I. BLOOD SAMPLE 1.

SAMPLE Matrix: blood Sample preparation: Condition a Bond-Elut C18 column with 2 volumes MeOH then 2 volumes water. Add 1 mL serum then 200 L 700 ng/mL promazine in MeOHrO. 1 M HCl 13:87 to each column, wash with 2 volumes water, wash with 2 volumes 0.1 M acetic acid, wash with MeOH/water, add 200 (JLL 10 mM ammonium acetate in MeOH, wait for 30 s, elute with vacuum, repeat elution process two more times. Combine eluates and evaporate them to dryness at 56-8 under compressed air. Reconstitute with 200 L mobile phase, vortex 10 s, inject 75-100 L aliquot. (MeOH/water was 500 mL MeOH:water 65: 35 plus 25 L concentrated HCl.)

HPLCVARIABLES Column: 250 X 4.6 5 jim Supelco silica Mobile phase: EtOH:MeCN:t-butylamine 98:2:0.05 (Mix 1 gallon EtOH with 77 mL MeCN and 1.9 mL t-butylamine.) Flow rate: 2 Injection volume: 75-100 Detector: UV 254

CHROMATOGRAM Retention time: 3.6, 3.3 (cis isomer) Internal standard: promazine (5.2) Limit of detection: 2 ng/mL

OTHER SUBSTANCES Extracted: amitriptyline, desipramine, desmethyldoxepin, imipramine, nortriptyline, protriptyline Simultaneous: N-acetylprocainamide, procainamide, zimeldine, morphine, codeine, trifluoperazine, 2-hydroxydesipramine, desmethylchlordiazepoxide, buprion, diazepam, demoxepam, chlordiazepoxide, propoxyphene, dextropropoxyphene, cocaine, oxapam, trimipramine, mianserin, trimeprazine, loxepin, fluphenazine, methadone, trifluopromazine, phenteramine, chlorimipramine Noninterfering: thiopropazine Interfering: thioridazine, hydroxyamoxapine, meperidine, chlorpromazine, disopyramide,

amphetamine, 2-hydroxyimipramine, iprindole, pyrilamine, promethazine, prolixin, amoxapine

KEYWORDS serum; normal phase

REFERENCE Beierle,F.A.; Hubbard,R-W. Liquid chromatographic separation of antidepressant drugs: I. Tricyclics, Ther.DrugMonit, 1983, 5, 279-292.

2. SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 1 mL 450 mM NaOH + 5 mL hexane:isopropanol 95: 5, shake for 5 min, centrifuge. Remove 4 mL of the organic layer and add it to 50 L 200 mM HCl, shake for 2 min, centrifuge. Inject a 20 L aliquot of the aqueous layer.

HPLC VARIABLES Column: 100 X 3 octyl CP-tm-Spher C8 glass column (Chrompack) Mobile phase: MeCN:500 mM NaH2PO4 35:65 adjusted to pH 2.2 with phosphoric acid Flow rate: 0.8 Injection volume: 20 Detector: UV 210

CHROMATOGRAM Retention time: 3.3 Limit of detection: 10 ng/mL

OTHER SUBSTANCES Simultaneous: nortriptyline, amitriptyline

KEY WORDS serum

REFERENCE

Van Damme,M.; Molle,L.; Abi Khalil,F. Useful sample handlings for reversed phase high performance liquid chromatography in emergency toxicology, J.Toxicol.Clin.ToxicoL, 1985, 23, 589-614.

3. SAMPLE Matrix: blood Sample preparation: Condition a 1 mL Analytichem cyanopropyl SPE cartridge with 1 mL water and 1 mL MeOH, do not allow to dry. Add 1 mL serum + 250 (xL 0.2 g/mL imipramine in 50 mM sodium n-heptanesulfonic acid to the SPE cartridge, wash with 1 mL water, 1 mL MeOH:water 50:50, air dry cartridge, elute with 1 mL MeOH:triethylamine 99.2:0.8, evaporate eluate to dryness under a stream of nitrogen at 40, reconstitute residue with 250 L mobile phase, inject a 50 L aliquot.

HPLC VARIABLES Column: 150 X 4.6 5(xm Spherisorb cyanopropyl Mobile phase: MeOH:20 mM phosphoric acid containing 0.05% N,N-diethyloctylamine 55: 45, pH was 2.4 Flow rate: 1.5 Injection volume: 50 Detector: UV 214

CHROMATOGRAM Retention time: 6 Internal standard: imipramine (9) Limit of detection: 2 ng/mL

OTHER SUBSTANCES Simultaneous: metabolites

KEYWORDS serum; SPE

REFERENCE

Emm,T.; Lesko,L.J.; Perkal,M.B. Simultaneous determination of doxepin and nordoxepin in serum using high-performance liquid chromatography, J.Chromatogr., 1987, 419, 445-451.

II. URINE BLOOD SAMPLE 1. SAMPLE Matrix: blood, urine Sample preparation: Add 500 L 3 M ammonia solution and 7 mL n-pentane:isopropanol 95:5 to 2 mL plasma containing 200 ng/mL nortriptyline or 2 mL urine containing 400 ng/mL nortriptyline, shake in an overhead shaker for 20 min, let stand for 10 min. Transfer the upper organic layer to a tube containing 1 mL 100 mM HCl, shake for 20 min, let stand for 5 min. Aspirate the organic phase to waste, wash the remaining aqueous layer with 3 mL pentane by shaking for 10 min. Add 500 L3 M ammonia solution and 6 mL apentane:isopropanol 95:5 to the washed aqueous layer, shake for 20 min, evaporate the organic layer under a stream of nitrogen at 65, reconstitute the residue with 160 L mobile phase, inject a 60 L aliquot.

HPLC VARIABLES Column: 150 X 4.5 3 m Spherisorb silica Mobile phase: MeOH:hexane:nonylamine 5:95:0.3 Flow rate: 1 Injection volume: 60 Detector: UV 254

CHROMATOGRAM Retention time: 4.0 (cis), 4.5 (trans) Internal standard: nortriptyline (10)

OTHER SUBSTANCES Extracted: metabolites, N-desmethyldoxepin

KEYWORDS dog; human; plasma; normal phase

REFERENCE

Yan,J.; Hubbard,J.W.; McKay,G.; Midha,K.K. Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by highperformance liquid chromatography, J.Chromatogr.B, 1997, 691, 131-138.

III. HAIR SAMPLE 1. SAMPLE Matrix: hair Sample preparation: Wash hair in water, rinse 3 times with MeOH, dry, weigh. 5-25 mg Washed hair + 1 mL 1 M NaOH, heat at 70 for 30 min, adjust pH to 9.5-10. 1 mL Extract + 1 |xg protriptyline + 1 mL water + 1 mL 200 mM sodium carbonate buffer, mix, extract with hexane:butanol 95:5 for 20 min. Remove the organic layer and add it to 100 JULL 0.2% orthophosphoric acid, mix for 20 min, inject a 30 JULL aliquot of the aqueous layer.

HPLC VARIABLES Guard column: 15 X 3.2 7 u-m Newguard RP-18 Column: 100 X 4.6 Spheri-5 RP-C18 Mobile phase: MeCN:buffer 40:60 (Buffer was 1.2 L 100 mM pH 7.0 NaH2PO4 + 30 Ml diethylamine.) Flow rate: 2 Injection volume: 30 Detector: UV 214

CHROMATOGRAM Internal standard: protriptyline (4)

OTHER SUBSTANCES Extracted: amitriptyline, clomipramine, desipramine, dothiepin, haloperidol, imipramine, mianserin, nortriptyline

KEYWORDS may be interferences

IV. SOLUTION SAMPLE 1. SAMPLE Matrix: solutions

HPLC VARIABLES Guard column: 30 X 2.1 Spheri-5 RP-8 Column: 220 X 2.1 Spheri-5 RP-8 Mobile phase: Gradient. A was 0.08% diethylamine and 0.09% phosphoric acid in water, pH 2.3. B was MeCN:water 90:10 containing 0.08% diethylamine and 0.09% phosphoric acid. A:B 95:5 for 2 min, to 0:100 over 15 min (?), maintain at 0:100 for 5 min. Column temperature: 50 Flow rate: 0.5 Detector: UV 200

CHROMATOGRAM Retention time: 13

OTHER SUBSTANCES Simultaneous: desmethyldoxepin, desipramine, nortriptyline, imipramine, amitriptyline Also analyzed: amphetamine, chlordiazepoxide, chlorpromazine, desalkylflurazepam, diazepam, diethylpropion, ephedrine, fenfluramine, nordiazepam, flurazepam, oxazepam, mesoridazine, phentermine,

methamphetamine,

norchlordiazepoxide,

phenylpropanolamine, prazepam, promazine, thioridazine, thiothixene, trifluoperazine

REFERENCE Rainin Catalog, Cl-94, 1994, p. 7.24.