li__PM_;_I:_‘C-Aflfigl/l)l”{l:S/l§:{l:§: a powerful
`tool for integratihg drug
`metabolism into the drug
`discovery process
`
`Walter A. Korfmacher, Kathleen A. Cox, Matthew 8. Bryant,
`John Veals, KWOl{€l Ng, Robert Watlrins and Chin—Chung Lin
`
`HPLC combined with atmospheric pressure ionization
`
`employed in selected cases for quantitative analysis. More
`
`(APB mass spectrometry {MS} has become a very useful
`
`tool in the pharmaceutical industry. The technique of
`
`recently, with the introduction of commercially available
`I-IPLC combined with atmospheric pressure ior1izati0n/
`
`tandem mass
`
`spectrometry (HPLCEAPI/MS/MS), both
`
`HPLC~APl/MS/MS is becoming very important for both
`
`metabolite identification and quantitative analyses are being
`
`drug discovery and drug development programs. in the
`
`drug discovery area, it has three major uses: (‘ll rapid,
`
`quantitative method development,
`
`(2) metabolite
`
`. identification, and l3l multi—drug analysis. The sensitivity
`
`of the API source and the selectivity provided by tandem
`
`mass spectrometry (MS/MS) enable rapid, quantitative
`
`performed routinely using this new technique.
`Until recently, most analytical methods for the determi-
`nation of a candidate drug were based on either GC or HPLC
`methods. The introduction of HPLGAPI/MS/MS systems has
`provicled new opportunities for rapid method development
`
`and metabolite identification, and this has helped to inte-
`
`grate cling metabolism into the drug discovery process.
`Recent articles have described HPLC/AP]/MS/MS technolu
`
`' method development
`
`for drugs in plasma. Early
`
`ogy and provided some examples of its utility for specific
`
`information on the metabolism of candidate drugs can
`
`guide structural modifications, thereby improving the
`
`activity and/or bioavailability.
`
`assaysl"“. in this report, the utility of HPLC-API/MS/MS sys-
`tems for integrating drug metabolism into the drug discov-
`
`ery process will be discussed. A comparison will be made
`EXHIBIT
`with GC and HPLC methodologies.
`
`Ex. 1020
`
`he Lise of mass spectrometry (MS)
`
`to support
`
`metabolism studies of drugs in development
`
`is
`
`well documented“? In the past, various ‘off-line’
`MS methods were employed for metabolite iclenti—
`
`fication; these procedures were typically reserved for can(li~
`
`date drugs in the development stage. In addition. GC-MS was
`
`Rapid method development
`Currently, various techniques are available to generate large
`
`numbers of compounds for biological screening, such as
`combinatorial synthesis and isolation from natural sources.
`
`The active compounds identified in these screens often
`require pharmacokinetic and in Uitro metabolic evaluation.
`Over 100 Compounds may have to be screened for their
`
`Walter A. Korfmacher*, Kathieen A. Cox, Matthew 3. Bryant, John Veals, Kwokei N9 and Chin-Chung Lin, Department of Drug
`Metabolism and Pharmacokinetics, and Robert Watkins, Department of Pharmacology, Schering—Plough Research Institute, 2015
`Galloping Hill Road, Kenilworth, NJ 07033-0539, USA. *tel: +1 908 2983183, fax: +1 908 2983966, email: walter.l<orfmacber@spcorp.com
`
`532
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`.
`,
`,
`Sun-Amneal-lPR2016-011_0 -
`:
`3
`EllS7liJli21-5
`Copyright © Elsevter Science Ltd. All rights reserved. 1359-64 6;‘87lM7.00. PilEx. 020 p.1of6
`
`DDT Vol. 2. No. 12 December 199?
`
`

`
`
`
`REVIEWS
`
`in vftro stability or in viva pharmacokinetic parameters before
`a lead compound can be chosen. These in vivo and in ultra
`
`Sample clean—up involves protein precipitation. This only
`requires 20-40 til of plasma or serum. Current API source
`
`studies require large numbers of samples in various biologi-
`
`cal matrices that need to be subjected to quantitative analy-
`
`designs are well suited to accept hundreds of injections of
`this type of sample, with minimal source cleaning needed
`
`sis; rapid pharmacokinetic evaluation is therefore required.
`
`between sample sets. in addition, HPLC method develop-
`
`in the past, standard methods used various sample clean-
`
`ment is less demanding because of the added selectivity
`
`up methods (for example liquid/liquid phase extraction and
`
`solid-phase extraction) and GC or HPLC techniques for sepa-
`ration and detection of compoundsll. As shown in Figure 1,
`
`these sample preparation methods typically required a sam~
`ple volume of 200-1000 pl of plasma or serum. The sepa-
`ration method depended on the nature of the compound,
`
`and 21 significant amount of time was required to obtain the
`proper analytical conditions necessary to resolve the various
`
`provided by the mass spectrometer. Compounds with dif~
`ferent molecular weights do not need to be resolved.
`
`Typically, 1 or 2 days is sufficient to develop a method for a
`
`compound that is part of an ongoing series of compounds
`being screened for their pharmacokinetic parameters.
`
`By using one HPi.C—APl/MS/l\rIS system, one operator can
`typically develop methods for
`two compounds and the
`methods used to analyze one set of samples for each com-
`
`components in the matrix. Often, a derivatization step was
`
`necessary to obtain enhanced sensitivity. This multi—step
`
`pound per week. This can result in a rapid turnaround of
`samples so that the required pharmacokinetic information
`
`procedure could take 1—5 weeks to develop.
`
`However, rapid method development (Figure l} is poss—
`ible when samples are analyzed by I-lPLC—API/MS/'l\/IS.
`
`Standard sample
`preparation
`
`Flaplcl sample
`preparation
`
`200-1 .000 pl plasma
`
`40 ill plasma
`
`i i
`
`Add internal standard
`l
`Liquid/liquid or solid-phase
`extraction
`
`Add 100 ttl CH3CN
`+ internal standard
`l
`VOIIGX
`
`Eveporate to dryness
`l
`Add solvent
`
`Centrifuge
`l
`Transfer to microvlals
`
`Vortex
`
`Analyze by HPLC-APIIMSIMS
`
`Centrifuge
`l
`Transfer to microvials
`
`l
`Analyze by HPLC-UV or G0
`
`Method development
`time: 1-3 weeks
`
`Method development
`time: t—2 days
`
`Figure 1. COllijf)6ll‘iSO?l ofsran.a‘am' scmzple
`prepmiwion versus rapid sample preparation.
`
`can be provided to the drug discovery team in a timely man»
`
`ner. Evolving strategies (see below) may help to improve
`this throughput in the near future.
`The two main reasons why I-IPLC-API/MS/MS allows
`
`rapid method development are the inherent selectivity and
`sensitivity of the instrumentation. The selectivity of HPLC-
`
`API.fMS/MS is on account of the specificity provided by tan-
`dem mass spectrometry (MS/MS)“. When used for quantita~
`
`live analysis, tandem mass spectrometry with a triple stage
`
`quadrupole mass spectrometer is used as follows. A specific
`
`ion is selected in the first quadrupole. typically the proton-
`ated molecule {MH*) for the compound of interest. The
`selected ion then enters the collision cell
`i’the second
`
`l
`
`quadrtlpole) of the instrument and fragments into one or
`more smaller (product) ions. These fragment ions are ana-
`
`lyzed in the third quadrupole and detected. Because of the
`mass selectivity provided by a tandem mass spectrometer,
`
`the high clironmtographic resolution typically required for
`UV or fluorescence detection is not necessary. Usually,
`
`extensive sample clean-up procedures are not required. The
`sensitivity of the HPLGAPI/MS/MS system is provided in
`
`part by the AP] source. For typical pharmaceutical mol~
`ecules, assays can be readily developed for concentrations
`in the range of 10-25 ng/"ml using only 40 pl of plasma.
`Thus, preconcentration or derivatization techniques are
`generally not required.
`
`Serial bleeding in rats
`
`Previously, one tat per time point was the standard pro
`
`cedure for pharmacokinetic studies in the rat. Currently.
`
`using HPLC—APl/‘MS/lvlS, methods can be developed based
`
`DDT Vol. 2, No. 12 December 199?
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-lPR2016-01104- Ex. 1020, p. 2 of 6
`
`533
`
`

`
`REVIEWS
`
`
`
`on the analysis of 40 ul of plasma or serum (Figure 1)”. or
`
`even on 10-20 1.1] of plasma or serum if lower sample volumes
`are necessary”. Because of these small sample volume re-
`
`quirements, it is possible to analyze samples from rats that
`are subjected to serial bleeding. In serial bleeding, a single
`
`rat is dosed with the test compound and sampled at mul-
`
`tiple time points (typically five to eight). At each time point
`
`a small volume of blood is removed (typically 200 iii). For
`
`three rats can he dosed. The advantages of
`mean data,
`serial bleeding in rats are:
`
`0
`
`-
`
`Fewer rats are needed to obtain pharmacokinetic data
`on a compound.
`
`Smaller amounts of the test compound are needed for
`closing.
`
`- There is less variability in the data.
`
`- The entire set of pharmacokinetic parameters (Cum, area
`
`under curve, tm) are available for each animal.
`
`Figure 2 shows data obtained from (a) a serial bleeding
`
`study and (b) a multi-rat study for the same compound
`dosed orally at 10 mg/kg. It can be seen that
`there is 21
`
`better correlation between the individual data and the mean
`data For the serial bleeding study than for the multi-rat study.
`
`Metabolite identification
`
`Previously, metabolite identification was resen-'etl for com—
`
`pounds in the development phase. One reason for this was
`that the standard method for metabolite identification relied
`
`on radiolabeled drug. As shown in Figure 3, the process of
`
`..E‘.
`
`10000
`
`(ng/ml)
`Concentration
`
`1000
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`1000
`
`(ngirnl)
`Concentration
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Time after dosing (h)
`
`Figure 2. Compcmlson Qfdrifa obfaiinedfrom (1.?) ct
`serial bleedmg starch» versus (b) (if imiiti-mi studyfor
`the some compormd dosed omlly at 10 mg/Reg.
`
`synthesizing the drug and collecting, purifying. and analyz-
`ing the metabolites typically takes 2-4 months. This time
`
`these parameters can be changed by improving the metabolic
`stability of the compound. in order to irnprove metabolic
`
`frame is not acceptable by current drug discovery standards.
`In the drug discovery phase. I-IPLCAAPIFMS/JVIS can be used
`
`it is very important to know how a compound is
`stability.
`nietaholized. The goal of Cling discovery is to progress a
`
`for metabolite identification. As shown in Figure 3, exten-
`
`sive metabolite identitication can be completed in 1 week.
`In many cases,
`1 day is sufficient
`to obtain useful infor—
`
`lead compound into a final candidate drug that can be
`placed in the development stage. The traditional roie of
`
`drug nietabolism in drug discovery was often limited and, in
`
`mation on the plasma metabolites of a drug dosed in an
`
`the past. consisted mainly of producing a pharmacokinetlc
`
`experimental animal, A more extensive look at other fluids
`
`profile of lead compounds. Early metabolite identification
`
`(urine, bile) or other tissues (e.g. brain. heart) could take 1
`or 2 weeks.
`in either case, significant amounts of infor-
`
`can provide information on how to improve the metabolic
`
`stability of the lead structure. in this way, future lead com-
`
`mation on the metabolism of a compound can be obtained
`in a relatively short period of time.
`
`pounds might be a metabolite identified from the previous
`lead drug or an analog of the previous drug designed to
`
`Metabolite identification in drug discovery provides early
`information that can lead to structural changes in the current
`
`block the major route of metabolism. in either case, metaba
`olite information in early drug discovery may lead to a much
`
`lead compound,
`
`improving such pharmacokinetic paramv
`
`eters as oral bioavailability, half~lil"e (13,? 3, or C;m. Often
`
`faster progression from the early lead dmg to the final
`candidate drug.
`
`534
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-lPR2016-01104- Ex. 1020, p. 3 of 6
`
`DDT Vol. 2, No. 12 December 1997
`
`

`
` REVIEWS
`
`
`Standard metabolite
`identification
`
`Metabolite identification
`using HPLC-APUMS/MS
`
`Full scans
`(find MH+)
`
`Make radiolabeled drug
`1
`Collect HPLC peaks
`l
`Purify
`
`40 iii plasma
`1
`Add 100 ill CH3CN
`l
`Vortex
`
`Suspected metabolites
`\
`
`Neutral loss scans
`//
`Product ion scene
`I
`l
`Parent ion scans
`
`Figure 4. Getzeml stmtegvfor the -use ofia-ndem mass
`.*§p£’Cf1'0i?T9f?jJ (MS/MS_)for the detection and
`i'deritifi‘cati'on of metabolites.
`
`to a precursor ion scan except that the characteristic frag-
`ment selected is a neutral species rather than an ionic
`
`species. Conjugated species such as glutathione or sulfate
`
`give characteristic neutral losses when fragmented in a mass
`spectrometer;
`therefore,
`these metabolites can often be
`detected with a neutral loss scan.
`
`In addition to the identification of metabolites of lead
`
`Analyze via MS
`
`Centrifuge
`
`AnalyzebyHPLC-AP]/MS/MS
`
`Time: 2-5 days
`
`Time: 2-4 months
`
`I
`
`llI
`
`Figure 3. Cbnyaarisotz ofstandard metabolite
`z'dem:f1'cntz'on procedures versus metabolite detection
`and identification using HPI.C~APl/MS/MS.
`
`The methodology for using tandem mass Spectrometry’
`
`(MS/l\'IS) to identify metabolites is well docunientedlri”-"1;
`however, the application of this methodology to drug dis-
`
`. compounds, it is very important to give some estimate of the
`levels of these metabolites compared with the dosed drug. If
`
`covery is a relatively recent development. Figure 4 shows a
`
`general strategy for the use of MS/MS for the detection and
`identification of metabolites Without the need for a radio-
`
`labeied drug. A full—scan mass spectrum displays all masses
`‘detected over the desired mass range. This provides no
`
`structural information on the compounds detected, so there
`could be substantial contributions from unrelated com-
`
`pounds, solvent
`
`ions or chemical noise. Structural
`
`infor-
`
`mation can be obtained on masses of interest by subjecting
`them to MSWIS. The most common type of MS/MS experi-
`ment is a product ion scan. In this case, ions of interest are
`
`isolated and fragmented into several product
`
`ions. These
`
`at metabolite is minor (1% or less of the closed drug), it may
`not be important to block its route of metabolism. If the
`
`metabolite is major (>209/o of the dosed drug),
`
`it may
`
`become the new lead drug, or modification of the site of
`
`metabolism may lead to a better compound. The best way to
`
`quantitate a metabolite is to compare its response with an
`
`authentic standard of the metabolite (as is done with the
`closed drug). I-Iowever, a metabolite stanclarcl is not usually
`available, and the only estimate that can be made is to use
`the calibration curve for the dosed drug to estimate the level
`
`of the metabolite. It is known that MS responses for various
`compounds can be quite different; but, in many cases, the
`
`information
`ions provide characteristic structural
`product
`about the original ion. For example, a suspected metabolite
`
`ion would fragment into product ions that either resemble
`
`response of the metabolite will be within a factor of two to
`three of the dosed clmg. Thus, while this method of estimai;~
`ing the level of one or more metabolites is not idea], it can
`
`ion
`the original parent drug, or differ from the product
`masses by the mass of the metabolite modification. A second
`
`provide useful information.
`
`Figure 5 shows a mass chromatograrn of drug B dosed at
`
`M5/l\*lS mode is a precursor ion scan. Here, a Cl1fl1‘:1Cl§€i'lS[lC
`product ion is specified and all ions that fragment to form
`
`10 mg/kg orally in the rat. The top trace shows the internal
`
`standard, which is a structurally similar analog that is used
`
`this product are monitored. in this manner, a characteristic
`fragment of the parent drug can be selected to screen for
`
`for quantitation. The second trace shows the peak for drug
`B. The third trace shows three M+16 metabolites that were
`
`possible metabolites that might produce the same fragment.
`The third i\-iS,’MS mode is a neutral loss scan. This is similar
`
`monitored for this compound. The bottom trace is the
`
`reconstructed ion chromatograrn which is the sum of the top
`
`DDT Vol. 2, No. 12 December 1997
`
`Sun-Amneal-lPR2016-01104- Ex. 1020, p. 4 of 6
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`
`7
`
`535
`
`

`
`
`
`100 m/z = 183.2; DA of 45B.50> 457.50
`
`(a)
`
`50
`0
`
`mo mlz = 411.2; DA OT 437.50> 438.50
`
`(b)
`
`to
`
`(d)
`
`50
`0
`
`mo
`50
`0
`
`m/z = 427; DA of 453.50:- 454.50
`V
`A B
`
`C
`
`100 RIC
`
`50
`0
`
`
`
`6.42? x E“
`
`1.334 x E5
`
`1:40
`
`3:20
`
`5:00
`
`6:40
`
`8:20
`
`‘Fume (min)
`
`Figure 5. Mass Cbi‘OiI'E6?fOgT6Ifl’lSfl‘0fiI the HPLC'—AP1/M.$(IlfS aizcnfnris oftm
`extract ofa ratplasnza sczmplejrom at 10 mg/leg om! dost’:-zg srtmfr:
`(ct) infernal sfomdard, (b) dosed drug, (C) peaks (ciesfgrrrtfecl A,B mid C)
`from three M+16 metabolites (£0 reconstmcted ion cbromafogrorm (RfC)_.-
`DA = daughter‘ ion.
`
`- analysis of multiple studies and
`0 multi-drug study analysis.
`
`the analytical chal-
`In either case,
`lenge is the same.
`
`In multiple analysis, spiked plasma
`(or serum, etc.) calibration samples
`
`are prepared with more than one
`analyte. The simplest example is two
`
`analytes, although the analysis of
`
`samples containing as many as 10 or
`
`20 analytes has been reported”. The
`
`basic principle is the same regardless
`of the number. Figure 7 shows the
`
`results of the analysis of four drugs
`plus one internal standard in a single
`HPLC«APl/MS/MS assay,
`in which
`
`each analyte has a unique MS/lViS
`
`‘analytical wintlow’. The four drugs
`are compounds with similar struc»
`
`tures that are spiked at 50 ng/ml each
`
`into rat plasma, resulting in Four cali~
`biation curves (one for each of the
`
`four drugs) For the single analytical
`
`tun. Thus, in the same analytical run,
`samples from one or more single
`
`:I-—- Parent
`—-A-— ivi+16(B)
`-we»-~ MWB2
`--- ~--:u1+1a(A)
`__.o__
`M-14
`
`1000
`
`Concentration
`
`(ng/ml) "time after dosing (h)
`
`Figure 6. Resrrltsfrom tbe arzcrlj-sis ofsamples
`obtwizedfrom CI discovery drug that was dosed orally
`at 10 mg/leg; the dosed compozmd levels are compared
`with estimateci memrbo/ire levels. 7799 metabolites are
`
`
`
`i'de11f1‘jied b_)'fbet'rrm1ss dtfiererzcefrortt the drug. 7799
`two 1'l4’+16 rrretaboifies are disfmgmsbed as A and B.
`
`three traces. Each mass chromatographic trace is normalized
`to the highest peak. This figure shows the excellent selec-
`
`tivity of the HPLCJAPI/MS/MS technique;
`
`this is typical of
`
`the type of data that can he obtained by this procedure.
`
`Figure 6 shows an example of a discovery drug that was
`
`extensively metabolized. The orally dosed drug (parent)
`reached a maximum level of less than 100 ng/ml. Three of
`its metabolites reached levels that were estimated to be sig-
`
`nificantly higher than the parent drug. A fourth metabolite
`showed very low levels. This information provided impor-
`
`tant clues concerning what part of the molecule needed be
`
`modified in order to improve the metabolic stability of the
`compound.
`
`Multiple analysis
`
`Another advantage of the HPLGAPI/MS/MS technology is
`
`that
`
`it can be used for multiple analyses, which can be
`
`defined as the determination of more than one compound in
`
`one chromatographic assay. Use of this capability is a recent
`development for HPLC—APi/IVJS/MS. This feature can be
`
`utilized in at least two different ways:
`
`536
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:24)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-lPR2016-01104- Ex. 1020, p. 5 of 6
`
`DDT Vol. 2, No. 12 December 1997
`
`

`
` REVIEWS
`
`RIC; DA of 590.29
`
`(a) 100 10
`
`.
`
`100
`
`RIC‘ DA of 560.29
`‘
`
`]_0
`
`(bl
`
`RIC; DA of 544.29
`
`(c) 100
`0
`
`M} 100 FiiC;DAof540.29
`0 l
`
`RIG: DA of 610.29
`
`.
`
`(e)
`
`‘O0 |
`
`0
`
`100 RIG
`
`0
`
`(r)
`
`K
`
`.
`Q
`
`.
`L
`
`5
`
`.
`
`1:40
`
`3:20
`
`5:00
`
`6:40
`
`‘lime (min)
`
`t8.477 x E5
`
`[2384 " E4
`
`]7.112xE3
`
`5
`
`[son x E
`
`8:20
`
`Figure 7. Mass cbroiizatogratnsfrom the HPLC-APMlvI.Sf.-1i‘5‘ analysis of an
`extracl of at am plnsnm sample spiked I(’l'Ibf0IH'd1‘[JgS (traces rt, 59. (f, e) at 6!
`level o_/‘50 ng/mi’ ecicb, plus fbe inter-mil srrmdm-‘d (C); rmcef is the
`r'econsfruct€d ion cbromarogram (RIC); DA = a'au.gbterioi-1.
`
`;
`
`utility of multi-drug studies is
`unclear.
`
`still
`
`Conclusion
`
`I-iPLC—API/MS/MS has been shown to
`
`have an important role in integrating
`drug metabolism into the drug dis-
`
`covery process. The technique has
`three primary uses:
`
`iapid method development,
`-
`- metabolite identification,
`
`0 multi-component analysis.
`
`in addition, because of its sensitivity,
`
`the technique can be used for the
`analysis of the low volume samples
`obtained from serial bleeding studies
`
`with rats. These capabilities guaranv
`tee that HPLC-API/MS/MS will con—
`
`tinue to be the technique of choice
`for drug discovery method develop-
`
`ment and study sample analysis for
`the foreseeable future.
`
`REFERENCES
`Kt:l:rJrie.l’.'=1nt|'l".tng.L (1993) :1 mil. Chem. 22. 97?.A—986A
`Gelpi. l‘-. (1993).]. Cbi‘onrc?tngi'.'!U3. $9430
`Covey. T.R.. Lee. ED. and Hcniun. _I.l'). i i986) Aiml. Cbtmr. "38,
`24554.-ibU
`
`I 2 5
`
`Korfnr.1clier.\V.A. ea‘ :11. (1995) in Em:_rciopen'!a ofArm:_'1-(ital Science,
`pp. 3itI2‘—5G34. 1-‘ic.iticniic Press
`Knifmaclier. \\’’.A. at at (I990) liiomezi. !;‘nivi'ro.vi. .-ling Spectrum. 19,
`]91—ll.ll
`
`\.I\
`
`Baillc. TA. l 1992) Int]. .-limit 3Pé’CfJ'0Hi.IOii' Process. I'll-1.-‘J19.
`25‘%5]vi
`.»\.lli.’l1. CD. (‘I Hi’. (1995) JCC-GC1-i. 510-514
`8 ¥'iiln1ei;D:\. and \-bllmer. D.L. (1996) LGGC 14. 236-212
`
`Dulik. D.:\l. at at (1996) in .-mm Spectmiiietty in the Biological Sciatica:
`lfliitlingame. A. and Can. SA. eds). pp. -123--i29. i-human-.1
`Olah. '1‘.\‘. et mi. (1991) j, Pimrm. Bionreri. Atmi’. 12. ?05—Ti2
`Cunshinzer. M. En’ iii. (1995) J. Cbn!Jim1.'Dgt‘. 666. 117426
`Biynnt. M5. at (if. (199?) J. C.‘£n'oma.'agr. 7.77. 61-66
`Chilton. A. at m’. (199?) Proceedings ofthe 45:1: ASMS Conference mi Mas:
`Specri-iiiiimjv and Allie-(I '1"q0:'o‘.
`l—'3 June (in press)
`(is iiuffmiinn, E. l_1996)_]. .-'|I(1S.\‘ Spectrum. 31. I.?,9»i5T'
`i-lallm.
`Ix’. (19963 I’i'0ce£-riiirgs r.f.'he 4-ftb ASMS ConjL=riwc.e on .-liars:
`bpecii-oiiiivii_1' mid .-tilted Topics, I246 May. p. 1465
`i'lerin:tn, _l. at til. (199?)_.v'. riled. Chem. 40. 829-331
`OI-.111. T.\’. e.’ at (1997) Rapid Commmi. .-liass Specrmiii. ii. 17-23
`
`dose studies can be analyzed by monitoring the four ana-
`
`iytes and one internal standard. Alternatively, samples from
`
`a multi—di'ug study (i.e. the four drugs closed in one animal)
`can also be analyzed using this technique.
`In a multi~drL1g study, more than one compound is dosed
`
`simultaneously in a laboratoiy animal”-17. These studies
`
`are also referred to as combinatorial pharmacokinetic
`studies. ‘cassette closing’ or ‘N-in~one‘ dosing studies“’. The
`
`atlvantage oi‘ a multi—diug study is that more compounds
`can be closed in a shorter time with fewer animals used.
`
`Another advantage is that the sample analysis is more effi-
`
`cient. On the other hand. a disativantage is that dnig—tlrug
`interactions mzu;
`lead to misleading pharmacokinetic
`
`resultslfi. One way to niinimize potential drugwliug inter»
`actions is to reduce the dosing level. A recent approach is
`to dose at 10/ Ir: mg/kg, where TI is the number of coniv
`
`pounds being dosed. Another disadvantage is that method
`
`development and sample calculation time is increased over
`the more common single dosing/single assay studies. A third
`
`clis-advantage is that metabolite identification is not practical
`
`with multi—clrug studies. A metabolite may have the same
`
`mass as another drug in the assay. For these reasons, the
`
`'6
`>-'*-I
`
`DDT Vol. 2, No. 12 December 1997‘
`
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