Poster 8B-31IUPAC

Seventh International Congress of Pesticide ChemistryHamburg, Federal Republic of Germany

August 5-10, 1990

DESIGN OF AN HPLC SYSTEMUSING POST-COLUMN DERIVATIZATION

and FLUORESCENCE DETECTIONOPTIMIZED FOR THE HIGH SENSITWITY ANALYSIS

of CARBAMATE PESTICIDES

McDonald, P.D.*; Sims, A.E.; Leveille, W; Wildman, W.J.;Hansen, J.; Zener, V.; Morawski, J.

Waters Chromatography Divivision, Millipore Corporation34 Maple St., Milford, MA 01757 USA

Use of liquid chromatography with post-column derivatization andfluorescence detection for the analysis of carbamate pesticides was firstreported by Moye and co-workers in 1977 (1). More recentlv thistechnique has formed the basis for U.S.E.P.A. Methods 531 and 531.1 fordrinking water analysis as well as the A.O.A.C method for crop residueanalysis.

We will describe the design and development of an integrated system forcarbamate analysis. Some special features of this system include:

O a special column and separation conditions which yieldoptimum selectivity, for the eleven analytes specified in theEPA methods;

[3 new post-column derivatization hardware componentsincluding a proprietary reaction coil and patentedcountercurrent heat exchanger designed for maximumsensitivity and reliability;

[J a new, highly sensitive fluorescence detector with a xenonsource.

The ability of this newly developed chromatographic system to exceed theEPA performance standards and lower detection limits forN-methylcarbamate and N-methylcarbamoyloxime pesticide analysis byan order of magnitude will be demonstrated (2).

(1) Moye, HA; Scherer, S.J.; St. John, P.A., Anal. Lett., 10, 1049-1073 (1977).(2) McDonald, PD; Leveille, W; Sims, AE; Wildman, WJ; Zener, V; Scarchilli, AD,

Advances in Water Analysis and Treatment, Proceedings Volume, Water QualityTechnology Conference, Philadelphia, PA, November 12-16, 1989, American WaterWorks Association, Denver, CO, 1990, pp. 631-649.

i

. GoalI II I

To increase the sensitivity

and the reliabilityof an HPLC method

with on-line post-columnderivatization and

fluorescence detection

for the high sensitivity analysis

of carbamate pesticides.

ApproachI

Improve reversed phase

gradient LC separation.

Optimize post-column reactionconditions

Integrate improvements in

post-column reactor design into

a complete analytical system.

Optimize detection parameters.

ExperimentalCarbamate Analysis System

including:WatersModel 600EMultisolventDeliverySystemController;

Fluid Handling Unit with integral post-col'un_ reaction system;Post-column Reagent Delivery. System;

Temperature Control Module & Column Heating Chamber;Model 470 Scanning Fluorescence Detector

Carbamate Analysis Column3.9 x 150 ram, 4gin spnerical

octadec'ylsilyl-bonded silica packing

Milli-Q ®Water System

HPLC grade solvents, highpurity reagents & standards

LC SeparationCompare EPA Method 531 &

NPS Method 5 separations withour improved ternary gradient.

Baseline resolution of all analytesusing small volume, high efficiencycolumn with optimum selectivity;

Resolution maintained with sampleinjections of one milliliter of water;

Two-fold increase in sensitivity withternary gradient using acetonitrile;

Twenty-two minute analysis permits' two sample-per-hour throughput.

EPAMethod 531IIII I I IIIIIII IIII I I IIII

DeuteriumSource235 nmex _./419 nm em_.

5 Methanol/Acetonitrile [4:1]20% to 70% in water

Lineargradientt_Bondapak"rMC18packing in

23 RadiaI-Pak®Cartri_dge7 9

6 1- Aldicarb sulfoxide2 - Aldicarb sulfone

' 3 - Oxamyl

L 4 Methomyl

5 3-Hydroxycarbofuran6 Aldicarb7- Carbofuran8 - Carbaryl

k._., _, . _ .., 9 1-NaphtholI I I I

0 S I0 Ib

M_nule2$

EPA-NPSMethod 5

DeuteriumSource230 nm ex _./418 nmem _.

Methanol/Water15:85 to 100:0 in 32 min

, LineargradientUltrasphere®ODS 5 _m

4.6 x 250 mm

2 _ 1 - Aldicarb sulfoxide2 - Aldicarb sulfone

Decomp. Product of 13-Hgdroxycarbofuran

7 - Aldicarb8- Propoxur

I 9 Carbofuran_ 10 - Carbaryl

,o,_.,._,._,,...,e,i 11 1-Naphthol12 Methiocarb13 - Internal Std.

@1990M_o_e Co_o_rallon

Analytes in EPA Method 531.1 "II III I IIIIII IIIII III IIIII

| O CH 3 2 O CH 3 3 O 4II I 11 I II

CH3-S-C-CH=N-ORI CHa-IIS-?-CH:N-OR (CH3)2N-C--C:N--OR, CH3-? :N-OR

CH3 O CH 3 SCH 3 SCH 3

Aldicarb sulfoxide Aldicarb sulfone Oxamyl Methomyl[Standak_) (Vydate_) (Lannate_)

H 0 CHa-S--C-CH:N-OR O O

3 CH3-/xCH3 C 3

3-Hydroxycorbofuran Aldicarb Propoxur Carbofuran[Temik®) (Baygorx_) (Furadarx_)

H3 --C-NH--CH 3

Carbaryl 1-Naphthol Methiocarb(Sevin_) (Mesurol_)

Optimized HPLC Separation-- - IIIIIIIII I II I IIII III I IIII ||1111 I I II III III III I II I

Carbamate Analysis Column Model 470 FluorescenceDetector0.39 x 15 cm 339 nm ex _/445 nmem

Water/Methanol/AcetonitrileComplexgradient

9 25 ppb Standards400 IlL Injection Volume

2 5 10 ng of each analyte oncolumn34 6 7 ] 11 1 - Aldicarb sulfoxide

2 - Aldicarb sulfone3 - Oxamyl

10 4 - Methomyl5 - 3.Hydroxycarbofuran6 - Aldicarb

__ 78- PropoxurCarbofuran

L _ L_ 9 - Carbary!, _ -" -- _- 10 - 1-Naphthol

0 5 10 15 20 min 11 - Methiocarb

" One mL Sample CapabilityI II I I IIIIII IIII II III IIII I

CarbamateAnalysis Column Model 470 FluorescenceDetector0.39 x 15 cm 339 nmex _./445 nmem

Water/ Methanol/AcetonitrileComplexgradient

1 ppb Standards1000 I_LInjectionVolume

9 1 ng of each analyteon column

: 2 - Aldicarb sulfone

- OxamylMethomyl- 5 - 3-Hydroxycarbofuran........ "-- -- -- -- 6 Aldicarb0 5 10 15 20 min 7 - Propoxur

8 Carbofuran9 - Carba.o,I

10 - 1.Naphthol11 - Methiocarb

Higher Sensitivity with Ternary Gradient

Water/Methanol/Acetonitrile Water/Methanol only

5 Compareresponsefor last

9 sevenanalytes.6

3 1 1 2

Post-Column ReactionConditions have been

optimized for two-stepsequence.

Hydrolysis is done at lowertemperature & reagent concentrationthan previously published methods:

0.0125 M. NaOH30 seconds at 80° C.

OPA/ME: "more is not better"

OPA/ME mixed with analyte streamat 80° C.; cooled to ambient

temperature in < one second prior todetection.

Reaction SequenceIIIII III II IIIIIIII III I II IIII I II I II II II I

Step 1" Hydrolysis

0it Aq. alkali

R-O-C-NH-CH3 A _ CH3NH2 + R-OH + H2CO 3N-Methylcarbamate Methylamine

Step 2: Derivatization of methylamine with 2-mercaptoethanol ando-phthalaldehyde

O + HS-CH2CH2OH S-CH 2CH2OH

"C-H Aq. alkaliC-H + H2NCH3 " -CH3IIO

ImprovementsLower OPA concentration &

rapid reaction reducesbackground noise &

fluorescence quenching.

Lower temperature reducesdegradation of OPA reagent &

isoindole product-background noise is lessened &

higher amount of derivativereaches detector cell.

Reactor DesignHigher flow rate- 1.5 mL/min

3:1 & 4:1 dilution ratios of

first & second step reagentsolutions, respectively.

Proprietary knitted opentubular hydrolysis coil.

Patented countercurrent heat

exchanger used to preheatreactant & reagent streams, cool

derivative stream- consistent

reactions; less degradation ofOPA & isoindole once formed.

RXNTM1000Reaction CoilI I IIIIIIIII IIII II II I I

[3 Proprietary knitted open tubular design

CI Compact linearshape

El 30-second reaction time ~1'80°turns, l..90_ _Jout-of-plane

[3 Minimal bandspreading

- typical crfor entire system < 50 _L

CHEXTMCountercurrent Heat Exchanger*

Solder sheathover gent #1

Copperplate

gent #2

Steel wire wraover

Stainless steeltubing imn out

ictor out

"US Patent No 4,284352

: Fluid Path SchematicI1| I I IIIII II II II

Two-StageSystem Controller Post-Column

v r.............................., Reactor ... RXN1000ReactionCoil..... ".....( II [ _..+,,,,,,,.-.+.._.....,,..,..,,,,,,,,,,,._;,,.+MUlllSOlVenl" I _ _ _ / I_ _ _.- i_--CHEX Heat ExchangerDelivery 12,_I_ [

syst"e_ ' ' f_ h ,_ *_"_"_ DataF,u,_.o_.n0i co,umn''_oo.'' r.....S*o.on

Unit _'i _-_ /'"°"1/I"OPA t _ Waste

water--_d VJI I:ft_.__,,,o,ib--11' _IMeOH__l "r I I t ! _ _ I I "'1

ACN_ Injector ; Reagent Fluorescence

I-Joeve°.te.,oTemperature SystemControl

Detection Parameters

Xenon source for higherintensity at optimum ;_.

Excitation at --340 nrn is more

selective, gives less interferencethan--230 nrn exc. used with

deuterium & pulsed Xe sources.

On-line, stop-flow ;_scans usedto select best compromise inemission & excitation _,'s for

maximum sensitivity.

Detector Optimization

Emission

Begin WavelengthScan : Scan

, / _,maxh,V 447 nm \

il i/ _J _ End

ih _ _.____LLSconI I I I I _

Inject 349 400 500 600 nm

Detector Optimization

ExcitationI Wavelength! Scan

i 't '

i

BeginScan

_,max

J 337 nm EndScan

_j L__J ,..

I I I I

210 300 400 440 nm

Method Results

Excellent reliability &reproducibility.

Precision & limits of detection

superior to standard methods -LOD's lowered by as much as

30-40x.

Signal-to-noise measured byinjecting one mL of

10 part-per-trillion standardmixture.

System PerformanceII

Waters CAS Linearity & Retention Time ReproducibiltyRetention Time

Unearity Retention Time ReproducibilityPeak #/Analyte (R 2 ) (min) (% RSD)

1. Aldicarb sulfoxide 1.000 4.42 0.272. Aldicarb sultone 1.000 5.35 0.21

3. Oxamyl 1.000 6.04 0.254.Methomyl 1.000 6.87 0.23

5.3-Hydroxycarbofuron 0.996 11.37 0.076.Aldlcarb 0.998 13.13 0.09

7.Propoxur 0.998 16.31 0.118.Carbofuran 0.999 16.92 0.I0

9. Carbaryl 1.000 19.03 0.0810. 1-Naphthol 0.999 20.19 0.041I. Methiocarb 1.000 2_2.09 0.03

l.ineartty: Peak area vs. ng on column. All linesintersect origin: 400 I_Linjection volume, 0.25 to100nanograms of each analyte Injected on column.Retention times: Average of 16 replicates, 1000 I_Linjection volume, I ppb: 24 hour systemshutdown inbetween each set of 8 runs.

Comparison with EPA Method i

IIH |lJll Jl II IIIIII Bill

Typical Recovery, Precision & Detection Limits

Recovery Precision Detection Limit% % RSD i_g/L (ppb)

Peak #/Analyte EPA Waters EPA Waters EPA Waters

1. Aldicarb sulfoxide 47 101 21 3.2 2.0 0.22. Aldicarb sulfone 83 100 20 1.3 2.0 0.2

3. Oxamyl 82 106 17 2.7 2.0 0.254. Methomyl 102 100 18 3.5 0.5 0.35. 3-Hydroxycarbofuran 108 100 29 1.9 2.0 0.26. Aldicarb 107 102 7 2.9 1.0 0.3

7. Propoxur 101 99 32 1.1 1.0 0.38. Carbofuran 90 99 12 1.9 1.5 0.3

9. Carbaryl 97 102 23 1.9 2.0 0.210, 1-Naphthol -- 106 -- 6.6 - 0.6

1I. Methiocarb 82 103 19 2.2 4.0 0.3

Samcxe:SpkeaReagentWaterLoll3).ConcJflo_: EPA/NPSMe_ 5: Spike ieve_ at EDL.range from 0.5 to 4.0 v,g/f,..:7 or 8 repllcat_; Inlectton volume: 400 _LL:SIN - 5: I.Waters CAS: Solke levelsall 1,0Izgli-: 8 reDllcates: Inlectlon volume: 1000Id-:470 detector: ex-339 nmlem-445 nm:SIN - 15:1at cletectton llmlt llsteclalDove.NOTE: Resultssr_ownare for a single operatorllaOoratoqy: your resultsmay van/. clepenalng upon operatlng concllttoas.Refer to EPAmetrK)cl fo_acceptance criteria.

Acknowledgments| II

We would like to thank the

following individuals for theirhelp and encouragement:

Uwe Neue, William Carson,Tad Dourdeville- Waters;

and especially:Prof. H. Anson Moye- Univ. of

Florida, GainesviUe,Richard Krause- US FDA,

Washington, DC,Prof. Heinz Engelhardt- Univ.

of Saarbriicken, FRG, uponwhose pioneering work this

method is based.