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A high performance liquid chromatography–inductively coupled plasma-mass spectrometry interface employing desolvation for speciation studies of platinum in …

A high performance liquid chromatography–inductively coupled plasma-mass spectrometry interface employing desolvation for speciation studies of platinum in …
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  RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom.  2003;  17 : 1855–1858Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1125 High-performance liquid chromatography/inductivelycoupled plasma mass spectrometry with iodine-specificdetection for profiling the metabolites produced in theearthworm  Eisenia veneta  by exposure to 2-fluoro-4-iodoaniline Catherine J. Duckett 1 , Ian D. Wilson 2 *, Heather Walker 3 , Fadi Abou-Shakra 3 ,John C. Lindon 1 and Jeremy K. Nicholson 1 1 Biological Chemistry, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building,South Kensington, London SW7 2AZ, UK 2 Department of Drug Metabolism and Pharmacokinetics, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK 3 Micromass UK Limited, Atlas Park, Simonsway, Manchester M22 5PP, UK Received 21 May 2003; Revised 26 May 2003; Accepted 26 May 2003 High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) provided a rapid and specific means for profiling the iodine-containing metabolites pro-duced by the earthworm  Eisenia veneta  following exposure to 2-fluoro-4-iodoaniline. Profiles wereobtained, using gradient reversed-phase HPLC, from extracts of whole earthworms and from coleo-mic fluid with as little as 25ng/peak of iodine detected. The use of ICPMS in this way provides aconvenient means of determining the metabolic fate of iodinated compounds without the need for radiolabelled compounds. Copyright # 2003 John Wiley & Sons, Ltd. The advent of robust methods for the hyphenation of induc-tively coupled plasma mass spectrometry (ICPMS) to high-performance liquid chromatography (HPLC) has opened upa range of new analytical opportunities in elemental specia-tion. 1 In the biomedical field, there have been a numberof publications exemplifying the use of HPLC/ICPMS forxenobiotic and drug metabolism studies in animals andhumans. 2–9 Traditionally, applications of ICPMS have beenmainly concerned with metals or metal-containing species, butadvancesinthetechnologysurroundingthereaction/col-lision cell of the ICPMS system,which decreases the interfer-encesassociatedwiththeargonplasma,havemadeitpossibletodetectlowatomicnumberelements,atlowconcentrations,andtoanalysethehalogensmorereadily.Asaresult,thereisincreasedpotentialforapplicationsintheanalysisofpharma-ceuticals where halogens are common substituents of drugcandidates.HPLC/ICPMShasthusbeenappliedtothedetec-tionofbromine-, 4–6 sulfur-andchlorine-containing 7 xenobio-ticsandtheirmetabolitesinraturine,combinedinsomecaseswithorthogonalaccelerationtimeofflightmassspectrometry(oa-TOFMS) to provide structural information. In a similarway, Marshall  et al . have used bromine-detected HPLC/ICPMS to examine bromobradykinin metabolism in humanand rat plasma samples. 8 In addition to sulfur, bromine andchlorine, iodine-detected HPLC/ICPMS has been success-fully applied to the separation and quantification of iodine-containing pharmaceuticalstandardsinasyntheticmixture, 9 to the analysis of thyroid gland protease digests 10 and urineandserumsamples 11 foriodinespeciationandquantificationof thyroid hormones. Iodine-ICPMS has also been used todetermine total plasma iodine in patients with thyroid disor-ders. 12 However, to date, no work has been reported on theprofiling of the metabolites of an iodine-substituted xenobio-tic in a complex biological matrix.This work reports a novel application of HPLC/ICPMS totheinvestigationofamodelecotoxinintheearthworm,usingiodine detection. Earthworms are used extensively as modelorganisms in ecotoxicological studies, yet knowledge isgenerally lacking about their defence mechanisms againstxenobiotics 13 and, although there is good evidence for P450cytochromes in these organisms, 14 relatively little is knownabout how earthworms metabolise organic chemicals. EXPERIMENTALChemicals 2-Fluoro-4-iodoaniline (purity 98%) was obtained fromSigma-Aldrich Company Ltd. (Gillingham, Dorset, UK) andusedasreceived.Solventsforchromatographywereofhigh-purity HPLC grade and were obtained from Fisher ScientificUK Ltd. (Loughborough, UK). Copyright # 2003 John Wiley & Sons, Ltd. * Correspondence to : I. D. Wilson, Department of Drug Metabo-lism and Pharmacokinetics, AstraZeneca, Mereside, AlderleyPark, Macclesfield SK10 4TG, UK.E-mail: ian.wilson@astrazeneca.com  Procedures A filter-paper toxicity test was conducted following OECDguidelines. 15 After a 24h depuration period,  Eisenia veneta earthworms were exposed to the model ecotoxin, 2-fluoro-4-iodoaniline, at 5 m g cm  2 , on filter paper (55cm 2 ) withinglass vials. The earthworms were exposed to the xenobioticfor 72h, after which coelomic fluid was extracted, 16 and thewormsfreeze-driedovernight.Atissueextractwaspreparedas follows: four earthworms of approximately equal sizeweregrounduptogetherinamortarandpestlewithaspatulaof sand (Prolabo, Fontenay, France) and approx. 2–3mLmethanol, then sonicated for 30min. The solution was fil-tered,theresiduewashedwithmethanol(2mL),andthesol-vent removed under a stream of N 2  before storage at  40 8 C.Prior to analysis, samples were reconstituted in MeOH, andcentrifuged to remove particulate matter and precipitatedproteins (10min, 17000  g ). Coelomic fluids did not undergoany sample preparation. Analytical methods AWatersAlliance2690HPLCsystemwithaUV-diodearraydetector(Waters,Milford,MA,USA)wascoupledtoaMicro-mass Platform ICP mass spectrometer (Micromass UK Ltd.,Manchester, UK) tuned to monitor  m/z  127. The flow of thegases and the operating and acquisition conditions of theICPMSsystemaredescribedinTable1.Thechromatographicseparation was carried out using a Supelco Discovery 1 C185 m m 4.6  150mm column (Bellefonte, PA, USA). The flowratewas0.8mLmin  1 ,whichwassplitto400 m Lbeforeenter-ing the mass spectrometer. A linear reversed-phase gradientmethod was employed based on 0.01M formic acid (solventA)andmethanol(solventB),asfollows,0to10min95%A,10to 40min 5 to 80% B, maintained from 40 to 50min, 50 to55min 80 to 5% B, remaining at 95% A/5% B until 60min.Typically, 50 or 100 m L injections of coelomic fluid or me-thanol tissue extract samples were made, with direct post-column injection through a rheodyne loop injector of theexternalstandardat  54min(3.2 m giodinein50 m L)preparedas a solution of 2-fluoro-4-iodoaniline in methanol. MassLynx TM software (Micromass UK Ltd.) was used for instru-ment control, data acquisition and handling. RESULTS AND DISCUSSION The metabolism of anilines as model ecotoxins has been pre-viously investigated in earthworms using HPLC/NMR,HPLC/MS and  19 F spectroscopy 17–19 with a similar protocolto that given above in the Experimental section. Where themetabolic fate of a compound is being investigated in anorganismforthefirsttimethereisconsiderablevalueinhav-ing a sensitive and specific means of detecting compound-related material. Such studies are therefore usually per-formed using a radiolabelled form of the compound withalloftheproblemsthatthisentails.Theabilitytouseaspecificmeans of detection based on the presence of iodine in thecompound under investigation is consequently a consider-able benefit.This is demonstrated here by the analysis of earthwormtissue extracts and coelomic fluids, which showed complexUV diode-array chromatograms, and relatively simple  127 Itraces. Thus Fig. 1 shows the HPLC/UV trace (A) andHPLC/ 127 I-ICPMS trace (B) of methanolic extracts of wormtissue obtained after a 72-h exposure to 2-fluoro-4-iodoani-line. Figure 2 shows the corresponding HPLC/ 127 I-ICPMStrace of coelomic fluid (B) taken from a different wormexposed to the same compound.Analysis both of the methanolic tissue extracts and of thecoelomic fluid showed a number of iodine-containing peakseluting between 24 and 47min, principally in the region 28–34min. The whole tissue extract shows a complex pattern of metabolites together with a major peak, at 39.3min, corre-spondinginretentiontimetounchanged2-fluoro-4-iodoani-line(confirmedbystandardaddition).Inthechromatogramsobtained for coelomic fluid two major peaks were detected, Table 1.  Instrument operating conditions for ICPMS Cooling gas 17.34L/min RF power 1700WIntermediate gas 0.69L/min Acquisition mode SIRNebuliser gas 0.63L/min Dwell time 0.2sHelium gas 8.1mL/min Masses monitored 127Hydrogen gas 6.0mL/min Total analysis time 60minHexapoleauxiliarygas4mL/min Figure 1.  (A) HPLC/DAD chromatogram of worm tissueextract and (B) HPLC/ICPMS  127 I-detected chromatogramsof worm tissue extract after 72h exposure to 2-fluoro-4-iodoaniline. The peak labelled ‘standard’ corresponds to3.2 m g of  127 I. Copyright # 2003 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2003;  17 : 1855–1858 1856 C. J. Duckett  et al.  one of which corresponded in retention time to 2-fluoro-4-iodoaniline,andanearlierelutingpeak,whichcorrespondedin retention time to one of the major peaks in the tissueextract, eluting at 29.3min. Further work is now required todetermine the identity of these iodine-containing metabo-lites, but the potential for profiling using HPLC/ICPMS isclear.In addition to enabling qualitative metabolite profiling to be undertaken, the presence of iodine in these metabolitesalso provides the potential for quantification. Whilst for thepreliminary work reported here we have not attempted toproduce fully validated methodology, an attempt has beenmade to provide a semi-quantitative analysis of the samples.Based on the analytical data obtained during this study wewere able separate and quantify peaks containing as little as25ng of iodine (and peaks containing smaller amounts weredetectable) providing the basis for a sensitive method formetabolicprofilingofiodine-containingspecies.Thislevelof sensitivity is consistent with the operating specifications of the instrument for an organic solvent matrix. In order toenable approximate quantification of the 2-fluoro-4-iodoani-line containing components in the sample, injection of an‘external’ standard, post-column, into the ICPMS system foreach run was made. The instrumental software incorporatesan integration function such that the area under each peak ismeasured and simple calculation gives the iodine concentra-tion of each peak relative to the standard. Assuming themaximum level of exposure attainable to be 275 m g of thesubstituted aniline (5 m g cm  2 , on 55cm 2 of filter paper),the coelomic fluids from different worms were found tocontain between 5 and 13% of the initial dose. Based on thetotalrecoveryofiodinatedmaterialdetectedinthecombinedtissue extract, each worm contained an average of 19% of theinitial dose. Whilst such calculations demonstrate thepotential of the technique to provide quantification, it isapparentthatanyattempttodeterminetheexactexposureof the worms to 2-fluoro-iodoaniline in this type of experimentis difficult. In addition, there are a number of potentialopportunities for loss of either volatile metabolites or theparent compound during the workup procedure. Thepreparation of the methanolic tissue extracts involvesfreeze-drying, extraction and filtration, and then subsequentsolvent evaporation by N 2 , each of which has its ownpotential for losses. Had it been the aim of this study todemonstrate accurate quantification, the addition of asuitable internal standard would clearly have been neededat the beginning of the procedure. However, unchangedparent compound was the major component of the iodinetrace obtained for the tissue extract samples, showing thatsuch putative losses were not total. For the coelomic fluid,wheresamplepreparationwasminimal,theprofileshowninFig. 2 can be expected to be more representative of the  in vivo metabolic profile.These results demonstrate, for the first time, the use of HPLC/ICPMS to obtain a metabolite profile of a xenobioticdosed to earthworm, and, additionally, it is the first knownexample of HPLC/iodine detection/ICPMS of a xenobioticwithin a biological matrix. No doubt such profiling could becarried out using conventional mass spectrometric techni-ques with selectivity for iodine-containing compoundsobtained by applying neutral loss scanning for 127Da.However, the ICPMS-based detection of iodine guaranteesdetection ofalliodine-containing speciesand,asitreliesonaresponse for the element itself rather than the molecule,provides the potential for quantification of unknowns with-out the need for authentic standards. In contrast, quantifica-tion of unknowns in HPLC/MS is not practicable.As with previous investigations, 2 the increasing methanolcontent of the chromatographic gradient was not found tointerferewiththesensitivityofdetectionanddidnotproduceincreasing background response. Additionally, spectro-scopicinterferencessuchasionisedargonpolyatomicspecieswere reduced by addition of hydrogen into the collision cell,and tuning was carried out to minimise oxide levels such asCdO (128) and CdOH (129) during a blank injection.While iodine is a relatively uncommon substituent fordrugs and pesticides, and there is a greater potential withsuchelementsasCl,P,SandBr,thisstudyhasshownthatthetechnique (HPLC/ICPMS) could be extended to a widernumberofapplicationsandisarapidandspecificmethodforthe detection of metabolites and a means to quantify them.Further work, involving the analysis of the earthwormsamples by HPLC/MS for metabolite identification and  19 F NMR spectroscopy for profiling and quantification, isunderway. Figure 2.  (A) HPLC/DAD chromatogram of coelomic fluidand (B) HPLC/ICPMS  127 I-detected chromatograms ofcoelomic fluid after 72h exposure to 2-fluoro-4-iodoaniline.The peak labelled ‘standard’ corresponds to 3.2 m g of  127 I. HPLC/ 127 I-ICPMS for xenobiotic metabolism studies in worm 1857 Copyright # 2003 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2003;  17 : 1855–1858  Acknowledgements The authors thank Micromass UK Ltd. for access to ICPMSinstrumentation. TheRoyal Society of Chemistry (AnalyticalDivision), EPSRC and AstraZeneca are acknowledged forfinancial support to Catherine Duckett. Also, we acknowl-edge the provision of earthworm samples, by Dr. J. Bundy(NERC, Environmental Diagnostics Programme). REFERENCES 1. Michalke B.  Trends Anal. Chem . 2002;  21 : 154.2. Duckett CJ, Bailey NJC, Walker H, Abou-Shakra F, WilsonID,LindonJC,Nicholson JK. Rapid Commun. Mass Spectrom .2002;  16 : 245.3. Smith CJ, Wilson ID, Abou-Shakra F, Payne R, Grisedale H,Long A, Roberts D, Malone M.  Chromatographia  2002; 55 (Suppl. 1): S151.4. NicholsonJK,LindonJC,ScarfeG,WilsonID,Abou-ShakraF, Castro-Perez J, Eaton A, Preece S.  Analyst  2000;  125 : 235.5. NicholsonJK,LindonJC,ScarfeG,WilsonID,Abou-ShakraF, Sage AB, Castro-Perez J.  Anal. Chem . 2001;  73 : 1491.6. Abou-Shakra F, Sage AB, Castro-Perez J, Nicholson JK,Lindon JC, Scarfe GB, Wilson ID.  Chromatographia  2002; 55 (Suppl. 1): S9.7. Corcoran O, Nicholson JK, Lenz EM, Abou-Shakra F,Castro-Perez J, Sage AB, Wilson ID.  Rapid Commun. MassSpectrom . 2000;  14 : 2377.8. Marshall P, Heudi O, Mckeown S, Amour A, Abou-ShakraF.  Rapid Commun. Mass Spectrom . 2002;  16 : 220.9. Axelsson BO, Jorten-Karlsson M, Michelson P,Abou-ShakraF. RapidCommun.MassSpectrom .2001; 15 :375.10. Takatera K, Watanabe T.  Anal. Chem . 1993;  65 : 759.11. Michalke B, Witte H, Schramel P.  Biol. Trace Elem. Res . 2000; 78 : 81.12. Allaine P, Berre S, Krari N, Laine-Cessac P, Le Bouil A,Barbot N, Rohmer V, Bigorgne JC.  J. Clin. Pathol . 1993;  46 :453.13. Stenersen J. In  Ecotoxicology of Earthworms , Intercept Ltd.:Andover, 1992; 129–138.14. Lee RF.  Comp. Biochem. Physiol. C  1998;  121 : 173.15. OECD,  Guideline for Testing of Chemicals , no. 207,  Earth-worm, Acute Toxicity Tests , Organisation for Economic Co-operation and Development: Paris, 1984.16. Bundy JG, Osborn D, Weeks JM, Lindon JC, Nicholson JK.  FEBS Lett . 2001;  500 : 31.17. Lenz EM, Lindon JC, Nicholson JK, Weeks JM, Osborn D. Xenobiotica  2002;  32 : 535.18. Bundy JG, Lenz EM, Osborn D, Weeks JM, Lindon JC,Nicholson JK.  Xenobiotica  2002;  32 : 479.19. Lenz EM, Lindon JC, Nicholson JK, Weeks JM.  Ecotoxicol.Environ. Saf  . 2003;  54 : 157.1858 C. J. Duckett  et al. Copyright # 2003 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2003;  17 : 1855–1858
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