LC-MS/MS Analysis of Mercapturic Acids: Addressing ... · -3-HPMA (italic) differ by more than 10%...

INTRODUCTION• 3-hydroxypropylmercapturicacid(3-HPMA),

2-Cyanoethylmercapturicacid(CEMA),3-hydroxypropyl-1-methylmercapturicacid(HBMA)(Figure1)areurinarymarkersofexposuretoacrolein,crotonaldehydeandacrylonitrile,respectively.

• Theseglutathioneconjugateshavebeenshowntobemarkersoftobaccosmokeexposure.

• Selectivityoftheanalysisistypicallynotadequatelyaddressedinamajorityofpublishedmethodspotentiallyresultinginsignificantlyelevatedvaluesofsomebiomarkers.

• AselectiveLC-MS/MSmethodforsimultaneousanalysisofCEMA,3-HPMAandHBMAinhumanurinewasvalidatedusingRPUHPLC.

• Thechromatographicseparation,selectionofMRMtransitionsandthechoiceofstablelabeledinternalstandardswerecriticalfactorsintheselectivityandreproducibilityofthevalidatedmethod.

Figure 1. Chemical structures of mercapturic acids (3-HPMA, CEMA and HBMA)

Sample pRepaRaTION• Aliquot(0.100mL)ofhumanurinewasspikedwithinternal

standardsanddilutedwith1%formicacid• Sampleswereloadedona96-wellOasisHLBplate,washed

withformicacidanddilutemethanol• Elutionwasperformedwith20%acetonitrile

INSTRUmeNTaTION• Electrosprayionization(ESI)datawereacquiredbymultiple

reaction-monitoring(MRM)innegativemodeonanABSCIEXAPI4000tandemmassspectrometer

• Theacquisitiontimewaslessthan5minutes

LC-MS/MS Analysis of Mercapturic Acids: Addressing Selectivity and Matrix Effect Issuesalan Dzerk, Veni N. lapko, Ridha Nachi, Kirk Newland and Curtis SheldonCelerion, lincoln, Ne, USa

aNION exChaNge COmpaRISON meThOD• HPLCwitha3x50mmThermoBiobasicAXcolumnwas

operatedisocraticallyinadualcolumnalternatingbackflushconfigurationatambienttemperature

• Mobilephasewasamixtureofacetonitrileandammoniumformatebuffer,pH2.5

Figure 2. Anion exchange chromatography

Rp-C18 COmpaRISON meThOD •HPLCwitha3x50mmKromasilC18columnwasoperatedin

gradientmodeatambienttemperature•Mobilephaseswere0.1%formicacidandacetonitrile/formicacid

Figure 3. RP-C18 Chromatography

TheextrapeakpresentintheRP-C18HBMAtransitionwasinitiallyassumedtobethediastereomerpresentinthereferencestandard(Urisub),however,thepeakwasnotobservedintheISchannel.InvestigationemulatingthechromatographyofCarmella,etal.(Chem.Res.Toxicol.,Vol.22,No.4,2009),demonstratedthediastereomerswereco-elutingatthelowermobilephasepHbeingusedintheRP-C18method.

Figure 4. HBMA Diastereomers and extra peak-Phenomenex Synergi Max-RP

Upper-15mMAmmoniumacetate(native)/MeoHgradientLower-15mMAmmoniumacetate(pH5.3)/MeoHgradient

ValIDaTeD Rp-UhplC meThOD•AWatersAcquityUPLCwitha2.1x150mmC18analytical

columnwasoperatedingradientmodeat50oC•Mobilephaseswere0.1%formicacidandacetonitrile/formic

acid(Figure5)

Figure 5. Validated RP-UPLC Chromatography

ReSUlTSThesuccessfulresolutionofinterferencesonHBMAchannelwasobtainedusingaRPUPLCcolumnunderacidicconditions,withoutseparationofR,RandR,S-enantiomers.

Selectivityoftheassaywasevaluatedcomparingquantitationinmultipleurinelotsfromsmokersandnon-smokersusing2MRMtransitionsforeachanalyteunderdifferentchromatographicconditions

Major3-HPMAMRMtransitiondidnotprovideadequateselectivityusingion-exchangeorRPHPLCasshowninTable1.WhenRPUPLCwasusedbothtransitionswereselectiveduetoimprovedchromatographicresolution

Table 1. 3-HPMA quantitaton testing using 2 MRM transitions: (major MRM, 220a91 and second MRM, 220a89). Data used are means of duplicates using d4-3-HPMA as IS. Over-estimation of 3-HMPA concentrations by major MRM are shown in bold. RP-C18 concentrations were similar to AX and omitted for simplicity.

Table 2. 3-HPMA Quantitaton testing using 2 MRM transitions: (major MRM, 220a91and second MRM, 220a89). Data used are means of duplicates using 13C315N-3-HPMA as IS.

• Theuseof13C315N-internalstandardsimprovedquantitation

forRPUPLCanalysiscomparedtodeuteratedinternalstandards(Table3)

Endogenous level (ng/mL) Difference (%)

Lot# Major MRMAX

Second MRMAX AX RP-C18

1 1440 1380 104 104

2 3550 2270 156 1533 578 546 106 1074 1680 1630 103 1045 2770 1120 247 2466 324 327 99.1 1377 209 148 141 1008 327 328 100 1359 77.4 68.2 113 136

10 1130 972 116 10211 141 124 114 11712 250 244 102 14613 239 225 106 11814 1160 1130 103 11515 1180 1160 102 10016 1130 1040 109 10417 1520 1410 108 10118 55.9 32.0 175 10319 539 288 187 11020 4640 4830 96.1 107

Endogenous level (ng/mL) Difference (%)

RP-UPLCLot# Major MRM

RP-UPLCSecond MRM

RP-UPLC

1 791 799 99

2 1135 1155 943 33.1 32.6 1024 247 262 945 19.8 22.4 886 1110 1170 957 1735 1855 948 409 440 93

Table 3. Performance of 13C315N- (A), and d6-3-

HPMA (B), as internal standards in spiked quantification test in human urine. Data quatitating >10% different than expected are in bold. Basal level concentrations for lot 2, 7 and 8 using d6-3-HPMA (italic) differ by more than 10% from those concentrations determined with 13C3

15N-3-HPMA as Internal Standard

The validated analytical rangewas from 20.0 to 5,000 ng/mLfor3-HPMAandHBMAandfrom0.200to150ng/mLforCEMAusingUrisubascalibrationmatrix

• LLOQand3xLLOQconcentrationQCsampleswerepreparedinUrisub

• LowendogenousconcentrationQCsamples(QCA)werepreparedbypoolingpre-screenednon-smokers’urinewithlowconcentrationofthemercapturicacids

• Smokers’urinelotswereusedtopreparehighendogenousconcentrationQC(QCC)

• Theextractionrecoveryofmercapturicacidswasnotlessthan80%

• Theinter-batchprecision(%C.V.)andaccuracy(%Bias)ofquality-controlsamplesisshownin(Table4)

A

Lot#Basal Level

(ng/mL)

ExpectedConc.

Basal+20(ng/mL)

Calc.Conc.

(ng/mL)% Dev.

ExpectedConc.

Basal+3750(ng/mL)

Calc.Conc.

(ng/mL)% Dev.

Lot#Basal Level

(ng/mL)

ExpectedConc.

Basal+20(ng/mL)

Calc.Conc.

(ng/mL)% Dev.

ExpectedConc.

Basal+3750(ng/mL)

Calc.Conc.

(ng/mL)% Dev.

1 813 833 768 -7.8 4563 4660 +2.12 1160 1180 1150 -2.5 4910 4840 -1.43 31.1 51.1 53.1 +3.9 3781 3900 +3.14 225 245 256 +4.5 3975 4010 +0.95 22.1 42.1 39.3 -6.7 3772 3950 +4.76 1320 1340 1280 -4.5 5070 5160 +1.87 1960 1980 1850 -6.6 5710 5660 -0.98 440 460 446 -3.0 4191 4130 -1.5

B

1 795 815 828 +1.6 4545 4080 -10.22 902 922 860 -6.7 4652 3500 -24.83 30.7 50.7 49.1 -3.2 3781 3390 -10.34 227 247 268 +8.5 3977 4140 +4.15 21.3 41.3 38.4 -7.0 3771 3790 +0.56 1230 1250 1110 -11.2 4980 4260 -14.57 2330 2350 2300 -2.1 6080 6970 +14.68 536 556 572 +2.9 4286 5270 +23.0

A. 3- HPMA

LLOQ QC20.0

ng/mL

QC S60.0

ng/mL

QC A114

ng/mL

QC B296

ng/mL

QC C616

ng/mL

QC D 2470

ng/mL

Inter-run Mean (ng/mL) 18.9 55.9 112 292 607 2460

% CV 11.5 6.1 2.6 3.1 3.4 3.0

% Bias -5.5 -6.8 -1.8 -1.4 -1.5 -0.4

B. HBMA

LLOQ QC20.0

ng/mL

QC S60.0

ng/mL

QC A81.7

ng/mL

QC B265

ng/mL

QC C315

ng/mL

QC D 3710

ng/mL

Inter-run Mean (ng/mL) 17.4 56.4 82.2 265 322 3670

% CV 8.2 6.7 4.8 2.9 6.1 4.6

% Bias -13.0 -6.0 0.6 0 -2.2 -1.1

C: CEMA

LLOQ QC0.200 ng/mL

QC S0.600 ng/mL

QC A0.500 ng/mL

QC B2.53

ng/mL

QC C44.0

ng/mL

QC D 118

ng/mL

Inter-run Mean (ng/mL) 0.191 0.609 0.492 2.49 42.6 114

% CV 10.1 5.5 5.0 4.1 3.5 5.0

% Bias -4.5 1.5 -1.6 -1.6 -3.2 -3.4

Table 4. Inter-Batch Precision (%CV) and Accuracy (%Bias) of Quality Control Samples (Validation)

CONClUSIONSAnLC-MS/MSmethodforsimultaneousanalysisofCEMA,3-HPMAandHBMAwithimprovedselectivityhasbeendevelopedandvalidated.

aCKNOwleDgmeNTTheauthorsthankAlyssaPerry,ChrisKafonek,LeannaSchlesiger,JasonWenzl,BrandonRetke,MarzukiMohamed,JosephSilvaandWendiBarntsfortheircontributions.