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Amitriptyline
Molecular formula: C20H23N Molecular weight: 277.4 CAS Registry No.: 50-48-6 (amitriptyline), 549-18-8 (amitriptyline hydrochloride)

SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 500 jxL 750 mM pH 10 sodium bicarbonate/carbonate buffer + 50 |xL IS in EtOH: water 50:50 + 8 mL heptane: isoamyl alcohol 98:2, shake at 250 cycles/min for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Remove the organic layer and add it to 150 |xL 22 mM pH 2.5 KHaPCVphosphoric acid buffer, shake at 250 cycles/min for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Discard the organic layer, inject a 65 |xL aliquot of the aqueous layer. HPLCVARIABLES Column: 250 X 4.6 Supelco C18 Mobile phase: MeCN: buffer 45:55 (Buffer was 44 mM KH2PO4 containing 1.5 mL/L triethylamine, adjusted to pH 2.5 with phosphoric acid.) Flow rate: 1.5 Injection volume: 65 Detector: UV 240 CHROMATOGRAM Retention time: 11.5 Internal standard: 1 - (3- (dimethylamino)propyl) -1 - (p-chlorophenyl) -1,3-dihydroisobenzofuran-5-carbonitrile (LU 10-202) (Lundbeck, Copenhagen) (8.33) OTHER SUBSTANCES Extracted: citalopram, nortriptyline Simultaneous: chlorprothixene, clomipramine, clozapine, flupenthixol, haloperidol, levomepromazine, perphenazine, zuclopenthixol Noninterfering: benzodiazepines Interfering: didesmethylclomipramine, levomepromazine KEYWORDS serum REFERENCE
Olesen, O.V.; Linnet, K. Simplified high-performance liquid chromatographic method for the determination of citalopram and desmethylcitalopram in serum without interference from commonly used psychotropic drugs and their metabolites. J.Chromatogr.B, 1996, 675, 83-88

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma -I- 500 |xL 1 M sodium carbonate, vortex for 30 s, add 5 mL diethyl ether, vortex for 2 min, centrifuge at 4000 rpm for 10 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 250 fxL mobile phase, vortex for 30 s, centrifuge at 12000 rpm for 5 min, inject an aliquot of the supernatant. HPLCVARIABLES Column: 300 X 3.9 10 |xm jxBondapak C18

Mobile phase: MeOH: water 62:38 adjusted to pH 3.5 with phosphoric acid Column temperature: 40 Flow rate: 1.2 Detector: UV 262 CHROMATOGRAM Retention time: 6.1 Internal standard: amitriptyline OTHER SUBSTANCES Extracted: pheniramine Simultaneous: chlorpheniramine, diazepam, diltiazem, flurbiprofen, ibuprofen, itraconazole, ketoprofen, mebeverine, metoclopramide, phenylbutazone KEYWORDS dog; plasma; amitriptyline is IS REFERENCE
El-Sayed, YM.; Niazy, E.M.; Khidir, S.H. High-performance liquid chromatographic method for the quantitative determination of pheniramine in plasma. J.Liq.Chromatogr., 1995, 18, 763777

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + doxepin + NaOH -f hexane: isoamyl alcohol 98:2, extract. Remove the organic phase and add it to 0.03% phosphoric acid, extract, inject an aliquot of the aqueous phase. HPLCVARIABLES Guard column: C18 Column: 100 X 8 10 jxm Resolve C8 (Waters) Mobile phase: MeCN: MeOH: 56 mM ammonium acetate :1 M ammonium hydroxide 100: 10:4.5:2.6 Flow rate: 2.5 Detector: UV 220 CHROMATOGRAM Retention time: 17.8 Internal standard: doxepin (11.6) OTHER SUBSTANCES Extracted: fluoxetine, norfluoxetine, nortriptyline KEYWORDS plasma REFERENCE
el-Yazigi, A.; Chaleby, K.; Gad, A.; Raines, D.A. Steady-state kinetics of fluoxetine and amitriptyline in patients treated with a combination of these drugs as compared with those treated with amitriptyline alone. J.Clin.PharmacoL, 1995, 35, 17-21

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 240 CHROMATOGRAM Retention time: 9.23 Limit of detection: <120 ng/mL OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amisulpride, amodiaquine, amoxapine, aspirin, astemizole, atenolol, benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bisoprolol, bromazepam, brompheniramine, bumadizone, 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, clozapine, cocaine, codeine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fluphenazine, flurbiprofen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, 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, nimodipine, nitrazepam, nitrendipine, nizatidine, nomifensine, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, 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, tofisopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: etodolac, fluoxetine, nortriptyline, tioclomarol 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: 1 mL Serum + 500 |JLL 250 |xg/mL protriptyline hydrochloride + 1 mL 500 mM NaOH + 4 mL toluene: n-hexane: isoamyl alcohol 77:22:3, mix for 10 min, centrifuge at 3000 rpm for 5 min. Remove the upper organic layer and evaporate it to dryness under a stream of air at 40, reconstitute the residue in 200 |JLL MeOH, inject a 50 |xL aliquot. HPLCVARIABLES Column: 150 X 4.7 5 jxm Supelcosil LC-PCN cyanopropyl Mobile phase: MeCN.MeOH.10 mM pH 7.2 potassium phosphate buffer 60:15:25'(Prepare buffer by mixing 194 mL 1.36 g/L KH2PO4 with 274 mL 1.74 g/L K2HPO4.) Flow rate: 2 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 3.8 Internal standard: protriptyline (8.1) OTHER SUBSTANCES Extracted: norcycyclobenzaprine, nortriptyline Interfering: cyclobenzaprine KEYWORDS serum REFERENCE
Wong, E.C.C; Koenig, J.; Turk, J. Potential interference of cyclobenzaprine and norcyclobenzaprine with HPLC measurement of amitriptyline and nortriptyline: resolution by GC-MS analysis. J.Anal.ToxicoL, 1995, 19, 218-224

SAMPLE Matrix: blood Sample preparation: Automated SPE by ASPEC system. Condition a C18 Clean-Up SPE cartridge (CEC 18111, Worldwide Monitoring) with 2 mL MeOH then 2 mL water. 1 mL Plasma + 1 mL 400 ng/mL protriptyline in water, vortex, add to SPE cartridge, wash with 3 mL water, wash with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 IxL 0.1 M ammonium acetate in MeOH. Add 0.5 mL 0.5 M NaOH and 4 mL 50 mL/L isopropanol in heptane to eluate, mix thoroughly. Allow 5 min for phase separation. Remove upper heptane phase and add it to 300 jxL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase. HPLCVARIABLES Guard column: LC-8-DB (Supelco) Column: 150 X 4.6 LC-8-DB (Supelco) Mobile phase: MeCN: buffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted to pH 5.5 with glacial acetic acid.) Flow rate: 2 Injection volume: 100 Detector: UV 228

CHROMATOGRAM Retention time: 5.6 Internal standard: protriptyline (4) OTHER SUBSTANCES Extracted: chlordiazepoxide, chlorimipramine, chlorpromazine, desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl, flecainide, fluoxetine, flurazepam, haloperidol, hydroxyethylfiurazepam, ibuprofen, imipramine, lidocaine, maprotiline, methaqualone, mexiletine, midazolam, norchlorimipramine, nordoxepin, nordiazepam, nortriptyline, norverapamil, pentazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine, trazodone, verapamil Noninterfering: acetaminophen, acetylmorphine, amiodarone, amobarbital, amphetamine, bendroflumethiazide, benzocaine, benzoylecgonine, benzthiazide, butalbital, carbamazepine, chlorothiazide, clonazepam, cocaine, codeine, cotinine, cyclosporine, cyclothiazide, desalkylflurazepam, diamorphine, dicumerol, ephedrine, ethacrynic acid, ethanol, ethchlorvynol, ethosuximide, furosemide, glutethimide, hydrochlorothiazide, hydrocodone, hydroflumethiazide, hydromorphone, lorazepam, mephentermine, meprobamate, methamphetamine, metharbital, methoxsalen, methoxyphenteramine, methsuximide, methylcyclothiazide, metoprolol, MHPG, monoacetylmorphine, morphine, normethsuximide, oxazepam, oxycodone, oxymorphone, pentobarbital, phencyclidine, phenteramine, phenylephrine, phenytoin, polythiazide, primidone, prochlorperazine, salicylic acid, sulfanilamide, THC-COOH, theophylline, thiazolam, thiopental, thioridazine, tocainide, trichloromethiazide, trifluoperazine, valproic acid, warfarin Interfering: acetazolamide, methadone, norfluoxetine, temazepam, trimipramine KEYWORDS SPE; plasma REFERENCE
Nichols, J.H.; Charlson, J.R.; Lawson, G.M. Automated HPLC assay of fluoxetine and norfluoxetine in serum. Clin.Chem., 1994, 40, 1312-1316

SAMPLE Matrix: blood Sample preparation: Condition a 1 mL BondElut C18 SPE cartridge with 1 mL 1 M HCl, 1 mL MeOH, 1 mL water, and 1 mL 1% potassium carbonate. 700 JJLL Serum + 50 |xL 5 |xg/mL trimipramine in 5% potassium bicarbonate + 700 |xL MeCN, vortex, centrifuge at 1500 g for 5 min, add supernatant to SPE cartridge (at ca. 1 mL/min). Wash with 2 mL water and 1 mL MeCN, elute with 250 |JLL MeOH: 35% perchloric acid 20:1 by gravity (10 min) then centrifuge for 20 s to remove rest of eluant, inject a 50 jxL aliquot of the elu HPLCVARIABLES Guard column: 15 mm 7 jxm Brownlee RP-8 Column: 150 X 4.6 5 \xm Ultrasphere Octyl Mobile phase: MeCN: water 37.5:62.5 containing 0.5 g/L tetramethylammonium perchlorate and 0.5 mL/L 7% perchloric acid Flow rate: 1.5 Injection volume: 50 Detector: UV 215 CHROMATOGRAM Retention time: 8.3 Internal standard: trimipramine (9.6) Limit of quantitation: 5 ng/mL

OTHER SUBSTANCES Extracted: clomipramine, desipramine, doxepin, fluoxetine, fluvoxamine, imipramine, nortriptyline, protriptyline Interfering: desmethyltrimipramine, maprotiline KEYWORDS SPE; serum REFERENCE
Gupta, R.N. An improved solid phase extraction procedure for the determination of antidepressants in serum by column liquid chromatography. J.Liq.Chromatogr., 1993, 16, 2751-2765

SAMPLE Matrix: blood Sample preparation: 500 |xL Serum + 250 |xL 100 mM lauryl sulfate, centrifuge at 2500 g for 8 min, inject a 250 |JLL aliquot of the supernatant onto column A with mobile phase A, elute with mobile phase A for 6 min, backflush contents of column A onto column B with mobile phase B for 4 min. Elute column B with mobile phase B for 6 min and conduct analysis. When not in use flush column A with mobile phase A. Every eight injections backflush column A with MeCN: water 70:30. HPLCVARIABLES Column: A Guard-Pak 10 |xm Resolve CN (Waters); B 150 X 3 7 jim Separon SGX CN (Tessek) Mobile phase: A MeCN: water 3:97; B MeCN: buffer 26:74 (Buffer was 50 mM phosphoric acid, 50 mM ammonium phosphate, and 28 mM diethylamine, pH 2.55.) Flow rate: 1 Injection volume: 250 Detector: UV 210 CHROMATOGRAM Retention time: 12 Limit of detection: 20-25 ng/mL OTHER SUBSTANCES Extracted: nortriptyline KEYWORDS column-switching; serum REFERENCE
Dolezalova, M. On-line solid-phase extraction and high-performance liquid chromatographic determination of nortriptyline and amitriptyline in serum. J.Chromatogr., 1992, 579, 291-297

SAMPLE Matrix: blood Sample preparation: For each 1 mL plasma or serum add 10 jxL 14 |xg/mL trimipramine in MeOH. Inject serum or plasma directly onto column A with mobile phase A, elute with mobile phase A to waste. After 15 min elute column A onto column B (foreflush) with mobile phase B. After 2 min remove column A from the circuit, elute column B with mobile phase B, monitor the effluent from column B. Re-equilibrate column A with mobile phase A. HPLCVARIABLES Column: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 jxm Hypersil CPS CN + 250 X 4.6 5 \xm Nucleosil 100 CN

Mobile phase: A MeOH: water 5:95; B MeCN: MeOH: buffer 578:188:235 (Buffer was 10 mM K2HPO4 adjusted to pH 6.8 with 85% phosphoric acid.) Flow rate: 1.5 Injection volume: 100 Detector: UV 214 CHROMATOGRAM Retention time: 8.54 Internal standard: trimipramine (6.5) Limit of detection: 5-10 ng/mL (1 ng/mL with 3 injections) OTHER SUBSTANCES Extracted: metabolites, desipramine, fluvoxamine, imipramine, maprotiline, nortriptyline Noninterfering: chlordiazepoxide, clobazam, clozapine, diazepam, flurazepam, fluspirilene, haloperidol, nitrazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidol Interfering: clomipramine, doxepin KEYWORDS plasma; serum; column-switching REFERENCE
Hartter, S.; Hiemke, C. Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum. J.Chromatogr., 1992, 578, 273-282

SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 3 (xL 20 ng/mL clobazam in methanol -I- 1 mL saturated sodium borate (pH adjusted to 11 with 6 M NaOH) + 5 mL n-hexane, mix for 2 min, centrifuge at 3000 g for 10 min. Separate organic phase, evaporate to dryness under a stream of helium at 30, reconstitute in 20 jxL mobile phase, inject a 10 |xL aliquot. HPLCVARIABLES Guard column: 20 mm 40 jxm Pelliguard LC-8 (Supelco) Column: 150 X 4.6 Supelco 5 |xm C8 Mobile phase: MeCN: buffer 50:50 (Buffer was 10 mM NaH2PO4 + 1.2 mL/L butylamine, pH adjusted to 3 with phosphoric acid.) Flow rate: 1 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 9 Internal standard: clobazam (5) Limit of detection: 10 ng/mL OTHER SUBSTANCES Extracted: clomipramine, desipramine, imipramine, nortriptyline Simultaneous: alprazolam, clonazepam, diazepam, flunitrazepam, haloperidol, lorazepam, maprotiline, nitrazepam, triazolam KEYWORDS serum

REFERENCE
Segatti, M.P.; Nisi, G.; Grossi, R; Mangiarotti, M.; Lucarelli, C. Rapid and simple high-performance liquid chromatographic determination of tricyclic antidepressants for routine and emergency serum analysis. J.Chromatogr., 1991, 536, 319-325

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 20 |xL 10 |Jig/mL clovoxamine + 120 |xL 2 M NaOH + 4 mL heptane: isopropanol 98:2, shake for 30 min, centrifuge at 3000 g for 10 min. Remove the organic layer and add it to 100 |xL 100 mM HCl, shake for 20 min, centrifuge at 3000 g for 10 min, inject a 20 |xL aliquot of the aqueous layer. HPLCVARIABLES Column: 120 X 4.6 5 |xm Nucleosil C8 Mobile phase: MeCN:buffer 36:64 (Buffer was 16 mM KH2PO4 adjusted to pH 2.5 with concentrated phosphoric acid.) Flow rate: 1 Injection volume: 20 Detector: UV 215 CHROMATOGRAM Retention time: 5.5 Internal standard: clovoxamine (3.3) OTHER SUBSTANCES Extracted: chlorimipramine, desipramine, doxepin, fluvoxamine, imipramine, nortriptyline, trimipramine KEYWORDS plasma REFERENCE
Foglia, J.P.; Birder, L.A.; Perel, J.M. Determination of fluvoxamine in human plasma by high-performance liquid chromatography with ultraviolet detection. J.Chromatogr., 1989, 495, 295-302

SAMPLE Matrix: blood Sample preparation: Inject 200 |xL serum onto column A and elute with mobile phase A for 10 min then back-flush column A onto column B with mobile phase B for 4 min. Elute column B with mobile phase B and monitor the effluent. Remove column A from circuit and wash with MeCN: water 60:40 for 6 min then with mobile phase A for 10 min. HPLCVARIABLES Column: A 40 X 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh) Mobile phase: A 50 mM pH 7.5 potassium phosphate; B MeCN: 100 mM pH 2.7 potassium phosphate 32.5:67.5, containing 0.2 g/L sodium 1-heptanesulfonate Flow rate: 1 Injection volume: 200 Detector: UV 210 CHROMATOGRAM Retention time: 17 Limit of detection: 10 ng/mL

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