Clozapine

Molecular formula: C18H19CIN4 Molecular weight: 326.8 CAS Registry No.: 5786-21 -0

SAMPLE Matrix: bile, blood, urine Sample preparation: 1 mL Bile, plasma, or urine + 20 JXL 40 [xg/mL clozapine in EtOH + 200 |xL 2.5 mM pH 6.0 sodium pentanesulfonate, mix for 3 s, add 6 mL diethyl ether, shake vigorously for 2 min, centrifuge at 2500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |JLL mobile phase, vortex for 30 s, inject a 25 JXL aliquot. HPLCVARIABLES Guard column: 25 X 4.6 30 |xm C18 (Merck) Column: 100 X 8 10 |xm ixBondapak C18 Mobile phase: MeOH: water: triethylamine 65:35:0.5, adjust pH to 6-7 with glacial acetic acid Flow rate: 2 Injection volume: 25 Detector: UV 254 CHROMATOGRAM Retention time: 9.7 Internal standard: clozapine OTHER SUBSTANCES Extracted: moricizine KEYWORDS plasma; rat; clozapine is IS REFERENCE
Yang, J.M.; Chan, K. Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography. J.Chromatogr.B, 1995, 663, 172-176

SAMPLE Matrix: blood Sample preparation: Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH and 1 mL buffer. Mix 100 (rat) or 250 (human) JULL plasma or serum with 100 |JLL 5 |jig/mL Nmethylspiperone, add to SPE cartridge, wash twice with 1 mL MeCN: buffer 28:72, elute with 1 mL MeOH: triethylamine 99.3:0.7. Evaporate the eluate to dryness under a stream of nitrogen at 50, reconstitute the residue in 100 JJLL mobile phase, inject a 55 |xL aliquot. (Buffer was 7.8 g K2HPO4 in 1 L water, pH 9.0.) HPLCVARIABLES Guard column: 50 mm long C18 (Waters) Column: 300 X 3.9 ixBondapak C18

Mobile phase: MeCN: buffer 36:64, pH adjusted to 3.7 with 10% phosphoric acid (Buffer was 7.8 g K2HPO4 in 500 mL water, adjust pH to 4.0 with phosphoric acid, make up to 1 L with water.) Flow rate: 1 Injection volume: 55 Detector: UV 254
CHROMATOGRAM

Retention time: 6.0 Internal standard: N-methylspiperone (11.2) Limit of quantitation: 60 ng/mL (rat); 15 ng/mL (human) OTHER SUBSTANCES Extracted: metabolites Simultaneous: amitriptyline, diazepam, nitrazepam, oxazepam, protriptyline
KEYWORDS

plasma; serum; human; rat; pharmacokinetics; SPE

REFERENCE
Fadiran, E.O.; Leslie, J.; Fossler, M.; Young, D. Determination of clozapine and its major metabolites in human serum and rat plasma by liquid chromatography using solid-phase extraction and ultraviolet detection. J.Pharm.Biomed.Anal, 1995, 13, 185-190

SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL BondElut C18 SPE cartridge with 1 volume of 1 M HCl, 2 volumes of MeOH, and 1 volume of water. 500 (xL Serum H - 50 |xL amoxapine in 1% potassium bicarbonate + 500 |xL MeCN, mix, centrifuge at 1500 g for 3 min, add the supernatant to the SPE cartridge, wash with 2 volumes of water, wash with 1 volume of MeCN, elute with 250 |JLL MeOH: 35% perchloric acid 100:1, remove all eluate by centrifuging at 1000 g for 30 s, inject a 15 jxL aliquot.
HPLCVARIABLES

Guard column: 15 X 3.2 7 jxm C8 (Applied Biosystems) Column: 150 X 4.6 5 |xm Ultrasphere Octyl Mobile phase: MeCN.0.1% tetramethylammonium perchlorate 27:73, adjusted to pH 4.2 with 10% perchloric acid Flow rate: 2 Injection volume: 15 Detector: UV 245
CHROMATOGRAM

Retention time: 7.3 Internal standard: amoxapine (15.8) Limit of quantitation: 15 ng/mL OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

serum; SPE; protect from light

REFERENCE
Gupta, R.N. Column liquid chromatographic determination of clozapine and N-desmethylclozapine in human serum using solid-phase extraction. J.Chromatogr.B, 1995, 673, 311-315

SAMPLE

Matrix: blood Sample preparation: 200 |xL Plasma or serum + 50 |xL 4 |xg/mL nortriptyline hydrochloride in 100 mM HCl + 100 |JLL 2 M pH 10.6 Tris buffer + 200 jxL MTBE, vortex for 30 s, centrifuge at 9950 g for 3 min, inject a 50 |xL aliquot of the organic layer.
HPLCVARIABLES

Guard column: 10 X 4.6 SCX-10C5 (Hichrom) Column: 150 X 4.6 Spherisorb S5 SCX (sulfopropyl-bonded silica) cation exchange Mobile phase: 35 mM ammonium perchlorate in MeOH adjusted to an apparent pH of 6.7 with 100 mM NaOH in MeOH Flow rate: 1.5 Injection volume: 50 Detector: UV 215
CHROMATOGRAM

Retention time: 6 Internal standard: nortriptyline hydrochloride (9) Limit of detection: 50 ng/mL OTHER SUBSTANCES Extracted: metabolites Simultaneous: amitriptyline, chlorpromazine, clomipramine, dothiepin, doxepin, fluoxetine, fluphenazine, haloperidol, imipramine, maprotiline, mianserin, norclomipramine, nordothiepin, nordoxepin, norfluoxetine, nortriptyline, paroxetine, remoxipride, sertraline, sulpride, thioridazine, trazodone Noninterfering: carbamazepine, clonazepam, diazepam, flunitrazepam, lorazepam, nordiazepam, theophylline Interfering: fluvoxamine
KEYWORDS

plasma; serum
REFERENCE
McCarthy, P.T.; Hughes, S.; Paton, C. Measurement of clozapine and norclozapine in plasma/serum by high-performance liquid chromatography with ultraviolet detection. Biomed.Chromatogr., 1995, 9, 36-41

SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol: n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 \xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLCVARIABLES

Column: 300 X 3.9 4 |xm NovaPack C18 Mobile phase: MeOH: THF: buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 269

CHROMATOGRAM Retention time: 6.53 Limit of detection: <120 ng/mL OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amisulpride, amitriptyline, amodiaquine, amoxapine, aspirin, astemizole, atenolol, benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinoxamine, carpipramine, carteolol, celiprolol, cetirizine, chlorambucil, chlordiazepoxide, chlormezanone, chlorophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, cocaine, codeine, colchicine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorubicin, debrisoquine, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, fiumazenil, flunitrazepam, fluoxetine, fluphenazine, fiurbiprofen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, Iabetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medifoxamine, mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapramine, metformin, methadone, methaqualone, methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine, mianserine, midazolam, minoxidil, moclobemide, moperone, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrexone, naproxen, nialamide, nicardipine, nifedipine, niflumic acid, nitrazepam, nitrendipine, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenobarbital, phenol, pimozide, pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, promethazine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine, quinupramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam, tenoxicam, terbutaline, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine, tiapride, tiaprofenic acid, ticlopidine, timolol, tioclomarol, tofisopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: bumadizone, cyamemazine, cyclizine, demexiptiline, diphenhydramine, histapyrrodine, medazepam, nimodipine, phenylbutazone, proguanil, trifluperidol KEYWORDS whole blood; plasma REFERENCE
Tracqui, A.; Kintz, P.; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic ScL, 1995, 40, 254-262

SAMPLE Matrix: blood Sample preparation: Inject plasma onto column A and elute to waste with mobile phase A, elute the contents of column A onto column B with mobile phase B, monitor the effluent from column B. HPLCVARIABLES Column: A 20 X 4.6 10 fxm CN; B 250 X 4.6 5 \xm Hypersil C18 ODS

Mobile phase: A water; B MeCN-.water 40:60 containing 0.4% TEMED adjusted to pH 6.5 with glacial acetic acid Detector: UV 254 CHROMATOGRAM Limit of detection: 10 ng/mL OTHER SUBSTANCES Extracted: metabolites KEYWORDS column-switching; plasma REFERENCE
Weigmann, H.; Hiemke, C. Automated determination of clozapine, N-desmethylclozapine and clozapineN-oxide by column switching and on-line high-performance liquid chromatography (Abstract 110). Ther.Drug Monit, 1995, 17, 410

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 100 jxL 4 |xg/mL protriptyline + 200 |JLL 2 M NaOH, vortex, allow to stand for 5 min, add 5 mL n-hexane: isoamyl alcohol 98.5:1.5, shake for 20 min, centrifuge at 3000 g for 5 min. Remove the organic phase and add it to 1 mL 100 mM HCl, shake for 5 min, centrifuge at 3000 g for 2 min. Remove the aqueous layer and add it to 200 JJLL 2 M NaOH, add n-hexane: isoamyl alcohol 98.5:1.5, extract, repeat extraction. Combine the organic layers and evaporate them at 45 under a stream of nitrogen, reconstitute in 100 |xL 100 mM HCl, inject a 80 |JLL aliquot. HPLC VARIABLES Guard column: 70 mm Whatman column survival kit 10 |xm ODS Column: 250 X 4.6 10 |jim Partisil 10 ODS-3 Mobile phase: MeCN: MeOH: buffer 24:12:64 (Buffer was 5 g Na2HPO4 (per L?) adjusted to pH 4.0 with phosphoric acid.) Flow rate: 2 Injection volume: 80 Detector: UV 230 CHROMATOGRAM Retention time: 9 Internal standard: protriptyline (15) Limit of detection: 2 ng/mL OTHER SUBSTANCES Extracted: metabolites KEYWORDS plasma REFERENCE
Chung, M.-C; Lin, S.-K.; Chang, W.-H.; Jann, M.W. Determination of clozapine and desmethylclozapine in human plasma by high-performance liquid chromatography with ultraviolet detection. J.Chromatogr., 1993, 613, 168-173

SAMPLE Matrix: blood

Sample preparation: 1 mL Serum + 1 mL 100 mM NaOH + 50 |JLL 4.098 |xg/mL trifluoperazine and 2.801 |xg/mL imipramine in EtOH: water 1:1, mix, add 5 mL heptane: isoamyl alcohol 99:1, shake at 250 shakings/min for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/ethanol. Remove the organic layer and evaporate it to dryness at 50 under a stream of nitrogen, dissolve the residue in 75 (JLL mobile phase, inject a 65 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 jxm Spherisorb S5W Mobile phase: MeOH: 50 mM pH 9.9 ammonium acetate buffer 85:15 Flow rate: 1.4 Injection volume: 65 Detector: UV 261
CHROMATOGRAM

Retention time: 2.3 Internal standard: trifluoperazine (3.5), imipramine (7.7) Limit of detection: 15 ng/mL Limit of quantitation: 30 ng/mL
OTHER SUBSTANCES

Simultaneous: metabolites, amitriptyline, carbamazepine, chlorprothixene, clomipramine, levomepromazine, nortriptyline Interfering: mianserine, perphenazine, zuclopenthixol
KEYWORDS

serum
REFERENCE
Olesen, O.V.; Poulsen, B. On-line fully automated determination of clozapine and desmethylclozapine in human serum by solid-phase extraction on exchangeable cartridges and liquid chromatography using a methanol buffer mobile phase on unmodified silica. J.Chromatogr., 1993, 622, 39-46

SAMPLE

Matrix: blood Sample preparation: Condition a 10 X 2 SPE cartridge packed with 40 |xm cyanopropyl bonded phase (J.T. Baker) with MeOH at 1.5 mL/min for 1 min, with water at 4.5 mL/min for 2 min, with 100 mM pH 8.0 ammonium acetate buffer at 1.5 mL/min for 2 min. 600 IxL Serum + 300 |xL trifluoperazine and imipramine in water, mix, add a 200 JJLL aliquot to the SPE cartridge, wash with 100 mM pH 8.0 ammonium acetate buffer at 1.5 mL/min for 0.5 min, wash with MeOH: water 9:91 at 1.5 mL/min for 1 min, elute the contents of the SPE cartridge directly onto the analytical column with mobile phase for 1 min.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Spherisorb S5W Mobile phase: MeOH: 50 mM pH 9.9 ammonium acetate buffer 85:15 Flow rate: 1.4 Detector: UV 261
CHROMATOGRAM Retention time: 2.3

Internal standard: trifluoperazine (3.5), imipramine (7.7) Limit of detection: 15 ng/mL Limit of quantitation: 30 ng/mL

OTHER SUBSTANCES Simultaneous: metabolites, amitriptyline, carbamazepine, chlorprothixene, clomipramine, levomepromazine, nortriptyline Interfering: mianserine, perphenazine, zuclopenthixol KEYWORDS serum; SPE REFERENCE
Olesen, O.V.; Poulsen, B. On-line fully automated determination of clozapine and desmethylclozapine in human serum by solid-phase extraction on exchangeable cartridges and liquid chromatography using a methanol buffer mobile phase on unmodified silica. J.Chromatogr., 1993, 622, 39-46

SAMPLE Matrix: blood Sample preparation: 600 |xL Serum + 100 jxL 300 |mg/mL triprolidine in 400 mM NaOH + 7 mL ethyl acetate, vortex for 1 min, centrifuge at 1000 g for 10 min. Remove the organic layer and add it to 150 |xL 100 mM HCl, vortex for 1 min, centrifuge, inject a 50 |xL aliquot of the aqueous phase. HPLCVARIABLES Column: 250 X 4.6 5 |jim Spherisorb-C6 Mobile phase: MeCN: 30 mM KH2PO4 45:55 containing 2 g sodium hexanesulfonate, pH adjusted to 2.7 with phosphoric acid Column temperature: 30 Flow rate: 1.5 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 4.5 Internal standard: triprolidine (5.5) Limit of detection: 3-4 ng/mL OTHER SUBSTANCES Extracted: metabolites Interfering: carbinoxamine, chlorpheniramine, cocaine, dipyridamole, oxazolam, trazodone KEYWORDS serum REFERENCE
Volpicelli, S.A.; Centorrino, R; Puopolo, RR.; Kando, J.; Rrankenburg, RR.; Baldessarini, R.J.; Flood, J.G. Determination of clozapine, norclozapine, and clozapine-N-oxide in serum by liquid chromatography. Clin.Chem., 1993, 39, 1656-1659

SAMPLE Matrix: blood Sample preparation: Add 10 |xL 20 \xg/mL oxaprotiline in MeOH to 990 (xL plasma or serum. Inject 100 JJLL plasma or serum onto column A with mobile phase A and elute to waste, after 15 min elute column A onto column B with mobile phase B for 2 min. Remove column A from circuit and re-equilibrate it with mobile phase A for 5 min. Chromatograph on column B with mobile phase B.

HPLCVARIABLES Column: A 20 X 4.6 10 [xm Hypersil MOS C8; B 20 X 4.6 5 ^m Hypersil CPS CN + 250 X 4.6 5 fxm Nucleosil 100 CN Mobile phase: A MeOH: water 5:95; B MeOH: MeCN: 10 mM pH 6.8 potassium phosphate buffer 188:578:235 Flow rate: 1.5 Injection volume: 100 Detector: UV 214 CHROMATOGRAM Retention time: 6.2 Internal standard: oxaprotiline (9.5) Limit of detection: 20 ng/mL OTHER SUBSTANCES Simultaneous: amitriptyline, clomipramine, desipramine, doxepin, fluoxetine, imipramine, maprotiline, metoclopramide, norfluoxetine, nortriptyline Noninterfering: carbamazepine, chlordiazepoxide, clobazam, diazepam, fiurazepam, fluspirilene, haloperidol, lorazepam, nitrazepam, nordiazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidol Interfering: fluvoxamine KEYWORDS plasma; serum; column-switching REFERENCE
Hartter, S.; Wetzel, H.; Hiemke, C. Automated determination of fluvoxamine in plasma by columnswitching high-performance liquid chromatography. Clin.Chem., 1992, 38, 2082-2086

SAMPLE Matrix: blood, saliva, tissue, urine Sample preparation: Homogenize (Polytron) tissue with 4 (whole brain) or 8 (brain striata) volumes of 100 mM pH 4.5 NaH2PO4 containing 0.5% NaF. Add 500 |xL brain homogenate or 500 JULL plasma, saliva, or urine containing 15 |ULL saturated NaF solution to 75 |xL 150 |xg/mL IS, add 50 fxL 50% perchloric acid, mix vigorously for 10 s, let stand at room temperature for 10 min, add 1 mL water, mix briefly, centrifuge at 10 at 2500 (?) for 30 min. Remove the supernatant and add it to 750 jxL saturated sodium carbonate solution, mix briefly, add 7.5 mL pentane: chloroform 95:5, rock gently for 10 min, centrifuge in a desk-top centrifuge for 2 min, freeze in dry ice/acetone for 2 min. Remove the organic layer and add it to 250 |xL 100 mM HCl, mix vigorously for 10 s, centrifuge in a desk-top centrifuge for 1-2 min, freeze in dry ice/acetone for 3-5 min, discard the organic layer. Allow the aqueous layer to thaw, remove any trace of organic solvent with a stream of nitrogen, inject a 75 (xL aliquot of the aqueous layer. HPLCVARIABLES Guard column: 15 X 3.2 7 |xm Brownlee RP-8 Column: 250 X 4.6 5 ^m Zorbax RX-C18 Mobile phase: MeCN: buffer 18:82 (Buffer was 100 mM K2HPO4 containing 0.5% triethylamine, adjusted to pH 2.7 with phosphoric acid.) Flow rate: 2 Injection volume: 75 Detector: UV 235 CHROMATOGRAM Retention time: 5.6 Internal standard: 2p-carbomethoxy-3p-(4-chlorophenyl)tropane (RTI-31) (Research Biochemical International, Natick MA) (11.4)

OTHER SUBSTANCES Extracted: chlordiazepoxide, cocaine, gepirone, methylphenidate, pentazocine, pseudococaine Simultaneous: acetaminophen, acetophenazine, amoxapine, amphetamine, atropine, benperidol, buspirone, caffeine, carbamazepine, chlorpheniramine, codeine, dextromethorphan, diazepam, diphenhydramine, flupenthixol, flurazepam, haloperidol, hydergine, hydrocodone, hydromorphone, lidocaine, loxapine, mepazine, meperidine, mesoridazine, methaquaalude, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethamphetamine, morphine, norcocaine, oxazepam, pentobarbital, phenylpropanolamine, procainamide, procaine, propyl benzoylecgonine, quinidine, quinine, salicylic acid, secobarbital, theophylline, trazodone, 3-tropanyl-3,5-dichlorobenzoate, vancomycin, WIN 35428 Noninterfering: amitriptyline, benztropine methanesulfonate, butaperazine, butriptyline, carphenazine, chlorpromazine, clomipramine, cyclobenzaprine, dextropropoxyphene, dronabinol, ephedrine, ethchlorvynol, fluoxetine, fluphenazine, imipramine, meprobamate, methadone, methamphetamine, nicotine, norfluoxetine, nortriptyline, PCP, phenothiazine, pseudoephedrine KEYWORDS rat; cow; plasma; brain REFERENCE
Bonate, P.L.; Davis, CM.; Silverman, P.B.; Swann, A. Determination of cocaine in biological matrices using reversed phase HPLC: Application to plasma and brain tissue. J.Liq.Chromatogr., 1995, 18, 3473-3494

SAMPLE Matrix: enzyme incubations Sample preparation: Inject a 20 |xL aliquot. HPLCVARIABLES Guard column: 30 X 2 5 |xm Ultracarb ODS 30 (Phenomenex) Column: 100 X 2 5 |xm Ultracarb ODS 30 (Phenomenex) Mobile phase: MeCN: water: acetic acid 75:25:1 containing 1 mM ammonium acetate Flow rate: 0.2 Injection volume: 20 Detector: UV 254; MS, Sciex API III triple quadrupole, IonSpray interface, ionizing voltage 5 kV, nebulizing gas air at 40 psi, orifice voltage 60 V, target gas argon 26 eV, post-column splitter to decrease flow to 20 jiL/min OTHER SUBSTANCES Extracted: metabolites, degradation products REFERENCE
Liu, Z.C; Uetrecht, J.P. Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells. J.PharmacoLExp.Ther., 1995, 275, 1476-1483

SAMPLE Matrix: microsomal incubations Sample preparation: Microsomal incubation + 2 mL ice-cold MeOH, let stand at 4 for 16 h, centrifuge. Remove the supernatant and evaporate it to dryness under a stream of nitrogen at 37, reconstitute the residue in 150 JULL MeOH: water 50:50, inject a 10 |xL aliquot. HPLCVARIABLES Column: 250 X 3.2 5 jxm Nucleosil C8

Mobile phase: Gradient. MeCN: 6 mM pH 3.5 ammonium formate from 10:90 to 25:75 over 15 min, to 55:45 over 20 min. Flow rate: 0.7 Injection volume: 10 Detector: UV 254; MS, Fisons Quattro II quadrupole, column effluent passed through a splitter and 40 |nL/min passed through a 150 cm X 75 (xm fused-silica capillary to the electrospray probe, nebulizing gas nitrogen at 12 L/h, drying gas nitrogen at 280 L/h, interface 60, capillary voltage 4 kV, con (orifice) voltage 30-80 V, photomultiplier 550 V, m/z 327 CHROMATOGRAM Retention time: 27 OTHER SUBSTANCES Extracted: metabolites KEYWORDS human; mouse; rat; liver REFERENCE
Maggs, J.L.; Williams, D.; Pirmohamed, M.; Park, B.K. The metabolic formation of reactive intermediates from clozapine, a drug associated with agranulocytosis in man. J.Pharmacol.Exp.Ther., 1995, 275, 1463-1475

SAMPLE Matrix: microsomal incubations Sample preparation: 1 mL Microsomal incubation + 2 mL ice-cold MeOH, let stand at 4 overnight, centrifuge. Remove the supernatant and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in EtOH .water 50:50, inject a 50 JULL aliquot. HPLCVARIABLES Column: 250 X 4.6 Nucleosil 5 C8 Mobile phase: Gradient. MeCN: 100 mM pH 3.8 ammonium acetate buffer from 20:80 to 40:60 over 15 min, return to 20:80 over 5 min. Flow rate: 1.5 Injection volume: 50 Detector: UV 254; Radioactivity CHROMATOGRAM Retention time: 13 OTHER SUBSTANCES Extracted: metabolites KEYWORDS liver REFERENCE
Pirmohamed, M.; Williams, D.; Madden, S.; Templeton, E.; Park, B.K. Metabolism and bioactivation of clozapine by human liver in vitro. J.Pharmacol.Exp.Ther., 1995, 272, 984-990

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 5 \xm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)

Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.)

Flow rate: 0.6 Injection volume: 25 Detector: UV 229

CHROMATOGRAM Retention time: 10.90 (A), 5.15 (B) OTHER SUBSTANCES Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone KEYWORDS some details of plasma extraction REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology. J.Chromatogr.A, 1995, 692, 103-119

ANNOTATED BIBLIOGRAPHY

Hubmann, M.R.; Waschgler, R.; Moll, W.; Conca, A.; Konig, P. Simultaneous drug monitoring of citalopram, clozapine, fluoxetine, maprotiline, and trazodone by HPLC analysis (Abstract 41). Ther.Drug Monit., 1995, 17, 393 [simultaneous citalopram, clozapine, fluoxetine, maprotiline, trazodone; LOQ 50 ng/mL]

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Schulz, E.; Fleischhaker, C; Remschmidt, H. Determination of clozapine and its major metabolites in serum samples of adolescent schizophrenic patients by high-performance liquid chromatography. Data from a prospective clinical trial. Pharmacopsychiatry, 1995, 28, 20-25 Lin, S.-K; Chang, W.-H.; Chung, M.-C; LamY.W.F.; Jann, M.W. Disposition of clozapine and desmethylclozapine in schizophrenic patients. J.Clin.Pharmacol., 1994, 34, 318-324 [plasma; protriptyline (IS); LOD 2 ng/mL] Weigmann, H.; Hiemke, C. Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection. J.Chromatogr., 1992, 583, 209-216 Lovdahl, M.J.; Perry, RJ.; Miller, D.D. The assay of clozapine and N-desmethylclozapine in human plasma by high-performance liquid chromatography. Ther.Drug Monit., 1991, 13, 69-72 Wilhelm, D.; Kemper, A. High-performance liquid chromatographic procedure for the determination of clozapine, haloperidol, droperidol and several benzodiazepines in plasma. J.Chromatogr., 1990, 525, 218-224 Humpel, C; Haring, C; Saria, A. Rapid and sensitive determination of clozapine in human plasma using high-performance liquid chromatography and amperometric detection. J.Chromatogr., 1989, 491, 235-239 Haring, C; Humpel, C; Auer, B.; Saria, A.; Barnas, C; Fleischhacker, W.; Hinterhuber, H. Clozapine plasma levels determined by high-performance liquid chromatography with ultraviolet detection. J.Chromatogr, 1988, 428, 160-166 Yin, J.L. [HPLC determination of clozapine in plasma]. Chung Hua Shen Ching Ching Shen Ko Tsa Chih, 1987, 20, 78-80 Wang, Z.R.; Lu, M.L.; Xu, P.P.; Zeng, YL. Determination of clozapine and its metabolites in serum and urine by reversed phase HPLC. Biomed.Chromatogr, 1986, 1, 53-57