LC-MS/MS Analysis of Mercapturic Acids: Addressing ... · -3-HPMA (italic) differ by more than 10%...
1
INTRODUCTION • 3-hydroxypropylmercapturic acid (3-HPMA), 2-Cyanoethylmercapturic acid (CEMA), 3-hydroxypropyl-1- methylmercapturic acid (HBMA) (Figure 1) are urinary markers of exposure to acrolein, crotonaldehyde and acrylonitrile, respectively. • These glutathione conjugates have been shown to be markers of tobacco smoke exposure. • Selectivity of the analysis is typically not adequately addressed in a majority of published methods potentially resulting in significantly elevated values of some biomarkers. • A selective LC-MS/MS method for simultaneous analysis of CEMA, 3-HPMA and HBMA in human urine was validated using RP UHPLC. • The chromatographic separation, selection of MRM transitions and the choice of stable labeled internal standards were critical factors in the selectivity and reproducibility of the validated method. Figure 1. Chemical structures of mercapturic acids (3- HPMA, CEMA and HBMA) SAMPLE PREPARATION • Aliquot (0.100 mL) of human urine was spiked with internal standards and diluted with 1% formic acid • Samples were loaded on a 96-well Oasis HLB plate, washed with formic acid and dilute methanol • Elution was performed with 20% acetonitrile INSTRUMENTATION • Electrospray ionization (ESI) data were acquired by multiple reaction-monitoring (MRM) in negative mode on an AB SCIEX API 4000 tandem mass spectrometer • The acquisition time was less than 5 minutes LC-MS/MS Analysis of Mercapturic Acids: Addressing Selectivity and Matrix Effect Issues Alan Dzerk, Veni N. Lapko, Ridha Nachi, Kirk Newland and Curtis Sheldon Celerion, Lincoln, NE, USA ANION EXCHANGE COMPARISON METHOD • HPLC with a 3x50 mm Thermo Biobasic AX column was operated isocratically in a dual column alternating backflush configuration at ambient temperature • Mobile phase was a mixture of acetonitrile and ammonium formate buffer, pH 2.5 Figure 2. Anion exchange chromatography RP-C18 COMPARISON METHOD • HPLC with a 3x50 mm Kromasil C18 column was operated in gradient mode at ambient temperature • Mobile phases were 0.1% formic acid and acetonitrile/formic acid Figure 3. RP-C18 Chromatography The extra peak present in the RP-C18 HBMA transition was initially assumed to be the diastereomer present in the reference standard (Urisub), however, the peak was not observed in the IS channel. Investigation emulating the chromatography of Carmella, et al. (Chem. Res. Toxicol., Vol. 22, No. 4, 2009), demonstrated the diastereomers were co-eluting at the lower mobile phase pH being used in the RP-C18 method. Figure 4. HBMA Diastereomers and extra peak- Phenomenex Synergi Max-RP Upper-15 mM Ammonium acetate (native)/MeoH gradient Lower-15 mM Ammonium acetate (pH 5.3)/MeoH gradient VALIDATED RP-UHPLC METHOD • A Waters Acquity UPLC with a 2.1x150 mm C18 analytical column was operated in gradient mode at 50 o C • Mobile phases were 0.1% formic acid and acetonitrile/formic acid (Figure 5) Figure 5. Validated RP-UPLC Chromatography RESULTS The successful resolution of interferences on HBMA channel was obtained using a RP UPLC column under acidic conditions, without separation of R, R and R, S-enantiomers. Selectivity of the assay was evaluated comparing quantitation in multiple urine lots from smokers and non-smokers using 2 MRM transitions for each analyte under different chromatographic conditions Major 3-HPMA MRM transition did not provide adequate selectivity using ion-exchange or RP HPLC as shown in Table 1. When RP UPLC was used both transitions were selective due to improved chromatographic resolution Table 1. 3-HPMA quantitaton testing using 2 MRM transitions: (major MRM, 220 a91 and second MRM, 220 a89). 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, 220 a91and second MRM, 220 a89). Data used are means of duplicates using 13C315N-3-HPMA as IS. • The use of 13 C 3 15 N-internal standards improved quantitation for RP UPLC analysis compared to deuterated internal standards (Table 3) Endogenous level (ng/mL) Difference (%) Lot# Major MRM AX Second MRM AX AX RP-C18 1 1440 1380 104 104 2 3550 2270 156 153 3 578 546 106 107 4 1680 1630 103 104 5 2770 1120 247 246 6 324 327 99.1 137 7 209 148 141 100 8 327 328 100 135 9 77.4 68.2 113 136 10 1130 972 116 102 11 141 124 114 117 12 250 244 102 146 13 239 225 106 118 14 1160 1130 103 115 15 1180 1160 102 100 16 1130 1040 109 104 17 1520 1410 108 101 18 55.9 32.0 175 103 19 539 288 187 110 20 4640 4830 96.1 107 Endogenous level (ng/mL) Difference (%) RP-UPLC Lot# Major MRM RP-UPLC Second MRM RP-UPLC 1 791 799 99 2 1135 1155 94 3 33.1 32.6 102 4 247 262 94 5 19.8 22.4 88 6 1110 1170 95 7 1735 1855 94 8 409 440 93 Table 3. Performance of 13 C 3 15 N- (A), and d 6 -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 d 6 -3-HPMA (italic) differ by more than 10% from those concentrations determined with 13 C 3 15 N-3-HPMA as Internal Standard The validated analytical range was from 20.0 to 5,000 ng/mL for 3-HPMA and HBMA and from 0.200 to 150 ng/mL for CEMA using Urisub as calibration matrix • LLOQ and 3xLLOQ concentration QC samples were prepared in Urisub • Low endogenous concentration QC samples (QC A) were prepared by pooling pre-screened non-smokers’ urine with low concentration of the mercapturic acids • Smokers’ urine lots were used to prepare high endogenous concentration QC (QC C) • The extraction recovery of mercapturic acids was not less than 80% • The inter-batch precision (% C.V.) and accuracy (% Bias) of quality- control samples is shown in (Table 4) A Lot# Basal Level (ng/mL) Expected Conc. Basal+20 (ng/mL) Calc. Conc. (ng/mL) % Dev. Expected Conc. Basal+3750 (ng/mL) Calc. Conc. (ng/mL) % Dev. Lot# Basal Level (ng/mL) Expected Conc. Basal+20 (ng/mL) Calc. Conc. (ng/mL) % Dev. Expected Conc. Basal+3750 (ng/mL) Calc. Conc. (ng/mL) % Dev. 1 813 833 768 -7.8 4563 4660 +2.1 2 1160 1180 1150 -2.5 4910 4840 -1.4 3 31.1 51.1 53.1 +3.9 3781 3900 +3.1 4 225 245 256 +4.5 3975 4010 +0.9 5 22.1 42.1 39.3 -6.7 3772 3950 +4.7 6 1320 1340 1280 -4.5 5070 5160 +1.8 7 1960 1980 1850 -6.6 5710 5660 -0.9 8 440 460 446 -3.0 4191 4130 -1.5 B 1 795 815 828 +1.6 4545 4080 -10.2 2 902 922 860 -6.7 4652 3500 -24.8 3 30.7 50.7 49.1 -3.2 3781 3390 -10.3 4 227 247 268 +8.5 3977 4140 +4.1 5 21.3 41.3 38.4 -7.0 3771 3790 +0.5 6 1230 1250 1110 -11.2 4980 4260 -14.5 7 2330 2350 2300 -2.1 6080 6970 +14.6 8 536 556 572 +2.9 4286 5270 +23.0 A. 3- HPMA LLOQ QC 20.0 ng/mL QC S 60.0 ng/mL QC A 114 ng/mL QC B 296 ng/mL QC C 616 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 QC 20.0 ng/mL QC S 60.0 ng/mL QC A 81.7 ng/mL QC B 265 ng/mL QC C 315 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 QC 0.200 ng/mL QC S 0.600 ng/mL QC A 0.500 ng/mL QC B 2.53 ng/mL QC C 44.0 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) CONCLUSIONS An LC-MS/MS method for simultaneous analysis of CEMA, 3-HPMA and HBMA with improved selectivity has been developed and validated. ACKNOWLEDGMENT The authors thank Alyssa Perry, Chris Kafonek, Leanna Schlesiger, Jason Wenzl, Brandon Retke, Marzuki Mohamed, Joseph Silva and Wendi Barnts for their contributions.
Transcript of 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.
![IL 1 - Mon · anticancer conjugates into clinical trial [1], most recently HPMA copolymer platinates, we have now designed several families of bioresponsive polymer-protein conjugates](https://static.fdocuments.us/doc/165x107/5f4d0dc1bd33b13fd85300f3/il-1-anticancer-conjugates-into-clinical-trial-1-most-recently-hpma-copolymer.jpg)
