Alprazolam

Molecular formula: C17H13CIN4 Molecular weight: 308.8 CAS Registry No.: 28981 -97-7

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 jxL 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 |jim 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 222 CHROMATOGRAM Retention time: 4.17 Limit of detection: <120 ng/mL OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, aconitine, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprenolol, amisulpride, amitriptyline, 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, cyproheptadine, eytarabine, dacarbazine, daunorubicin, demexiptiline, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazepam, diazoxide, diclofenac, dihydralazine, diltiazem, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin, doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fluvoxamine, glibenclamide, glibornuride, glipizide, glutethimide, haloperidol, histapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, loxapine, maprotiline, medazepam, medifoxamine, mefenamic acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapramine, metformin, methadone, methocarbamol, methotrexate, metipranolol, metoclopramide, mexiletine, mianserine, midazolam, minoxidil, moclobemide, moperone, morphine, nadolol, nalbuphine, nalor-

phine, naloxone, naltrexone, naproxen, nialamide, nicardipine, niflumic acid, nimodipine, nitrazepam, nitrendipine, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, promethazine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine, quinupramine, ramipril, ranitidine, reserpine, ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, 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, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: cycloguanil, debrisoquine, ketamine, lorazepam, methaqualone, metoprolol, nifedipine, piroxicam, sulindac KEYWORDS whole blood; plasma REFERENCE
Tracqui, A.; Kintz, R; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic Sci., 1995, 40, 254-262

SAMPLE Matrix: blood Sample preparation: 50 |xL Serum + 25 \xL 0.5 |xg/mL demoxepam in water + 100 |xL 1 M pH 9.0 borate buffer, mix well, add 2 mL diethyl ether, vortex for 40 s, centrifuge at 1100 g for 5 min. Remove ether layer and evaporate it at 40 under nitrogen. Take up residue in 50 JJLL mobile phase, inject an aliquot. HPLCVARIABLES Column: 150 X 2 5jxm Ultrasphere C18 Mobile phase: MeCN: MeOH: 43 mM pH 2.4 sodium acetate buffer 8:45:47 Flow rate: 0.3 Injection volume: 20 Detector: UV 230 CHROMATOGRAM Retention time: 7 Internal standard: demoxepam (4) Limit of detection: 5 ng/mL OTHER SUBSTANCES Extracted: metabolites, triazolam Simultaneous: chlorpromazine, clonazepam, diazepam, flurazepam, hexobarbital, oxazepam, phenobarbital, temazepam Noninterfering: amphetamine, buspirone, chlordiazepoxide, cocaine, cocathylene, flumazenil, midazolam, norcocaine KEYWORDS rat; serum REFERENCE
Jin, L.; Lau, CE. Determination of alprazolam and its major metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats. J.Chromatogr.B, 1994, 654, 77-83

SAMPLE Matrix: blood Sample preparation: Inject 100-200 |xL plasma onto column A with mobile phase A and elute to waste, after 5 min backflush the contents of column A onto column B with mobile phase B, after 5 min remove column A from the circuit, elute column B with mobile phase B, monitor the effluent from column B. Wash column A with MeCN: water 60:40 at 1 mL/min for 6 min then re-equilibrate with pH 7.5 buffer for 10 min. HPLCVARIABLES Column: A 45 X 4 12 [xm TSK-gel G 3 PW (Tosohass); B 75 X 4.6 Ultrasphere ODS C18 3 ixm Mobile phase: A 50 mM pH 7.5 phosphate buffer; B Gradient. X was MeCN. Y was 65 mM KH2PO4 + 1% diethylamine adjusted to pH 5.4 with phosphoric acid. X:Y 22:78 for 5 min, to 25:75 over 10 min, to 60:40 over 15 min. Flow rate: 1 Injection volume: 100-200 Detector: UV 230 CHROMATOGRAM Retention time: 23 OTHER SUBSTANCES Extracted: bromazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clotiazepam, desmethylclobazam, desmethyldiazepam, diazepam, estazolam, flunitrazepam, loflazepate, lorazepam, medazepam, nitrazepam, oxazepam, prazepam, temazepam, tetrazepam, tofisopam, triazolam Noninterfering: carbamazepine, phenytoin, ethosuximide, phenobarbital, primidone, valproic acid KEYWORDS plasma; column-switching REFERENCE
Lacroix, C; Wojciechowski, R; Danger, P. Monitoring of benzodiazepines (clobazam, diazepam and their main active metabolites) in human plasma by column-switching high-performance liquid chromatography. J.Chromatogn, 1993, 617, 285-290

SAMPLE Matrix: blood Sample preparation: 3 mL Plasma + 30 JJLL 10 |xg/mL triazolam in water, mix 1 min, allow to stand for 15 min at room temperature, add to 3 mL Extrelut SPE cartridge and allow to soak in for 10 min, elute with 20 mL dichloromethane. Evaporate eluant at 30 under reduced pressure, take up residue in 1 mL MeCN: water 5:95, stand for 15 min, centrifuge at 14000 g for 2 min, remove supernatant. Inject a 250 jxL aliquot of the supernatant onto column A with mobile phase A and elute to waste. After 7 min forward flush the contents of column A onto column B with mobile phase B. After 0.47 min remove column A from the circuit and elute column B with mobile phase B, monitor the effluent from column B. When not in use, flush column A with mobile phase A. Between injections clean column A with two injections of 250 |xL MeCN. HPLCVARIABLES Column: A 30 X 2.1 10 ^m MPLC cartridge PRP-I (Kontron); B 100 X 4.6 MPLC cartridge 5 jxm RP-8 Spheri-5 (Kontron) Mobile phase: A l L water + 20 mL MeCN + 50 |xL phosphoric acid (pH 3.2); B MeCN: buffer 40:60 (Buffer was 1 L water + 20 mL MeCN + 50 \xL phosphoric acid (pH 3.2).) Flow rate: A 0.3; B 1

Injection volume: 250 Detector: UV 230

CHROMATOGRAM

Retention time: 7.6 Internal standard: triazolam (7.0) Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES

Simultaneous: metabolites, bromazepam, clobazam, diazepam, lorazepam, oxazepam

KEYWORDS

plasma; SPE; column-switching; pharmacokinetics

REFERENCE
Rieck, W.; Platt, D. High-performance liquid chromatographic method for the determination of alprazolam in plasma using the column-switching technique. J.Chromatogr., 1992, 578, 259-263

SAMPLE

Matrix: blood Sample preparation: 1 mL Serum + 25 |xL 1 |xg/mL triazolam in toluene + 75 |xL 0.1% ammonium hydroxide, vortex 30 s, add 5 mL methylene chloride + 5 mL toluene, shake 15 min, centrifuge at 177 g for 10 min. Remove aqueous layer and freeze residual aqueous layer in dry ice-acetone for 30 s. Decant organic layer, dry under nitrogen at 50, vortex residue with 200 |xL mobile phase, inject a 125 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 \xm C18 (Supelco) Mobile phase: MeOH: buffer 40:60 (Buffer was 1 mM phosphate and 3 mM hexyltriethylammonium phosphate in water at pH 7.4.) Column temperature: 35 Flow rate: 2 Injection volume: 125 Detector: UV 221
CHROMATOGRAM

Retention time: 26 Internal standard: triazolam (30) Limit of detection: 1 ng/mL OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

serum
REFERENCE
Schmith, V.D.; Cox, S.R.; Zemaitis, M.A.; Kroboth, RD. New high-performance liquid chromatographic method for the determination of alprazolam and its metabolites in serum: instability of 4-hydroxyalprazolam. J.Chromatogr., 1991, 568, 253-260

SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 10 |xL 4 |xg/mL triazolam in methanol + 0.5 mL pH 9.12 saturated solution of sodium borate + 4.0 mL ethyl acetate: heptane 85:15, shake

10 min, centrifuge at 220 g for 10 min. Remove organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute in 100 |xL mobile phase, inject an aliquot.
HPLCVARIABLES

Column: 100 X 4.6 5 \xm C18 (IBM) Mobile phase: MeCN: 50 mM pH 6 potassium phosphate 30:70 (Between injections wash column with 10 mL MeCN: water 70:30 then 10 mL mobile phase.) Flow rate: 1.5 Detector: UV 214
CHROMATOGRAM

Retention time: 12 Internal standard: triazolam (13.2) Limit of quantitation: 2 ng/mL

OTHER SUBSTANCES

Extracted: metabolites Simultaneous: nitrazepam

KEYWORDS

plasma; pharmacokinetics
REFERENCE
Miller, R.L.; DeVane, CL. Alprazolam, a-hydroxy- and 4-hydroxyalprazolam analysis in plasma by highperformance liquid chromatography. J.Chromatogr., 1988, 430, 180-186

SAMPLE

Matrix: blood Sample preparation: 2 mL Serum or plasma + 100 (JLL 1 \g/mL IS in water + 0.5 mL water, vortex, extract with 10 mL toluene: isoamyl alcohol 99:1 for 10 min on a rotator, centrifuge for 5 min. Remove upper organic layer, evaporate under a stream of nitrogen at 37, take up in 150 jxL mobile phase, vortex for 2 min, add 0.5 mL hexane, vortex briefly, centrifuge for 5 min, discard upper hexane layer, inject a 100 |JLL aliquot of the lower layer.
HPLCVARIABLES

Column: 250 x 4 Bio-Sil ODS-10 (Bio-Rad) Mobile phase: MeCN :pH 4.5 50 mM phosphate buffer 30:70 (Buffer was 6.9 g KH2PO4 in 1 L water adjusted to pH 4.5 with orthophosphoric acid.) Column temperature: 45 Flow rate: 2.5 Injection volume: 100 Detector: UV 202
CHROMATOGRAM

Retention time: 8.4 Internal standard: U-31485 (6.9) Limit of detection: 1 ng/mL
OTHER SUBSTANCES

Extracted: metabolites, desipramine, protriptyline Noninterfering: N-acetylprocainamide, amitriptyline, caffeine, chlordiazepoxide, chlorpromazine, diazepam, flurazepam, lorazepam, oxazepam, prazepam, procainamide, propranolol, thioridazine Interfering: imipramine, nortriptyline, triazolam

KEYWORDS serum; plasma REFERENCE

McCormick, S.R.; Nielsen, J.; Jatlow, P. Quantification of alprazolam in serum or plasma by liquid chromatography. Clin.Chem., 1984, 30, 1652-1655

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 5 jmm 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: 6.40 (A), 6.44 (B) OTHER SUBSTANCES Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, 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, clozapine, 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, Ievorphanol, 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 in paper

REFERENCE Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology. J.Chromatogr.A, 1995, 692, 103-119

SAMPLE Matrix: urine Sample preparation: Heat 5 mL urine + 1 mL temazepam in MeOH with 1 mL (3-glucuronidase at 37 for 2.5 h, cool, adjust to pH 8.5 with saturated Na2CO3, extract with 10 mL dichloromethane. Evaporate, take up the residue in 200 |xL mobile phase, inject an aliquot. HPLCVARIABLES Column: 250 X 4.6 Brownlee 5 |xm RP-8 Mobile phase: MeCN: 10 mM KH2PO4: n-nonylamine 450:550:0.6 adjusted to pH 3.2 with phosphoric acid Flow rate: 1.6 Detector: UV 225 CHROMATOGRAM Retention time: 8 Internal standard: temazepam (7) OTHER SUBSTANCES Extracted: metabolites Interfering: triazolam REFERENCE Fraser, A.D. Urinary screening for alprazolam, triazolam, and their metabolites with the EMIT d.a.u. benzodiazepine metabolite assay. J.Anal.ToxicoL, 1987, 11, 263-266

ANNOTATED BIBLIOGRAPHY Hall, M.A.; Robinson, CA.; Brissie, R.M. High-performance liquid chromatography of alprazolam in postmortem blood using solid-phase extraction. J.Anal.ToxicoL, 1995, 19, 511-513 [SPE; blood] Goldnik, A.; Gajewska, M. Determination of estazolam and alprazolam in serum by HPLC. Ada Pol.Pharm., 1994, 51, 311-312 Bogusz, M.; Erkens, M.; Franke, J.P.; Wijskbeek, J.; de Zeeuw, R.A. Interlaboratory applicability of a retention index library of drugs for screening by reversed phase HPLC in systematic toxicological analysis. J.Liq.Chromatogr., 1993, 16, 1341-1354 [gradient; also acebutolol, acetazolamide, aminophenazone, amphetamine, aspirin, atenolol, caffeine, carbromal, clomipramine, codeine, cyclobarbital, diamorphine, diazepam, dibenzepine, diclofenac, droperidol, flunarizine, fluphenazine, flurazepam, hydrochlorothiazide, ibuprofen, lidocaine, lormetazepam, methadone, mianserin, mianserine, normethadone, oxycodone, perphenazine, phenacetin, phenazone, phenobarbital, phenylbutazone, promazine, propranolol, propyphenazone, salicylamide, strychnine, tetrazepam, theobromine, thiopental, tilidine, tolbutamide, trifluoperazine, trifluoropromazine, triflupromazine, vinylbital, warfarin] Atta-Politou, J.; Parissi-Poulou, M.; Dona, A.; Koutselinis, A. A simple and rapid reversed phase high performance liquid chromatographic method for quantification of alprazolam and a- hydroxyalprazolam in plasma. J.Liq.Chromatogr., 1991, 14, 3531-3546 [plasma; LOD 1 ng/mL; extracted hydroxyalprazolam; flunitrazepam (IS)] Noggle, F.T., Jr.; Clark, CR.; DeRuiter, J. Liquid chromatographic separation of some common benzodiazepines and their metabolites. J.Liq.Chromatogr., 1990, 13, 4005-4021 [also bromazepam, chlor-

diazepoxide, clobazam, clonazepam, clorazepate, demoxepam, diazepam, estazolam, fludiazepam, flunitrazepam, flurazepam, halazepam, lorazepam, midazolam, nitrazepam, nordiazepam, oxazepam, prazepam, temazepam, triazolam] Adams, W.J.; Bombardt, P.A.; Brewer, J.E. Normal-phase liquid chromatographic determination of alprazolam in human serum. Anal.Chem., 1984, 56, 1590-1594