtic cnaiiugc rube into the ncwtlIll 1Il3nifod under the C18 tubes to coUe the eluate
6 Plu 10 tnL ofmethanol3 mf Acetic AQd (9010 viv) solution iot0 the column Allow the eluate to pus through at l moderare drip md colleet Discrd the Cl g
column I0 Wbei elution is finished pbcc the tube conoining the extnct onto the N-Evzp at1d
blow it down to rpproximattly I mL l 1 Wah ~side$ oftbc nbcwitz oipproximalciy l mL nethmo ud blow doU to
approximately 02 t 05 mt Rep=c with mother 1 mL wash and blow down to dryncu Immcditely ~top the nitrogCl Dow and remove the tnbc frcip the N-Ewp
12 Add enctlv 20 mL ofHPLC or MilliQ wa~ to the tabc containing the mporued Cl8 ehutc Vonet mix for S seconds uid place into the ultrasonic bath Anow the umplc to remain in the bath (set it 30 to 45 degrees) for S to 10 minutesmiddot
IO Filter rpproximatefy 10 mL of this fiW volume solution throusb a 02 niic=ol AcroDisc filler into m appropriitc autosamplcr vial Up
11 This snqile Snow ICldy for L_~ISIMS analysis II cut be stored for up to 3 aleeks in a rcfrigcritor at 4 bullc
JJ Insrrumouarion
43 Descriotio
Tais method requires an LCMSMS instrument equipped with m eccrospray intctiie for the mtlysis of water md $Oil An au1osamplcr is also required for the umttcndcd analysis of multiple samples and clibritioo solutioll1 The ~ytictl d3ta in this method wou obtiined from a Micromw Qurro ll LCMS equipped vith th HP Series I 100 Modular HPLC System which incudes a dual chmncl ptmp auoumplcr vac-1um degwcr tempcruurc-ltoncrolled column compartment md a UYmiddot Vis d~~ctor (oot required for tbis method) The HPLC md Mau Spcetromecr opmcing parunetm for these systems are given in secion 43l Tae chromacogripiLic colWilll md mobile phases specified provide good peik shapci and resolution of the anal1es nu illows sufficieit time to switch mass spcenlmetric ic_quisition paruneters thc~ly illowing better opcimttition of cich clwmeL Tue HPLC solvctrit progr=t includes a period ofhigb-orJmic solveitshyflow to purge miaiX materiUs Crom the column md allows sufficient rc-equilibration time bewCQI runs Do not ilter these periods In genertl instrument response is good to excellent for the milytcs ca the positive ESI SR1 cwmes AJways use the combinirion o(HPLC coaditioas md mus spccromcter pinmecrs specified for ocb ouaii
43J Oaeranmiddotng Conditions ficromass Ouattra ff with HP Series I (00 HPLC
A)IALYnCL CONDmONS FOR SOIL AID WAER
High Performance Liquid Ciiromaroraphy (HPLQ ~fobil Puse A iqueous 001 M icric 3cid
~fobiie Phise 3 Actonitrile
DRAFT
- 41
138
DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
- 42 shy
139
DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
- 43 shy
140
bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
Mobile Pusc Prognm
OiKohmm Flow Rlu Injcdon Volume Cohmm
Mass Spectrolltd6 (MS) onizition Mode HYLltm DpiDary Voltigc Cone Volbge Collision Voltage
Gcpeg1 pmmetn CollDioo Gas~ MSl LWHM Rcsahmon MS21-WHM R=olution Som= Tcnper1mr=
Acauisition Funedoru
ES+ Stut 70min Eod 95 Int Chan Delay 002 sec
2 ESP+ Start 95min bl 120 lDt ClLm Delay 002 sec
3 ESP+ Stut 120min Eid 170
Im Om Delay 002 sec
DuPont-2548
Tmefmic) A 8 0 100 0 3 90 JO
10 50 so 150 25 75 ISI S 9S 200 5 95 201 0 100 300 0 100
lO mUmin split-51 Divirt ~9 155-JO mm O microL Zoriiu RX-CS 25 cD x -t6 me id S microm with guard column amcud o ~c
P~siti-e ESI 026 1Y 40 iCV 32 Volts 18
Positive ESt Modes 17-20clOc-3 both 10 both 12 85degC
set lS shown below
Pireot Omehter Dwell 1711 71l 012 sc
P=nt Dauehter Dwell
2SSJ 1571 012 sec 2672 1710 012 269J 1710 012
P=n Dauehter Dwell 2392 1570 008 2S32 171l 008
Approrim11c Retendon Times of the 7 Anilytes
Anilvte Acerolt Reelrion Ttme fminl Acouisition Func~onTri-sirion
i=eibolite G3170 8S9 l 1711 -710
mctlbolitc A 1068 2 2693-1710
mccbolite A- l 1162 2 2693-lilO
DRAFT 13
- 42 shy
139
DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
- 43 shy
140
bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
Duont Reort ~o 2192
mebolicc I 1127 2 26i2-lilO mctlboli1e C 1012 2 2553-1570 hctitinODC 1446 3 2532-1710
mecbolitc B ]J62 J 2391-1570
Alternaie tan Spemotnaa (lS) Condbioru Confvmariotr Cuvinels
If ccreluting matrix compooCll izuericc with lJf of the maltt= on their primary positive dccttospl3y MSMS clianncls We the follooving ceoo~y cooditiocs and ~troute cllawics tltgt both confum th3r these co-eluting pciks ire in fact intcicencs md to qiuntictc the malyteS AltCrllte cwuicJ cui be det=mined from cumining the full scia speel or the daughter ion specn o(och malyte shown in Figure 2 The alfematc efeerosprly ~tel (mmascd coUisioa ps energy specified will also significantly rednce or climimtc the ialtrfcrin cks co the origiml chanoels ~ analyu samples using hi 01ltematc clmmcls along with the originals far~
Ioaiz3tioo Mode Positive EST HVL= 01 KV Dpiilary Voltag )6 cv Cone Voltgcshy 25Volts Collision Voltage 30
Gemicromln~ Positive ESI Collision Gas Pr=slIre 17middot20ilOc-J MSJ LMIEfM ~lutiaa bothamp v1S2 LdEM ~oiudan both 12 Source Tcirpcmnre ssmiddotc
Acauisition Fanctions set for ccetd altauite MR1 tnruitioos
433 ClllbDtion Procdtttcs lcsttumcitltion perfonnanc was UUrlally Cliocd for cWbrition response tineuity by the following proedurcs
I The LCIMSIMS lmrrum=t wu ttmed izI porilive elecrrosptay mode ro optiatize the (Mshycyclohcanc moiet)r peU ~still showing the base pelk [MHf For the negajivc
clectrospcay compounds the mwment wu tw1ed to maximize the [M-1 ] peak 2 Fullmiddotscm cmcohmm amlysis oa Ot 4-pgm cilibritioa solution cootiining eicb othe
amlytcs ~cs performed to VCiy appropriate mh ruponses and abundance ritios 3 Analysis ofcWbracioa soJuQcm (so seOoQ 2J forpnpuation) aad coastructioo of~
5-poim olforuion curve was ~Onoed to verify liocuity (R1 2 098) over the walytici1 13Ilge ind ~uue ~tor rspoosc (low etltbritioa solution response 2 5l signal-toshyuoisc) middot middot
Routine rnmuf~ imtruman cWdegbrltion lid =iatetW1c proced~s should be followed as necessiry 10 oprimi pctfomunc It is ilso oessary Ugt asure th2t the eicctrospriy interfac components ~e cleui A dirty source cin ciuse drift in insttUrnellt ~use
_ J Sam~le 4nalvris
Initi1Uy it le1St I 3Dd prf~bly ~ o( l =Jait-conuining simple should be nude 10 equilibrue the ESI-LCMS system prior 10 rurmicg i sunple u-unlcu instrument p~rformmc illows otherwise
DRAFT 14
- 43 shy
140
bull
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
DuPont Repon ~o 2192
Calibncion solutions and samccs should be Utm31lly maJrd through the lndyTis set so test satm1les cin be qumtified using the 3v~gc ~onse ofbricUtiDg sundlrd (Le SDtldud S3mpie stJDdud sample stuldard etc) I instruulint peOrucc is sable t is prnnimiile to run 2 samples benveen each standud provided a stand2rd prccdes the st sample of the ser and foUow-s the bst umple of the set Ac 3
minittwm a check sample et1nsisring of i control fonilied u tbe LOQ and processed through the method should be nm with every sample set
JJ CalCJlhzzions
J_J_J Metlimb
The ppm found and pecit of applied recoveries m dctcmined from cikmiddottlacions which compau the peik ttea ~oMCS of C1Ch malytc to the r=sponsc oCbnditing mayticil stuldards lJsc 2l thc primzry MRi clwmel for qumtitation Do oqr use the Toal Ion Chronutomm Peik uei response values should be ~ in an EXCELbull sprodshm tcnplate designed to pctfonn the appropriaic calcnlations middot
The response factor (RF) is calculated far e3Ch analytc in all alilmtion stmdard analys~ _by diyjding the pcl ~Cy the malytc cooc=o1tion Oic may us either the aYeragc ~ase factur ofthl nCte set of nandarrls if the response lw beci scblc over the period owa1ym or one may we the ivmge oCthc closest two standuds wftieh bnclct the sample being quantitatcd if imtrumcnt drift has been noted In either C5e valid recovery data are genented if the ~cu relative modml deviation ooRSD) for the peik arci ttSpanses of the stmdards used is less than or equal to 200oVzlues for the amount (ppm) deteccd msamples are decmined by comparing the pe3k uD ofC3Ch szmple inalytc detected with the average ~rue poundactor and eoaccting far ~CSMight ofsample md fica1 volume The pc=1E or ipplicd reiovelcs for fortified ssmples are =Jcubtetl by divictmg ppm foUJld by ppm applied and multiplying by 100 middotbull
C (Cline of sample microgmt) bull Aro(sample)RF(avg)
ppb (s=ple) bull CW bull(IAF)bull FY
W bullweight orsamile (g I mL bullJg) AF aliquot mcion (vol iliquotltotal vol) FV bull toaI volume of fiml m3lysis solution (mL
Sample UlcuJations (Bnclrting Standmis) For a h)potheti~ analytc
20 mL (bull20g) wucr saznlc fonifjed it OtOOppb aliquot fraction - I FiIW Volumebull 2 mL
Response of067 ngiml Stdbull 700 arc counts Response of20 nglmL Stdbull 2200 area counts Response o(0100 ppb fottifiatiosi bull 1200 Uel coUJlts
RF(avg)- [(l0020 ngmL) + (00067 ngmL))l2 bull I072(mUng) Co~ samplcbull 1200il07(mUni) bull 1119 oiymL AFbull 20mU20mL bull 10
ppb samplebull (1I 19 (agmL)f03] bull(II)bull mL O l l l9 ngg Ol l 19 ppb recovery O l l l90I000 bull 100 bull 112~~
DRAFT 15
- 4-l shy
141
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
------ ~--------
DuPont-2548
DuPont-2548
DuPont Rpon No 2292
FIGURE 1
CHEMICAL STRUCTURES AND NAMES
f-l=zincne QfTtCnllbmiddot DPXs4 cemra Nare= S-TriAzine-24-1H3HCcre 3shycc0~ttrltamro-1gt
cc~~MJmiddot 51225-04-2
MVtdileA 01tpnt Cccbull No middot T3937 ~ltzm STriaiine-2A-1H3H)-cfione 5shydimethytamino-JLlrjdcrFco- ~exyl-1-meh)i
Cl$ gr ~lamiddot none
Maoite A1 0 bullPT c-re 11b bull G3453 - ~
Omira N37g S-Triozire-2411i3iltJcre (raSshymiddot~da)i-~
Mtabclite a D middotPnt Oxbull fii_ IN-AZSt8 c-etira Nare S-Triazirn24-1H3HltCt1e 3shyc~l~irelllylltlrinor C1-S Pecentryen r-n bull 56611-54-2
MitcColileC DbullPmt CXbull middot IN-r~ c=enjrf Nrre 5-Triatine-24-(1H3Hdcre 3shy(~~1-mgtlt1micro (rrettrltanror
DRAFT
- 51 shy
148
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
FIGURE 1 (CONTINUEgt)
CHEMICAL STRUCTURES AND NAMES
OB ~mec(11 tt 0 p-rj rx N-03454
~~ CeTic llarrr S-Triaine-24-1HlmiddotiC~ ~ (2~cilpoundlt)1)-1-mgtihygtS- (~re-
o~NSCS3I ex
lkadiieD p10nt Co(bull Nabull JN-82a8
middot CAS R=fstty Na middotnone
=~ IN-~936 Obullir Nam S-Triazine-246-1HP5Hshylrione 1-4 hydroxyCtohexyf-3-methyi
Mt~iteF D fTt Ccrje rn JNl3221
ea Narnr S-Triazine-24-1H2HCcre 3-cjd~ 6 a1Uio-1~
c+c Pfcentttyen middotrme
MtcbdileG Deurorrif CTbullmiddot IN-T4916
Cwrid NzrreS-Triazine-24- 1HHdcre ~C~lhjlanino
CAlt psy middotrcne
ORAFJ
- 52 shy
149
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
DuPocc Ron -lo 2292
FIGURE 1 (CONTINUEgt) CHEMICAL 5TRUCTIJRES AND NAMES
~ileH a cv ere t-h bull rN-iw111 Cfnioi Nrrre STriazire-24-1H3hdae 3 oC~ cs Rfdstcy rh bull rrre
MtDlite 1 D ext DTP th l1CS472 C=eiira rfrrg5-Triatire24-(1H3H)-Ccre 6drratyenaJiro) -1-mltyen-3-(4shy=d~) CA~ 9-tjSQrfbullbmiddot rcre
Mtadite G31i0 DexfCXPdeg 1N-G3170
c=mid Naxe STriazire-24-1Hh dcne1-ltrelh)j~ arirn
CAC pstcy fb rcre
rvttadile G31i0 n-gfuooside D fTt CaP Nomiddot IN-NCS33
DRAFT 24
- 53 shy
150
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
DuPont-2548
DuPont-2548
Modifications to Duoonbull-2550 to confinn bv LCSMS
If sample splitting is needed to allow confinnation by LCVS7S the l6xl00 mm silanized glass tube used in step 4A5 must be calibrated at 20 ci as well as 40 mL
Follow the method (Met-93 revision 4) through step 451 At sep 45 insert at end Adjust to 40 mL with methanol vortex to mix sonicate 5 minutes Readjust to 40 mL with methanol and vortex Relove 2 mL for LCMSlvS if reqcired
Evaporate to dryness Continue with method DuPont-2292 (soil md water LClvSMS method) final preparation step 429 CS Bond Elut CleanupFil11 Preparation step 8
Standards for LCMSlvS analysis may be prepared from standard solutions used for GCNPD analysis by evaporating the organic solvent and redissolvllig the residue in water with mixing and sonication
- 62 shy
159
