Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

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1 RAPID CMMUNICATINS IN MASS SPECTRMETRY Rpid Commun. Mss Spectrom. 6; : Pulished online in Wiley InterScience ( DI:./rcm.377 Accurte mss filtering of ion chromtogrms for metolite identifiction using unit mss resolution liquid chromtogrphy/mss spectrometry system Ming Gu *, Yongdong Wng, Xin-guo Zho nd Zhe-ming Gu Cerno Bioscience, Deerprk Drive, Monmouth Junction, NJ 88, USA XenoBiotic Lortories, 7 Morgn Lne, Plinsoro, NJ 836, USA Received Septemer ; Revised 3 Jnury 6; Accepted 4 Jnury 6 Accelertion of liquid chromtogrphy/mss spectrometric (LC/MS) nlysis for metolite identifiction criticlly relies on effective dt processing since the rte of dt cquisition is much fster thn the rte of dt mining. The rpid nd ccurte identifiction of metolite peks from complex LC/MS dt is key component to speeding up the process. Current pproches routinely use selected ion chromtogrms tht cn suffer severely from mtrix effects. This pper descries new method to utomticlly extrct nd filter metolite-relted informtion from LC/MS dt otined t unit mss resolution in the presence of complex iologicl mtrices. This pproch is illustrted y LC/MS nlysis of the metolites of verpmil from rt microsome incution spiked with iologicl mtrix (ile). MS dt were cquired in profile mode on unit mss resolution triplequdrupole instrument, externlly clirted using unique procedure tht corrects for oth mss xis nd mss spectrl pek shpe to fcilitte metolite identifiction with high mss ccurcy. Through the doule-filtering effects of ccurte mss nd isotope profile, conventionl extrcted ion chromtogrms corresponding to the prent drug (verpmil t m/z 4), demethylted verpmil (m/z 44), nd delkylted verpmil (m/z 9), tht contined sustntil flse-positive peks, were simplified into chromtogrms tht re sustntilly free from mtrix interferences. These filtered chromtogrms pproch wht would hve een otined y using rdioctivity detector to detect rdio-leled metolites of interest. Copyright # 6 John Wiley & Sons, Ltd. Identifiction of drug metolites in iologicl mtrices such s urine, plsm, ile, nd feces presents formidle chllenge for ionlyticl scientists. Effective nlysis of the metolites y liquid chromtogrphy/mss spectrometry (LC/MS) is often compromised y overwhelming interferences from the endogenous mtrix ions. These ions re notoriously undnt nd cn pper throughout typicl LC/MS run to n extent such tht the metolite profile cn e difficult to oserve in the totl ion current (TIC) chromtogrm or se pek chromtogrm. A gret del of effort hs een devoted to removl of the interferences physiclly prior to or during the LC/MS nlysis. ne pproch is to clen up the in vivo smples y solid-phse extrction (SPE) or liquid-liquid extrction (LLE) efore injection into the LC/MS system. These smple preprtion methods pper to e simple nd fesile, ut they lso pose risk of losing metolites, especilly those present t low concentrtions. Another pproch is to use neutrl loss (NL) or precursor ion (PI) scn modes with triple-qudrupole instrument to selectively detect certin types of metolites.,3 For exmple, ny glucuronide conjugtion of drug cn e identified using NL scn for 76 D, corresponding *Correspondence to: M. Gu, Cerno Bioscience, Deerprk Drive, Monmouth Junction, NJ 88, USA. E-mil: ming.gu@cernoioscience.com to the mss of the glucuronide functionl group. Both NL nd PI scns cn significntly filter out signls from the interfering endogenous ions to revel the trget metolites nd their profiles. However, this pproch requires prior knowledge of chrcteristic frgmenttions nd is not universlly pplicle to other metolites. Moreover, these two scn modes involve tndem mss spectrometry (MS/ MS) experiments in which the undnces of minor metolites my e too low to produce sufficient frgment ions from collisionlly induced dissocition for relile detection of the trget metolite types. The endogenous interferences cn lso e removed or reduced y post-cquisition dt processing. The most common utility used to serch for the metolites in complex mixtures is the extrcted ion chromtogrm (XIC); this pproch does detect the metolites with much improved selectivity compred with the TIC chromtogrm, ut mny flse positives cn e produced when the metolites re contined in complex smple mtrices such s ile nd feces. Bsed on the oservtion tht the prent drug nd its metolites hve similr mss defects, mss defect filtering 4, hs een used to successfully remove signls from mtrix ions nd to produce metolite profile similr to the trce otined from rdioctivity detector. Currently, this filtering process cn e performed only on the dt cquired using high-resolution instruments such s qudrupole Copyright # 6 John Wiley & Sons, Ltd.

2 time-of-flight (qtf) or Fourier trnsform mss spectrometers (FTMS), where the higher resolution llows for etter seprtion of ions on-the-fly nd the ccompnying high mss ccurcy enles the mss filtering in the postprocessing mode. The filtering performnce, however, depends on whether the endogenous ions or ny of their significnt isotopes hve mss defects similr to those of the drug under investigtion. Experienced users hve lso used isotope pttern nlysis for the identifiction of chlorine- nd romine-contining metolites, ut this method is generlly not pplicle to metolites tht do not contin such distinctive isotope ptterns. Erlier work 6,7 hs shown tht experimentl mss ccurcy follows the reltionship given s Eqn. (): s / p R ffiffiffi () S where R is the mss spectrl resolving power nd S is the ion signl level; this reltionship ssumes only ion counting noise. With internl mss clirtion, mss uncertinty of md hs een chieved using n enhnced resolution qudrupole instrument (FWHM ¼. D) when operted in selected rection monitoring (SRM) mode. 8 The SRM operting mode oviously resulted in high S vlue nd prtilly contriuted to the mss ccurcy improvement. When compring mss uncertinties chieved using n MS system tht cn operte in oth low- nd high-resolution modes, the resolving power t unit mss resolution (FWHM ¼. D) for smll molecule of mss D is only, resulting in theoreticl (Eqn. ()) loss of mss ccurcy y fctor of compred to tht otined using higher resolving power of ; this loss of mss ccurcy resulting from low resolving power cn, however, e prtilly or fully compensted y the higher signl ville when operting t lower resolution. It hs een shown tht it is possile to chieve high mss ccurcy of few md or even ppm using unit mss resolution systems in oth infusion mode nd on the chromtogrphic time scle. 9 Nonetheless, the higher resolution ville on qtf systems still holds n intrinsic dvntge over unit mss resolution systems in the physicl rejection of ions with msses close to those of the metolites of interest. n the other hnd, there is significntly more informtion ville from unit mss resolution dt thn wht is currently exploited. A complete isotope profile tht includes ll isotopes of metolite ion, even mesured under unit mss resolution, cn provide the criticl distinguishing power necessry to distinguish true metolites nd mtrix ions. This pper presents new method tht comines ccurte mss ion chromtogrms with profile mode mss spectrl filtering to successfully extrct metolite-relted informtion from complex LC/MS dt cquired using unit mss resolution triple-qudrupole mss spectrometer. Dt otined for verpmil metolites uried in strong signls from the ions of ile mtrix demonstrte tht this new pproch removes the ckground ions nd genertes simplified ion chromtogrms virtully free of flse-positive peks. Mss filtering of ion chromtogrms t unit mss resolution 76 EXPERIMENTAL Regents HPLC-grde wter nd cetonitrile (ACN) were purchsed from EM Science (Gistown, NJ, USA). Verpmil, erythromycin, promethzine, reserpine, Tyr-Tyr-Tyr, terfendine, uspirone, lopermide hydrochloride, potssium diphosphte, mgnesium chloride, nd NADPH were purchsed from Sigm (St. Louis, M, USA). Ammonium cette (Bio-Chemik grde) ws purchsed from Fluk (Ronkonkom, NY, USA). Mle Sprgue-Dwley (SD) rt liver microsomes were otined from XenoTech, LLC (Lenex, KS, USA). Instrumenttion The HPLC system consisted of Shimdzu (Columi, MD, USA) LC-ADvp pumps nd SIL-HTA utosmpler. An Advnced MAC-MD Anlyticl (Chdds Ford, PA, USA) ACE 3 C8 HPLC column (C8, 4.6 mm, 3 mm) ws used. The moile phses were A (.4% formic cid in wter) nd B (ACN). A 68-min grdient from % B to % B ws employed for the seprtion of verpmil metolites t flow rte of.7 ml/min during 3 min rmping. The HPLC eluent pssed through divert vlve nd ws then introduced directly into the source of the mss spectrometer. An API 4 Q TRAP (ABI-Sciex, Toronto, Cnd) mss spectrometer ws employed for MS nlysis. All dt were cquired in profile mode. The instrument ws operted in positive electrospry ioniztion (ESI) mode, using the first qudrupole scnning from m/z 8. The Ionspry voltge ws set to V. An ion source turo gs temperture of 8C ws used, with curtin gs ckpressure set to psi nd the declustering nd entrnce potentils t nd V, respectively. Smple preprtion All incutions were conducted in. M potssium phosphte uffer (ph 7.4), with sustrtes ( mm), mgnesium chloride (4 mm), rt liver microsomes ( mg/ml microsoml protein) nd NADPH ( mm). The verpmil ws preincuted for min t 378C, nd rections were initited y the ddition of NADPH. A ml volume of the incute ws removed fter h of incution. The rections were quenched with ml of ice-chilled cetonitrile. Microsoml proteins were pelleted y centrifugtion t 4 rpm ( g) for min t 48C. The superntnts were trnsferred into clen tues nd evported to dryness under gentle strem of nitrogen t room temperture. The residues were reconstituted with ml of % methnol in wter. Aliquots of incuted verpmil were spiked with lnk SD rt ile or urine mtrix to mimic rel in vivo smple. A clirtion stndrd mixture, contining erythromycin, promethzine, reserpine, Tyr-Tyr-Tyr, terfendine, uspirone, nd lopermide hydrochloride, ws mde in n ACN/ wter mixture (:) contining.% formic cid. Dt cquisition nd processing The procedure for dt cquisition nd processing is summrized in Scheme. To chieve high mss ccurcy using the Q TRAP instrument, cquisition of dt for the Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :

3 766 M. Gu et l. Loop Injection of Clirtion Stndrds Genertion of Externl Clirtion y MSIntegrity LC/MS of Blnk nd Verpmil Metolites Dt File Conversion from Wiff to MATLAB Appliction of Clirtion to LC/MS Dt Files Scheme. Dt cquisition nd processing for LC/MS experiments using Q TRAP qudrupole system. stndrd mixture is required for the highly ccurte mss spectrl clirtion lgorithm (HAMSCA). 7 This ws ccomplished y loop injection of the stndrd mixture with the moile phses A nd B t : rtio nd the flow rte t.7 ml/min. These loop injection conditions were similr to those used for LC/MS nlysis of verpmil metolites. (md) Error x x 4 After clirtion Before clirtion m/z Figure. () Mss spectrum of the clirtion stndrds. () Expnded view of wek pek efore nd fter clirtion. (c) Mss errors for the stndrd ions used for clirtion. c After the loop injections, sequence of smples tht included lnk, verpmil incute, nd the verpmil incutes spiked with ile or urine mtrix, ws nlyzed y LC/MS. All the dt cquired using the Q TRAP were converted into MATLAB (MthWorks, Ntick, MA, USA) dt formt y n Anlyst script Wiff to Mtl. The converted dt files were then processed y the softwre MSIntegrity TM (Cerno Bioscience, Monmouth Junction, NJ, USA). MSIntegrity TM fetures three mjor functions: mss spectr clirtion, ccurte mss nd isotope profile filtering, nd compound identifiction y ccurte mss. Depending on the stility nd the nture of given instrument, the clirtion cn e performed s externl clirtion lone, internl clirtion lone, or comintion of the externl nd internl clirtions. In this study, the profile mode mss spectrum of the clirtion stndrd mixture ws used to crete n externl clirtion tht ws susequently pplied to every individul scn in the LC/MS dt sets. Following the clirtion, list of possile iotrnsformtions is creted nd loded into MSIntegrity TM to perform the nlysis with defult vlues of md for ccurte mss filtering nd % reltive error for isotope profile filtering. The oserved potentil metolite peks in the resulting ion chromtogrm, clled the ccurte mss nd profile extrcted ion chromtogrm (AMPXIC), were further confirmed y ccurte mss mesurements in the mss spectrl dimension. RESULTS AND DISCUSSIN Verpmil is clcium chnnel locker commonly used to tret hypertension nd coronry rtery disese.,3 The in vitro metolism of verpmil incuted with humn or rt liver microsomes hs een studied extensively, nd results in severl metolites. The mjor metolites re demethylted verpmil rising from loss of methyl group from four possile -methyl groups nd one N-methyl group (see the moleculr structure of verpmil in Fig. (c)). Verpmil hs often een used s model system to vlidte LC/MS method development for metolite identifiction pplictions. 4 6 Its well-chrcterized metolites re thus used here s exmples to demonstrte the effectiveness of this new pproch for metolite identifiction. Mss spectrometer clirtion As result of limittions imposed y current clirtion technology nd mss spectrl pek nlysis lgorithms, qudrupole-sed instruments do not offer dequte ccurte mss cpilities which re incresingly populr s n dditionl confirmtion tool for metolite structurl nlysis. It is one of the mjor ojectives in this study to chieve high ccurcy mss mesurement cpility with these instruments of only unit mss resolving power. The keys to high mss ccurcy using such low-resolving power MS system re comprehensive clirtion nd roust pek mss ssignment (lso clled centroiding). Conventionl MS clirtion for qudrupole instruments concentrtes only on correction of the mss errors, nd does not ddress the issues of seline nd isotope interferences, noise filtering, nd pek shpe compenstion. All of these issues, Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :

4 Mss filtering of ion chromtogrms t unit mss resolution 767 x x 7 - x 4 7 XIC: VerpmilM3, D 3 c Verpmil Verpmil N N Min Figure. () TIC chromtogrm for verpmil incute spiked with ile mtrix. () XIC for m/z for verpmil incute spiked with ile mtrix. (c) AMPXIC for the prent drug (verpmil) for the incute spiked with ile mtrix. long with the lck of relile centroiding lgorithm, contriute to poor mss ccurcy on these systems where pek width (FWHM) cn e s wide s.7 D. The new clirtion pproch used here focuses on the clirtion of oth mss scle nd pek shpes with the dded enefits of noise filtering. 7 As result of this comprehensive clirtion procedure, the mss spectrl peks ecome well-defined symmetricl mthemticl functions menle to highly ccurte pek mss mesurement nd pek re integrtion. Figure illustrtes the processes nd the results of the externl clirtion. The clirtion stndrds consist of six smll drug molecules with monoisotopic MH þ ions t m/z.7, 8.43, 386.6, 47.3, 69.8, nd 76.49, s shown in the rw spectrum (Fig. ()). An externl clirtion cn e developed following the HAMSCA process 7 from this clirtion mixture. When this externl clirtion is pplied to wek pek in the mss spectrum shown in Fig. (), oth mss xis djustment nd pek shpe compenstion cn e chieved long with improved signl-to-noise rtio (S/N, Fig. ()). The mss clirtion residuls for stndrd ions re ll within md (Fig. (c)). It is importnt to point out tht the mss clirtion residuls shown in Fig. (c) re only mesure of goodness of fit during clirtion. To ssess the true mss ccurcy chievle, the clirtion thus uilt needs to e pplied toechmssspectrlscninnlc/msrunndthem/z vlues of ions other thn the clirtion ions clculted nd compred to their theoreticl vlues. The externl clirtion, creted s descried ove, covered sufficient m/z rnge from 6 76 nd permitted high mss ccurcy mesurements for the verpmil metolites on the LC/MS time scle. These mesurements re demonstrted for representtive metolites of verpmil in Tle. Although there ws time gp of out 6 h etween the dt cquisition of the clirtion stndrds nd tht of the metolites, the externl clirtion ppers to hve held s result of the good instrument stility. Regrdless of their m/z vlues, the ccurte msses for ll these metolites were within md of the theoreticl vlues. The use of the externl clirtion for ccurte mss mesurements offers numer of dvntges over the internl clirtion. First, the pproch hs multiple clirtion stndrds to ccount for ll possile metolites over the clirted m/z rnge. Second, externl clirtion does not introduce ion suppression or interferences. Most importntly, this pproch does not require dul ESI spryers or the use of post-column ddition of internl stndrds through syringe pump, which gretly simplifies experimentl procedures. Tle. Accurte mss mesuremnets of verpmil metolites Metolites Exct mss (D) Verpmil in ile, externl clirted Retention time (min) Mesured mss (D) Mss errors (md) Prent drug Demethylted Demethylted Demethylted Demethylted Demethylted Delkylted Stndrd mss errors (md).7 Verpmil in urine, externl clirted Prent drug Demethylted Demethylted Demethylted Demethylted Demethylted Delkylted Stndrd mss errors (md) 7.7 Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :

5 768 M. Gu et l. Accurte mss nd profile extrcted ion chromtogrm (AMPXIC) While recent developments in mss spectrometry technologies hve provided highly utomted dt cquisition procedures for high-throughput LC/MS nlysis, the lck of sophisticted dt-hndling tools hs mde dt processing ottleneck in the overll nlyticl process. This is especilly true when serching for metolites in LC/MS dt sets for in vivo smples, ecuse the metolite peks re often rely visile in the TIC chromtogrm s result of the presence of ckground ions tht dominte the mss spectr. Identifiction of individul metolites is thus time-consuming nd lor-intensive. With the present ccurte mss nd isotope profile filtering pproch, the dt processing cn e fully utomted to significntly remove the mtrix ions nd result in n AMPXIC tht is much more selective thn conventionl XIC. Unlike the isotope pttern nlysis method pplicle only for chlorinend romine-contining metolites, this new pproch is universlly pplicle to diverse rnge of metolite structures. The drstic reduction in mtrix ion signls y this pproch is demonstrted y LC/MS nlysis of verpmil in vitro incution spiked with ile mtrix s model for in vivo smples. As expected, while the TIC chromtogrm for the LC/MS seprtion (Fig. ()) shows intrctle signls rising from undnt mtrix ions, the XIC creted using the usul. D mss window (from m/z , Fig. ()) includes the prent drug (verpmil, m/z 4.9) t retention time of 33 min. Unfortuntely, mny other peks re lso oserved in this sme XIC. This poor selectivity in the XIC is lrgely the result of the conventionl mss ccurcy ville on such unit mss resolution system, nd the fct tht it does not exploit the informtion in the isotope profile. When the new filtering pproch ws pplied to the sme dt set, however, it rejected lmost ll the flse positives nd resulted in n AMPXIC (Fig. (c)) tht is much clener thn the conventionl XIC. This significnt improvement in selectivity is first ttriuted to the filtrtion y ccurte mss mesurements. Two peks (group, Fig. ()), t retention times of pproximtely 3 nd 4. min in the originl XIC for the prent drug, were filtered out since their m/z vlues, 4.64 nd 4.66, respectively, were more thn md wy from the m/z vlue of the MH þ ion of verpmil t 4.9. The minor peks, ppering in the regions leled s groups, 3, nd 4 in Fig. (), were lso eliminted s result of their lrge mss differences from the selected vlue (tht of the prent drug). Similrly, the AMPXIC identified ll five demethylted metolites nd one delkylted metolite (dt not shown), with virtully no flse positives nd very little if ny seline vrition in the AMPXIC (cf. Fig. 3() with Fig. 3()). Nerly ll of the interference peks in the corresponding XIC (Fig. 3()) disppered s result of the ccurte mss nd profile filtering. For exmple, t retention times of.6 nd 7.3 min (Fig. 3()), two well-defined peks corresponding to m/z vlues of nd 44.96, respectively, were filtered out completely. The effect of ccurte mss filtering cn e further exemplified y the profile for the prent drug in its AMPXIC x x Figure 3. () XIC for m/z for verpmil incute spiked with ile mtrix. () AMPXIC for demethyltion metolites of verpmil for the incute spiked with ile mtrix. (Fig. 4()). The pek ppers to e split into two seprted peks, reflecting the vrying mss ccurcy from different scns cross the prent drug LC pek. As the pek eluted from the column into the mss spectrometer, the signl for verpmil first rose, then sturted the detector, nd finlly declined. It is well known tht detector sturtion leds to poor mss ccurcy. Evidently, oth the rising nd the declining portions of the chromtogrphic pek gve very ccurte m/z vlues t 4.87 nd 4.96, respectively, leding to the enhnced AMPXIC signl. The sturted pek top resulted in n ssigned m/z of 4.384, lmost md off the correct vlue for verpmil (4.9), leding to depressed AMPXIC signl nd resulting in two pprent split peks for the sme MH þ ion. Secondly, isotope profile filtering lso plys n importnt role in the selectivity enhncement, s illustrted y the pek t retention time of 47. min in Fig. (). Although the pek ws eluted much lter thn the prent drug verpmil, nd therefore not likely to e metolite derived from verpmil, the pek will e used s n exmple to show the effective use of isotope profile filtering when the pek hs resonly ccurte mss ssignment nd would e expected to e retined sed on ccurte mss filtering lone. A striking difference cn e seen etween the conventionl XIC nd the AMPXIC in the time window from 4 to min (Figs. () nd (c)). Although it ws the second most undnt pek in the XIC, the pek completely disppered in the AMPXIC due exclusively to isotope profile filtering. The pek hd n ccurte m/z ssignment of 4.3 (Fig. ()), md less thn the known m/z vlue (4.9) of the MH þ ion of the prent drug (verpmil). n compring the mss spectr of rejected (Fig. ()) nd retined (Fig. ()) peks with the theoreticlly clculted isotope profile of verpmil (Fig. (c)), it is ovious tht the mss spectrl pek t m/z 4.3 from 3 C pek of n unknown component t m/z in Fig. () ws removed ecuse its isotope profile devites significntly from tht of verpmil. n the other hnd, the retined mss spectrum (Fig. ()) hd oth good mss ssignment nd pek profile consistent with tht of verpmil. Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :

6 Mss filtering of ion chromtogrms t unit mss resolution 769 x7 x Min Figure 4. () Expnded view of the XIC for m/z D for the verpmil incute spiked with ile mtrix. () Expnded view of the AMPXIC for the prent drug (verpmil) for the incute spiked with ile mtrix. Figure 6 demonstrtes nother exmple of profile-mode spectrl filtering when the monoisotopic mss of pek mtches ccurtely with tht of suspected metolite ion. In the XIC for demethylted verpmil from 3 to 3. min, smll pek (Fig. 3()) ws oserved to hve n ccurte m/z ssignment of 44.79, only 4 md from the clculted m/z of Aundnce Reltive 6 4 x x m/z c Figure. () Rejected pek with A þ ion of MH þ ion t m/z 4.3 from the verpmil incute spiked with ile mtrix. () Retined pek with monoisotopic m/z 4.88 from the verpmil incute spiked with ile mtrix. (c) Theoreticl mss spectrum of verpmil with monoisotopic MH þ ion t m/z 4.9. Aundnce Reltive x x m/z Figure 6. () Rejected pek t m/z from the verpmil incute spiked with ile mtrix. () Retined pek with monoisotopic MH þ ion t m/z from verpmil incute spiked with ile mtrix. (c) Theoreticl mss spectrum of demethylted verpmil with monoisotopic MH þ ion t m/z for demethylted verpmil; thus this pek would hve een retined sed on ccurte mss filtering lone. However, this pek ws successfully rejected y profile filtering nd did not pper in the AMPXIC. Comprison with the theoreticlly clculted spectrl profile (Fig. 6(c)) shows tht the mesured spectrum (Fig. 6()) evidently hs significntly more undnt M þ nd especilly M þ peks, nd is therefore rejected. Further confirmtion sed on MS/MS experimenttion is underwy in the next phse of this project. In contrst, the mss spectrum retined in the AMPXIC (Fig. 6()) hs not only n ccurte mss ssignment, ut lso spectrl profile consistent with tht theoreticlly predicted for demethylted verpmil. This doule-filtering effect, y oth the ccurte mss nd profile mtching, eliminted ll spurious peks nd retined the truly drug-relted chromtogrphic peks in the AMPXIC. When operted t the usul unit mss resolution, mss spectrometers such s triple qudrupoles re more likely suject to interferences thn higher resolution mss spectrometers, such s qtf nd FTMS. The presence of isoric interferences could compromise the mss ccurcy mesurement nd led to possile flse negtives. Mny of these isoric interferences, however, cn e identified nd mthemticlly resolved due to the comprehensive clirtion performed. This is the suject of seprte puliction under preprtion. When there is significnt overlp of isotope profiles etween n ion of interest nd other interferences, the AMPXIC will tend to filter out the ion due to the high selectivity. When interferences of this nture re severe enough to cuse flse negtives, MS/MS nlysis will e needed for further confirmtion. Also, in spite of the intrinsic c Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :

7 77 M. Gu et l. noise filtering cpility uilt into the HAMSCA clirtion, there my e cses where the S/N is not enough to provide relile identifiction, requiring the use of different smple preprtion, seprtion, or detection techniques. CNCLUSINS The comintion of ccurte mss nd profile mode filtering represents n innovtive pproch to fcilitte dt processing for the quick nd relile identifiction of metolites in complex mixture. The pproch is estlished on the solid foundtion of unique clirtion procedure tht corrects for oth mss error nd mss spectrl pek shpe vritions, nd chieves rndom noise filtering ll in the sme process. This pproch mkes it possile to chieve ccurte mss mesurements for the metolites in the ile mtrix on chromtogrphic scle using qudrupole instrument with unit mss resolution. With this dded cpility for ccurte mss mesurements on top of its verstile scnning functions, the widely ccessile triple-qudrupole instruments cn e mde more useful for metolite identifiction nd other qulittive nlyses. Given moleculr formule of expected drug metolites, AMPXICs otined through ccurte mss ssignment nd profile-mode filtering hve fr superior selectivity compred with conventionl XICs, resulting in much simplified ion chromtogrms similr to those otined from rdioctivity detector with rdioleling of the suspected metolite. While n AMPXIC cn pinpoint ll key metolite peks with virtully no flse positives, the possiility of flse negtives from severe interferences, requiring the use of MS/MS for further confirmtion, does exist. The pplicility of this method cn e expnded to other structure identifiction pplictions such s detection of degrdtion products nd impurities, confirmtion of protein modifictions, nd quick identifiction of trce levels of hzrdous chemicl or iologicl compounds. It hs the potentil to provide ccurte mss mesurements of MS/MS frgments for metolites or other structurl elucidtion pplictions, suject under ctive reserch. Future reserch would include emedding this pproch into the MS system to perform highly trgeted dt-dependent MS/MS for pplictions rnging from metolite identifiction to protein sequencing. REFERENCES. Korfmcher WA. Using Mss Spectrometry for Drug Metolism Studies. CRC Press: Wshington DC,.. Busch KL, Glish GL, McLuckey SA. Mss Spectrometry/Mss- Spectrometry: Techniques nd Applictions of Tndem Mss Spectrometry. VCH Pulishers: New York, Clrke NJ, Rindgen D, Korfmcher WA, Cox KA. Anl. Chem. ; 73: 43A. 4. Zhng H. Proc. st ASMS Conf. Mss Spectrometry nd Allied Topics, Montrel, Cnd, June 8, 3.. Zhng H. Rpid Commun. Mss Spectrum. 3; 38:. 6. Blom KR. Anl. Chem. ; 73: Gu M, Wng YD, Kuehl D. Spectroscopy My,. 8. Grnge AH, Winnik W, Ferguson PL, Sovocool GW. Rpid Commun. Mss Spectrom. ; 9: Gu M, Wng YD, Zho XG, Gu ZM. Proc. 3rd ASMS Conf. Mss Spectrometry nd Allied Topics, Sn Antoni, June 9, ; Poster No... Zho XG, Gu ZM, Gu M, Wng YD. Proc. 3rd ASMS Conf. Mss Spectrometry nd Allied Topics, Sn Antoni, June 9, ; Poster No Storm T, Hrtig C, Reemtsm T, Jekel M. Anl. Chem. ; 73: 89.. McTvish D, Sorkin EM. Drugs 989; 38: Brogden RN, Benfield P. Drugs 996; : Knthrj E, Tuytelrs A, Proost PEA, ngel Z, Assouw HPV, Gilissen RAHJ. Rpid Commun. Mss Spectrom. 3; 7: 66.. Sun L, Zhng SQ, Zhong DF. Act Phrmcol. Sin. 4; :. 6. Von Richter, Eichelum M, Schonerger F, Hofmnn U. J. Chromtogr. B ; 738: 37. Copyright # 6 John Wiley & Sons, Ltd. Rpid Commun. Mss Spectrom. 6; :