Characterization of olanzapine (LY170053) in human liver slices by liquid chromatography/tandem mass spectrometry

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 33, 1237È1245 (1998)

Characterization of Olanzapine (LY170053) inHuman Liver Slices by LiquidChromatography/Tandem Mass Spectrometry

A. T. Murphy,* B. G. Lake,a J. R. Bernstein, R. B. Franklin and T. A. GillespieDepartment of Drug Metabolism and Disposition, Lilly Research Laboratories, Eli Lilly and Company, Lilly CorporateCenter, Indianapolis, IN 46285, USA and a BIBRA, Toxicology International, Woodmansterne Rd., Carshalton, Surrey, SM54DS, UK

Olanzapine metabolism was investigated using incubation of olanzapine with human liver slices. The intent of theinvestigation was to identify olanzapine metabolites and determine if the human liver slice incubations could poten-tially produce quantities of the olanzapine glucuronides for future studies. Along with known Phase 1 olanzapinemetabolites, N-desmethyl-, 2-hydroxymethyl-, and 4º-N-oxide-, a new hydroxylated species was detected. Detec-tion of Phase 2 metabolites included known N-10-glucuronides, a quaternary glucuronide and a novel glucuronideconjugate. This investigation showed the feasibility of using human liver slices to produce sufficient quantities ofolanzapine glucuronides for further studies. 1998 John Wiley & Sons, Ltd.(

KEYWORDS: olanzapine ; LC/MS/MS; human; liver slice ; metabolism

INTRODUCTION

Olanzapine, a thienobenzodiazepine (LY170053 : 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine ; Fig. 1) is a novel antipsychoticagent. Olanzapine has high affinity for dopamine (D1,D2, D3, D4, D5), serotonin (5-HT2A, 5-HT2B, 5-HT2C),norepinephrine (a1-adrenergic), acetylcholine (m1, m2,m3, m4, m5) and histamine (H1) receptors.1h2 Severalpreclinical studies indicate that olanzapine has a greaterpotency in the antagonism of serotonergic vs. dopamin-ergic neurotransmission.1h4 Based on clinical studies,olanzapine has therapeutic efficacy at least comparable(and in most instances superior) to conventional anti-psychotic drugs, and has a low propensity to induceextrapyramidal symptoms.5h10 Furthermore, olanza-pine does not increase prolactin levels signiÐcantly morethan what is observed with placebo.11

Human metabolism studies have shown olanzapineto be extensively metabolized, with the N-10-glucuro-nide being the major plasma and urinary metabolite.12The N-10-glucuronide has not been detected in rodentsor rhesus monkeys. However, a trace of the N-10-gluc-uronide metabolite has been detected in the urine ofdogs dosed orally with the compound.

The site of formation of the olanzapine N-10-glucuro-

* Correspondence to : A. T. Murphy, Department of Drug Metabo-lism and Disposition, Lilly Research Laboratories, Eli Lilly andCompany, Lilly Corporate Center, Indianapolis, Indiana 46285, USA

E-mail : murphy–anthony–t=lilly.com

nide, as well as a number of other oxidative metabolites,is thought to be the liver, and to conÐrm this, a studywas designed to investigate the metabolism of olanza-pine in human liver slices. Liver microsome and liverslice incubations have been established as techniques forthe production of oxidative metabolites and glucuro-nides.13h14 Use of liver slices as a predictive metabolismproÐler has also been demonstrated.15 Another reasonfor wishing to pursue the in vitro metabolism of olanza-pine was the need to obtain signiÐcant quantities of theN-10-glucuronide for further studies. Production andextraction of the glucuronide from human urine or for-tiÐed human hepatic microsomes was prohibitivebecause of the low dose administered to humans.

Previous studies conducted in our laboratories havedetected conjugated olanzapine metabolites in vivousing LC/MS/MS.12 Reviews on metabolism studieshave established the utility of LC/MS and LC/MS/MSfor the detection of oxidative and conjugated metabo-lites.16h17 Ionspray tandem mass spectrometry hasproven to be a powerful analytical tool to detect andidentify compounds extensively metabolized in vivo.18SpeciÐcally, ionspray has been used to identify N-glucuronidated metabolites in vitro.19 In this investiga-tion we utilized ionspray tandem mass spectrometry todetect several metabolic products, speciÐcally the majorhuman plasma and urinary metabolite of olanzapine,the N-10-glucuronide, resulting from the incubation ofolanzapine in human liver slices. Radiolabeled olanza-pine (14C at positions 4 and 10a) was used for the invitro studies in order to proÐle and quantitate the incu-bation medium for metabolites of olanzapine.

CCC 1076È5174/98/121237È09 $17.50 Received 24 February 1998( 1998 John Wiley & Sons, Ltd. Accepted 24 September 1998

1238 A. T. MURPHY ET AL .

Figure 1. Chemical structure of 170053(olanzapine), 170238(4¾-N-oxide olanzapine), 301664(olanzapine-lactam), 170055(N-desmethyl-olanzapine), 301504(7-methoxy-4¾-N-desmethyl-olanzapine), 290079(7-methoxy-olanzapine), 290411(2-hydroxymethyl-olanzapine) and280810.

EXPERIMENTAL

Chemicals

Olanzapine (LY170053), N-desmethyl-olanzapine(LY170055), 4@-N-oxide of olanzapine (LY170238),olanzapine-lactam (LY301664), 2-hydroxymethyl-olan-zapine (LY290411), quaternary glucuronide of olanza-pine, and 14C-olanzapine were synthesized at Eli Lillyand Company. All standards were characterized byNMR, mass spectrometry, and infrared spectroscopy.Ammonium acetate was obtained from J. T. Baker, Inc.(Phillipsburg, NJ). HPLC grade solvents were obtainedfrom Baxter (Muskegon, MI).

Human liver slice incubations

Precision-cut human liver slices, prepared with a Krum-

dieck tissue slicer, were obtained from a healthy donorliver aged 6 years and 8 months. Slices were cultured(two slices per vial) in 1 ml culture medium using adynamic organ culture system. The culture mediumconsisted of RPMI 1640 containing 5% fetal calf serum,0.5 mM L-methionine, 1 lM insulin, 0.1 mM hydrocor-tisone 21-hemisuccinate, 50 lg/ml gentamicin and 2.5lg/ml fungizone. Following a thirty-minute incubation,the medium was changed to fresh medium containingeither 50, 250 or 500 lM 14C-LY170053 (speciÐc activ-ity 22.6 lCi/mg; radiochemical purity 97%; lot V86-d6ME-267) in 10 ll of n-propanol, and the liver sliceswere cultured for a period of 24 h at 37 ¡C. After the24-hour incubation, the medium samples were removedfrom the slices and frozen. The liver slices were washedwith 0.154 M KCl containing 50 mM Tris-HCl, pH 7.4,to remove traces of the medium and then snap-frozen.All samples were stored at [80 ¡C prior to transfer tothe Department of Drug Metabolism and Disposition,

( 1998 John Wiley & Sons, Ltd. J. Mass Spectrom. 33, 1237È1245 (1998)

CHARACTERIZATION OF OLANZAPINE BY LC/MS/MS 1239

Lilly Research Laboratories, where the samples werestored at [70 ¡C until analysis. Prior to analysis, thesamples were thawed, evaporated to dryness in a Speed-Vac (Savant Instruments, Hicksville,Concentrator}NY, USA), and reconstituted in 400 ll of 50 mMammonium acetateÈacetonitrile (90 : 10, v/v).

Control incubations were prepared using, LY280810(Fig. 1), a compound previously reported to be exten-sively glucuronidated in fortiÐed human microsomes.20The glucuronidation of LY280810 was examined at aconcentration of 500 lM using methanol as the solvent.Preparation of substrate concentrations, media, andsubsequent incubations were carried out by BIBRAToxicology International, Woodmansterne Road, Car-shalton, Surrey, SM5 4DS, UK. Under BIBRA Projectnumber 1496/1.

LC/MS

A Shimadzu (Shimadzu ScientiÐc, Koyto, Japan)SCL-10A system controller was used to control twoShimadzu LC-10AD pumps. Liquid chromatographic(LC) separations were achieved using an Inertsil ODS-2(5 l, 250] 4.6 mm i.d.) column (MetaChem Technol-ogies ; Torrance, CA, USA). An injection volume of 200ll was used for each analysis. The mobile phase consist-ed of 50 mM ammonium acetate (solvent A) and ace-tonitrile (solvent B) operated in a programmed gradientas follows : 10% solvent B for 2 min, linear gradientincrease to 60% solvent B at 22 min and hold at 60%solvent B until 30 min followed by a gradient decreaseto 10% solvent B at 40 min.

The Ñow rate was set at 1 ml min~1 with the eluantbeing split to allow 30 ll min~1 directly into the SCIEXAPI III tandem mass spectrometer via the articulatedionspray interface. An ionspray voltage of 5000 V wasused with an oriÐce potential of 80 V. Full scan datawere obtained from a mass range of 200 to 800 amu at arate of 1.66 s scan~1. For collisionally activated disso-ciation (CAD), the instrument was programmed toadmit protonated molecular ions into the Q2 region.CAD was performed using a mixture of argon-nitrogen(90 : 10, v/v) at 275 ] 1012 atoms cm~2 and a collisionalenergy of 30 eV.

Quantitation of major metabolites in media from liverslice incubations

The major metabolites produced from incubations ofolanzapine (250 lM 14C-olanzapine) with human liverslices were quantitated by evaporating the media todryness (Savant Speed-Vac) and adding 50 ll 50 mMammonium acetate, 50 ll acetonitrile and 40 ll 95%ethanol, to the dried residue. Using the same chromato-graphic conditions described previously, the extract wasinjected onto the Inertsil column and the eluant wasmonitored by UV and an on-line Raytest Ramona 90radioactivity detector (Raytest, Pittsburgh, PA, USA).Additionally, the eluant was collected every 20 s for 30mins. To these samples 10 ml of scintillation cocktail(Aquassure) was added and the radioactivity was esti-

mated using a liquid scintillation counter and externalstandardization to compensate for quenching.

RESULTS

Glucuronidation of LY280810

Both full scan and CAD ion spectra were obtained forthe liver slice incubations containing LY280810. Thefull scan and CAD product ion spectra (not shown) ofthe LY280810 glucuronide (m/z 439) conÐrmed the pro-duction of the glucuronide conjugate thus ensuring thatexperimental conditions were conducive to glucuronideformation at the time of the olanzapine incubation.

LC/MS and LC/MS/MS of standards

A standard solution mixture (10 lg/ml in 50 mMammonium acetate-acetonitrile 90 : 10, v/v) consisting ofolanzapine (LY170053), 2-hydroxymethyl-olanzapine(LY290411), 4@-N-oxide of olanzapine (LY170238), N-desmethyl-olanzapine (LY170055), 7-methoxy-4@-N-des-methyl olanzapine (LY310504), 7-methoxy olanzapine(LY290079), and olanzapine-lactam (LY301664), (Fig. 1)was prepared to test the suitability of the system forqualitative determination of olanzapine and potentialmetabolites. This standard solution was assayed byLC/MS and LC/MS/MS to determine retention times,protonated molecular ions, and product ions for each ofthe standards. The full scan TIC (total ionchromatogram) of the standard solution mixture (Fig. 2)shows the detection of each standard. The protonatedmolecular ions of each standard were then analyzed byCAD to determine the product ions. The product ionmass spectra for each standard peak are shown in Fig.3. For reference, the retention time, protonated molecu-lar ion, and product ions for each of the standards isshown in Table 1.

For comparative purposes, full scan and CAD massspectra were also obtained for a synthetic quaternaryglucuronide of olanzapine by direct infusion into the

Figure 2. Total ion chromatogram of full scan (200–800 amu) ofsynthetic standard mixture containing 290411(2-hydroxymethyl-olanzapine), 170238(4¾-N-oxide olanzapine), 170055(N-desmethyl-olanzapine), 301504(7-methoxy-4¾-N-desmethyl-olanzapine), 170053(olanzapine), 290079(7-methoxy-olanza-pine) and 301664(olanzapine-lactam).



Figure 3. Product ion mass spectra of (a) 290411(2-hydroxymethyl-olanzapine), (b) 170238(4¾-N-oxide olanzapine), (c) 170055(N-desmethyl-olanzapine), (d) 301504(7-methoxy-4¾-N-desmethyl-olanzapine), (e) 170053(olanzapine), (f) 290079(7-methoxy-olanzapine)and (g) 301664(olanzapine-lactam).

mass spectrometer. The CAD mass spectrum of the syn-thetic quaternary glucuronide of olanzapine is shown inFig. 4.

Fragmentation of olanzapine

The CAD mass spectrum of olanzapine [LY170053,Fig. 3(e)] shows formation of characteristic product ions

at m/z 58, 84, 169, 180, 186, 198, 213, 222, 239, 256 and282. A proposed fragmentation mechanism is shown onFig. 3(e). The product ions observed at m/z 58, 84, 256and 282 have been conÐrmed with deuterium labeling ofthe methyl group attached to the 4@-nitrogen of the pip-erazine ring. The product ions detected at m/z 58, 84,256 and 282 are presumably formed from internal a-cleavages and hydrogen rearrangements from within themethyl piperazine ring.21 The product ions at m/z 186,198 and 213 are substantiated by the product ion

Table 1. Summary of standard mixture analysis by LC/MS and LC/MS/MS, showing the retention times, protonatedmolecular ions and corresponding product ions of standards in the mixture

Protonated

Retention molecular

Compound Metabolite or time ion

No. Compound (min) (u) CAD product ions (u)

LY290411 2-Hydroxymethylolanzapine 12.7 329 84, 119, 155, 199, 229, 242, 254, 272, 298

LY170238 Olanzapine 4¾-N-oxide 14.0 329 56, 82, 107, 169, 179, 198, 213, 229, 242, 282

LY170055 N-Desmethylolanzapine 14.8 299 70, 169, 180, 186, 198, 213, 230, 239, 256, 282

LY310504 7-Methoxy-4¾-N-desmethyl 15.0 329 70, 212, 228, 243, 260, 286, 312

olanzapine

LY170053 Olanzapine 16.9 313 58, 84, 169, 180, 186, 198, 213, 222, 239, 256, 282

LY290079 7-Methoxyolanzapine 17.2 343 84, 212, 228, 243, 271, 286, 312

LY301664 Olanzapine lactam 18.3 231 59, 97, 106, 133, 143, 169, 186, 197, 214

— Quaternary glucuronide — 489 213, 234, 256, 282, 313, 454



Figure 3. Continued.

spectra of the other standards injected. The 2-hydroxymethyl olanzapine product ion spectrum[LY290411, Fig. 3(a)] shows a 16 amu increase in theexpected fragments when compared to the product ionspectrum of olanzapine i.e. m/z 298 vs. 282, m/z 272 vs.256 and m/z 229 vs. 213. The product ions for N-desmethyl olanzapine [LY170055, Fig. 3(c)] shows theexpected 14 amu decrease in the product ion where the4@-N-methyl group is lost i.e. m/z 70 vs. 84. Evidence tosupport the fragmentation which yields product ions atm/z 186, 198 and 213 is supported in the product ionspectrum of 7-methoxy olanzapine [Fig. 3(f )]. Theproduct ions of olanzapine at m/z 186, 198 and 213 cor-respond to ions at m/z 216, 228 and 243, detected forthe 7-methoxy olanzapine [Fig. 3(f )]. The 7-methoxyolanzapine would be expected to yield an additional 30

amu to the product ions formed due to the methoxygroup attachment on the benzodiazepine ring system.

LC/MS of human liver slice incubations with olanzapine

The full scan of the 500 lM 14C-olanzapine incubation(Fig. 5) shows the detection of a large peak (100% rela-tive intensity) at approximately 17.5 min. To identify thepresence of olanzapine and any potential metabolic pro-ducts, extracted ion proÐles were plotted. Peaksdetected in the extracted ion proÐles of the 500 lM 14C-olanzapine are shown in Fig. 6. The extracted ion pro-Ðles for each of the three incubate concentrationsshowed a protonated molecular ion at m/z 313(olanzapine) as the most intense peak. The extracted ion



Figure 4. Product ion spectrum of synthetic quaternary glucuro-nide of olanzapine.

proÐles for protonated molecular ions at m/z 315(unknown), m/z 299 (N-desmethyl olanzapine) and m/z505 (hydroxylated glucuronide of olanzapine) revealedonly one peak for each trace. Multiple peaks weredetected in each extracted ion proÐle for protonatedmolecular ions at m/z 311 (unknown), m/z 313(olanzapine), m/z 329 (hydroxylated olanzapine) and m/z489 (glucuronide of olanzapine).

Figure 5. Total ion chromatogram of full scan (200–800 amu)analysis of 500 lM 14C-olanzapine liver slice incubate.

Figure 6. Extraction ion profiles from 500 lM 14C-olanzapineliver slice incubation. Extracted ion profiles of (a) m /z 299, (b)311, (c) 313, (d) 315, (e) 329, (f) 355, (g) 489 and (h) 505.

LC/MS/MS of human liver slice incubations witholanzapine

CAD mass spectra were obtained for protonated molec-ular ions at m/z 299, m/z 311, m/z 313 and m/z 315.CAD mass spectra were obtained for four protonatedmolecular ions at m/z 329, and three protonated molec-ular ions at m/z 489. The protonated molecular ion atm/z 505 was not of sufficient concentration in any of theincubation samples to yield an interpretable CAD massspectrum for conÐrmation. A summary of retentiontimes, protonated molecular ions and product ions foreach of the metabolites detected in the 500 lM olanza-pine incubation is shown in Table 2.

The Ðrst two peaks to elute from the TIC of the CADanalysis of the 500 lM olanzapine incubation were twoof the three protonated molecular ions at m/z 489. TheCAD mass spectrum of the Ðrst (11.2 min) and second(11.5 min) protonated molecular ions at m/z 489 [Fig.7(a)È(b)] was consistent with the mass spectrum



Table 2. Summary of metabolites detected in olanzapine human liver slice incubations using LC/MS and LC/MS/MS, showing theretention times, protonated molecular ions and corresponding product ions of standards in the mixture

Retention Protonated

Compound Metabolite or time molecular ion

No. Compound (min) (u) CAD product ions (u)

— N-Glucuronide of olanzapine 11.2 and 11.5 489 84, 256, 282, 313

— Unknown 12.1 355 109, 141, 153, 169, 181, 197, 273

290411 2-Hydroxymethylolanzapine 12.4 329 84, 119, 155, 199, 229, 242, 272, 298

Hydroxyolanzapine 13.0 329 58, 84, 133, 146, 158, 169, 201, 210, 238, 254, 272

— Quaternary glucuronide of olanzapine 13.3 489 84, 256, 282, 313

170238 Olanzapine 4¾-N-oxide 13.4 329 82, 107, 169, 179, 198, 213, 229, 242, 282

— Hydroxyolanzapine (unknown) 15.0 329 58, 84, 133, 146, 159, 169, 201, 210, 238, 254, 272

— Hydroxyglucuronide of olanzapine 15.2 505 no CAD confirmation

170055 N-Desmethylolanzapine 15.2 299 70, 169, 180, 186, 198, 213, 230, 256, 282

170053 Olanzapine 18.5 313 84, 169, 180, 198, 213, 222, 256, 282

— Unknown 23.5 311 58, 84, 167, 180, 186, 198, 213, 241, 256, 282

observed for the proposed N-10-glucuronide of olanza-pine observed in human urine.12 The CAD mass spec-trum of the third protonated molecular ion [Fig. 7(c)]at m/z 489 (13.3 min) did not match the mass spectrumof the previous m/z 489 ions at 11.2 and 11.5 min.However, the third protonated molecular ion at m/z 489(13.3 min) more closely matched the mass spectrumobtained for the synthetic quaternary glucuronide (Fig.4). The product ion spectrum for the synthetic quatern-ary glucuronide standard (Fig. 4) shows characteristicrelative abundances for fragments at m/z 313, 282, 256and 234. The relative abundances of the product ionsobserved for the metabolite at m/z 489 (13.3 min) areconsistent with the relative abundances observed for thesynthetic quaternary glucuronide standard. The m/z 234ion is indicative of the synthetic quaternary glucuronideof olanzapine as shown in the proposed fragmentationin Fig. 7(c). The product ion at m/z 234 ion was notobserved in the CAD of the metabolite, but is shown inthe CAD of the synthetic quaternary glucuronide stan-dard. The relatively low concentration of the quaternaryglucuronide metabolite of olanzapine explains theabsence of the product ion at m/z 234.

The identity of the protonated molecular ion at m/z355 (detected at 12.1 min) was not able to be deter-mined. The protonated molecular ion of m/z 355 wasconsistent with acetylated olanzapine, but the CADmass spectrum did not match the spectrum for the syn-thetic N-10-acetyl olanzapine standard (not shown).

A total of four protonated molecular ions at m/z 329were detected in the 500 lM olanzapine incubation. TheCAD mass spectrum of the Ðrst protonated molecularion at m/z 329 (12.4 min) conÐrmed the identity of themetabolite to be 2-hydroxymethyl-olanzapine. Thesecond protonated molecular ion at m/z 329 (13.0 min),while yielding product ions which showed structural rel-evance to olanzapine, was not able to be conÐrmed withthe use of synthetic standards during this study. Thethird metabolite with a protonated molecular ion at m/z329 (13.4 min), detected in the 500 lM olanzapine incu-bation, matched the CAD mass spectrum of the 4@-N-oxide standard. The fourth protonated molecular ion atm/z 329 (15.0 min) showed a CAD mass spectrum whichdid not match any of the hydroxylated standards. Thestructural identity of the fourth protonated molecular

ion at m/z 329 (15.0 min) could not be determined fromthis investigation.

The CAD mass spectrum of the protonated molecularion at m/z 299, detected at 15.2 min, matched the massspectrum obtained for the N-desmethyl olanzapinestandard.

The protonated molecular ion at m/z 315, detected at18.2 min, matched the mass spectrum for 14C-olanzapine (not shown) and is therefore not a metabo-lite of olanzapine. A metabolite with a protonatedmolecular ion at m/z 315 would correspond tohydroxylated N-desmethyl olanzapine. The CAD massspectrum of a hydroxylated N-desmethyl olanzapinemetabolite would not yield the m/z 84 product ionobserved in the CAD spectrum of olanzapine. Further-more, a hydroxylated N-desmethyl olanzapine metabo-lite would be expected to elute prior to olanzapine.

The full scan and CAD mass spectrum of the proto-nated molecular ion at m/z 313, detected at 18.5 min,conÐrm the peak to be olanzapine. The CAD spectrumof the protonated molecular ion at m/z 313 peakmatched the spectrum obtained for the olanzapine stan-dard.

The CAD mass spectrum of the protonated molecularion at m/z 311 (23.5 min) showed a structural relation-ship to olanzapine. However, the full structural identityof the m/z 311 ion was not determined in this investiga-tion. The protonated molecular ion peaks at m/z 311,detected at 14.5, 18.3 and 20.1 min, were attributed tobackground and did not contain olanzapine relatedions.

Quantitation of 14C-olanzapine metabolites

Seven radioactive entities were observed following thecollection HPLC fraction (20 s each) over a 30 minuteperiod (not shown). Over this period, greater than 96%of the injected radiocarbon was accounted for and iden-tiÐed in the HPLC eluant. The largest peak correspond-ed to olanzapine and accounted for 82% of theradioactivity injected onto the column. Other peakscorresponded to the 2-hydroxymethyl-, the 4@-N-oxide-and N-desmethyl-olanzapine metabolites and accountedfor approximately 4.0, 2.4 and 1.6%, respectively, of the



Figure 7. Product ion mass spectra of metabolites at m /z 489 detected at (a) 11.2 min, (b) 11.5 min and (c) 13.3 min.

injected radioactivity. Another peak collected corre-sponded to the proposed N-10 glucuronide of olanza-pine and accounted for approximately 6.0% (majormetabolic product) of the injected radioactivity. Two ofthe seven peaks did not correspond to known stan-dards.

DISCUSSION

This report describes the Ðrst production of olanzapinemetabolites using human liver slices. LC/MS/MSanalysis of the liver slice media, in which 14C-olanzapine was incubated at three concentrations 50,

250 and 500 lM, indicated that metabolites were pro-duced which were similar to those found in vivo.Extracted ion proÐles for the 50 and 250 lM incubatesshowed lesser amounts of the same ions detected in the500 lM incubation. Extracted ion proÐles for the 500lM incubation sample showed the detection of severalpeaks not observed in the full scan TIC. The retentiontime of olanzapine, and the other known metabolites inthe media samples, was consistently shifted when com-pared to the standard solution. This retention shift waspresumably due to the di†erences in matrix composi-tion, but we did not spike a control matrix to conÐrmthis suspicion.

Among the Phase 1 metabolites produced were theN-desmethyl-, 2-hydroxymethyl-, 4@-N-oxide- analogs,



as well as a novel hydroxylated olanzapine compound.The N-desmethyl-, 4@-N-oxide- and 2-hydroxymethyl-olanzapine metabolites were conÐrmed by mass spectra.The novel hydroxylated species had not been previouslydetected in vivo, nor did it spectrally match any otherhydroxylated metabolite. The CAD mass spectrum ofthis fourth hydroxylated species showed structuralrelationship to olanzapine (i.e. similar CAD fragments),but the site of hydroxylation could not be determined.

Phase 2 metabolites included the N-10-glucuronidesand the 4@-N-quaternary glucuronide. Also, spectra wereobtained that supported the formation of a glucuronideconjugate of a hydroxylated-olanzapine analog. Nosulfate or glutathione conjugates were detected in theincubates.

The CAD mass spectra of the metabolites with proto-nated molecular ions at m/z 489 (11.2 and 11.5 min)were consistent with the mass spectrum observed for theproposed N-10-glucuronide of olanzapine in humanurine. The two metabolites with protonated molecularions at m/z 489 are considered to be stereoisomers, butcould not be di†erentiated from this study. The m/z 489peak at 11.2 min was twice the relative abundance ofthe 11.5 min peak in the 50, 250 and 500 lM olanzapineincubation samples. The CAD mass spectrum of the m/z489 peak at 13.3 min did not match the CAD massspectra of the peaks which eluted at 11.2 and 11.5 min.However, the CAD mass spectrum of the protonatedmolecular ion at m/z 489 (13.3 min) did match the CADspectrum of the synthetic quaternary glucuronide ofolanzapine. This is the Ðrst reported detection of thesynthetic quaternary glucuronide of olanzapine in vitro.

Previous metabolism studies have also utilized LC/MS/MS for the determination of quaternary ammoniumlinked glucuronides.22 The glucuronide data thereforeindicates that this liver slice technique can be used toproduce the glucurondes for future studies.

Also collected were CAD spectra of compounds,potentially related to olanzapine, with protonatedmolecular ions at m/z 311 and m/z 355. No chemicalstructures were postulated for these ions. However, theCAD mass spectra obtained for these compounds showseveral of the same product ions observed for olanza-pine, indicating potential structural relationships.

Potential metabolites included in the standardmixture, but not detected in the incubates include, 7-methoxy-4@-N-desmethyl olanzapine (LY310504), 7-methoxy olanzapine (LY290079) and olanzapine lactam(LY301664).

The proÐle of Phase 1 and Phase 2 metabolites pro-duced by the human liver slices closely resembles thosemetabolites observed in vivo and are therefore predic-tive of in vivo metabolism. Furthermore, these studiessuggest that the incubation of olanzapine with humanliver slices may be useful in obtaining sufficient quan-tities of the N-10-glucuronide(s) for use in furtherstudies.

Acknowledgements

The authors would like to thank Robert Barbuch for his interpretivecontributions on this and many other metabolic proÐling projects.

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