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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-

debrisoquine. terfenadine. Systematic toxicological analysis using HPLC/DAD. propafenone. zorubicine Interfering: cycloguanil. naltrexone. J. temazepam Noninterfering: amphetamine. temazepam. Determination of alprazolam and its major metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats. quinidine. naloxone. pentazocine. oxprenolol. plasma REFERENCE Tracqui. sulindac KEYWORDS whole blood. terbutaline. Mangin. vincristine. flumazenil. triprolidine. oxazepam. sulpride. tolbutamide.5 |xg/mL demoxepam in water + 100 |xL 1 M pH 9. nimodipine. 77-83 . 1995. oxazepam.phine. flurazepam. R. nortriptyline. cocathylene. tiapride. thioridazine. tiaprofenic acid. penfluridol. nizatidine. sotalol. quinupramine. norcocaine KEYWORDS rat. P. tioclomarol. trifluperidol. strychnine. vindesine. diazepam.. midazolam. ketamine. ticlopidine. reserpine. phenylbutazone. 1994. verapamil. pipamperone. tenoxicam. proguanil. tofisopam.. phencyclidine. 654. nifedipine. suriclone. zolpidem. procarbazine. serum REFERENCE Jin. piroxicam. nomifensine. prilocaine. methaqualone. sulfinpyrazole. pimozide. Kintz. L. tropatenine. yohimbine. nialamide. zopiclone. trazodone. metoprolol. phenol. tetracaine.Forensic Sci. trimipramine. protriptyline. Lau. Take up residue in 50 JJLL mobile phase. 254-262 SAMPLE Matrix: blood Sample preparation: 50 |xL Serum + 25 \xL 0.B.4 sodium acetate buffer 8:45:47 Flow rate: 0. HPLCVARIABLES Column: 150 X 2 5jxm Ultrasphere C18 Mobile phase: MeCN: MeOH: 43 mM pH 2. tianeptine.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. A. ramipril. triazolam. thiopental. centrifuge at 1100 g for 5 min. niflumic acid. lorazepam. omeprazole. hexobarbital. nicardipine. vortex for 40 s. chlordiazepoxide. secobarbital. vinblastine. prazepam. naproxen. phenobarbital. viloxazine. timolol.Chromatogr. quinine. pindolol. warfarin. toloxatone. procainamide. J. triazolam Simultaneous: chlorpromazine. cocaine.0 borate buffer. promethazine. propranolol. prazosin. pyrimethamine. inject an aliquot. sultopride. clonazepam. thioproperazine. penbutolol. buspirone. tetrazepam. ritodrine. 40. nitrendipine. phenobarbital. Remove ether layer and evaporate it at 40° under nitrogen. ranitidine. CE. trifluoperazine. opipramol. add 2 mL diethyl ether.. nitrazepam. mix well.

X was MeCN. loflazepate. Y was 65 mM KH2PO4 + 1% diethylamine adjusted to pH 5. Danger. clorazepate.4 with phosphoric acid. to 60:40 over 15 min. When not in use. B Gradient. C. diazepam. After 7 min forward flush the contents of column A onto column B with mobile phase B. elute column B with mobile phase B. elute with 20 mL dichloromethane. phenytoin. primidone. Flow rate: 1 Injection volume: 100-200 Detector: UV 230 CHROMATOGRAM Retention time: 23 OTHER SUBSTANCES Extracted: bromazepam. phenobarbital. 1993. B 1 . HPLCVARIABLES Column: A 45 X 4 12 [xm TSK-gel G 3 PW (Tosohass). tetrazepam. allow to stand for 15 min at room temperature.) Flow rate: A 0. Wojciechowski. monitor the effluent from column B. stand for 15 min. after 5 min backflush the contents of column A onto column B with mobile phase B.47 min remove column A from the circuit and elute column B with mobile phase B. Inject a 250 jxL aliquot of the supernatant onto column A with mobile phase A and elute to waste. valproic acid KEYWORDS plasma. after 5 min remove column A from the circuit. chlordiazepoxide.3. mix 1 min. nitrazepam. B MeCN: buffer 40:60 (Buffer was 1 L water + 20 mL MeCN + 50 \xL phosphoric acid (pH 3. desmethylclobazam. R. to 25:75 over 10 min.SAMPLE Matrix: blood Sample preparation: Inject 100-200 |xL plasma onto column A with mobile phase A and elute to waste. prazepam. Between injections clean column A with two injections of 250 |xL MeCN. triazolam Noninterfering: carbamazepine. flunitrazepam.5 buffer for 10 min. J. clonazepam. remove supernatant. centrifuge at 14000 g for 2 min. 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. Monitoring of benzodiazepines (clobazam. clobazam.6 Ultrasphere ODS C18 3 ixm Mobile phase: A 50 mM pH 7. add to 3 mL Extrelut SPE cartridge and allow to soak in for 10 min. desmethyldiazepam. 617. Wash column A with MeCN: water 60:40 at 1 mL/min for 6 min then re-equilibrate with pH 7. tofisopam.Chromatogn. take up residue in 1 mL MeCN: water 5:95. X:Y 22:78 for 5 min. HPLCVARIABLES Column: A 30 X 2.1 10 ^m MPLC cartridge PRP-I (Kontron).5 phosphate buffer. lorazepam. estazolam.2). medazepam. ethosuximide. diazepam and their main active metabolites) in human plasma by column-switching high-performance liquid chromatography. temazepam.2). B 75 X 4. clotiazepam. 285-290 SAMPLE Matrix: blood Sample preparation: 3 mL Plasma + 30 JJLL 10 |xg/mL triazolam in water. oxazepam. After 0. monitor the effluent from column B. Evaporate eluant at 30° under reduced pressure. flush column A with mobile phase A. column-switching REFERENCE Lacroix. P.

vortex 30 s. add 5 mL methylene chloride + 5 mL toluene.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 7.6 Internal standard: triazolam (7. Decant organic layer. High-performance liquid chromatographic method for the determination of alprazolam in plasma using the column-switching technique. V. oxazepam KEYWORDS plasma. D.0 mL ethyl acetate: heptane 85:15.. 1991. New high-performance liquid chromatographic method for the determination of alprazolam and its metabolites in serum: instability of 4-hydroxyalprazolam. Remove aqueous layer and freeze residual aqueous layer in dry ice-acetone for 30 s. pharmacokinetics REFERENCE Rieck. SPE. column-switching..R. 1992. HPLCVARIABLES Column: 150 X 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. S. 253-260 SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 10 |xL 4 |xg/mL triazolam in methanol + 0. diazepam.Chromatogr.5 mL pH 9.0) Limit of quantitation: 1 ng/mL OTHER SUBSTANCES Simultaneous: metabolites. centrifuge at 177 g for 10 min.D. RD. inject a 125 |xL aliquot. lorazepam. J. Zemaitis..Chromatogr. 568. 259-263 SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 25 |xL 1 |xg/mL triazolam in toluene + 75 |xL 0. vortex residue with 200 |xL mobile phase. Cox.A. shake 15 min. 578. Kroboth. shake .1% ammonium hydroxide.4. bromazepam.Injection volume: 250 Detector: UV 230 CHROMATOGRAM Retention time: 7.. J...12 saturated solution of sodium borate + 4. Platt. M. W. dry under nitrogen at 50°. clobazam.

9) Limit of detection: 1 ng/mL OTHER SUBSTANCES Extracted: metabolites.5 Injection volume: 100 Detector: UV 202 CHROMATOGRAM Retention time: 8.5 mL water. Remove organic layer and evaporate it to dryness under a stream of nitrogen. protriptyline Noninterfering: N-acetylprocainamide. take up in 150 jxL mobile phase. procainamide. Alprazolam. prazepam. add 0. thioridazine Interfering: imipramine. R. centrifuge at 220 g for 10 min. inject a 100 |JLL aliquot of the lower layer.10 min. desipramine.4 Internal standard: U-31485 (6. Remove upper organic layer. vortex briefly.) Column temperature: 45 Flow rate: 2..5 mL hexane. oxazepam.5 50 mM phosphate buffer 30:70 (Buffer was 6. vortex. CL. reconstitute in 100 |xL mobile phase.5 with orthophosphoric acid.9 g KH2PO4 in 1 L water adjusted to pH 4. HPLCVARIABLES Column: 100 X 4. vortex for 2 min.and 4-hydroxyalprazolam analysis in plasma by highperformance liquid chromatography. triazolam . DeVane. lorazepam. amitriptyline. a-hydroxy. diazepam. inject an aliquot. centrifuge for 5 min. HPLCVARIABLES Column: 250 x 4 Bio-Sil ODS-10 (Bio-Rad) Mobile phase: MeCN :pH 4. propranolol. 180-186 SAMPLE Matrix: blood Sample preparation: 2 mL Serum or plasma + 100 (JLL 1 \±g/mL IS in water + 0. flurazepam.2) Limit of quantitation: 2 ng/mL OTHER SUBSTANCES Extracted: metabolites Simultaneous: nitrazepam KEYWORDS plasma. J. discard upper hexane layer. extract with 10 mL toluene: isoamyl alcohol 99:1 for 10 min on a rotator. nortriptyline. centrifuge for 5 min. chlorpromazine. evaporate under a stream of nitrogen at 37°.5 Detector: UV 214 CHROMATOGRAM Retention time: 12 Internal standard: triazolam (13. caffeine. pharmacokinetics REFERENCE Miller. 430.Chromatogr. 1988..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.L. chlordiazepoxide.) Flow rate: 1.

pentobarbital. hydroxyzine. diclofenac.. sulindac. brompheniramine. remoxipride. buspirone. disopyramide. fluvoxamine. promethazine. verapamil. theophylline. chlorcyclizine. atropine. carbamazepine. nalbuphine. guaifenesin.40 (A). tocainide. nadolol. piroxicam. propiomazine. P. trimethoprim. propofol. pentazocine. butalbital. cyclobenzaprine. fluoxetine. azatadine. cisapride.Chem. sulfinpyrazone. metolazone. ethidium bromide. thiothixene. plasma REFERENCE McCormick. midazolam. chloroquine. racemethorphan. Quantification of alprazolam in serum or plasma by liquid chromatography. nifedipine. diazepam. desipramine. loratadine. trazodone. protriptyline. mefenamic acid. homatropine. promazine. diphenidol. diphenoxylate. risperidone. moclobemide. chlorpheniramine. diltiazem. acetophenazine. methocarbamol. temazepam. cimetidine. mephenytoin. scopolamine. phentolamine. naproxen. chlorpromazine. caffeine. phenolphthalein. spironolactone. hydrocodone. naloxone. adjust pH to 3. hydralazine. phenobarbital. haloperidol. atenolol. ibuprofen. prochlorperazine. ranitidine.4 with dilute phosphoric acid. pindolol. ethopropazine. amobarbital. imipramine. J. metronidazole.44 (B) OTHER SUBSTANCES Also analyzed: acebutolol. diflunisal. doxepin. thiethylperazine. clonazepam. methoxamine. Nielsen. xylometazoline. ketoconazole. pramoxine. quazepam. hydromorphone. make up to 1 L. 30. oxycodone. pentoxifylline. droperidol. tetracaine. nortriptyline. methyldopa. bupivacaine. amoxapine. butabarbital. prazosin. pheniramine. metoclopramide.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. phenytoin. chlordiazepoxide. sertraline. clonidine. baclofen. mesoridazine. methotrexate. mazindol. 1984. furosemide. diphenhydramine. amitriptyline. chlorpropamide. 6. trimeprazine. secobarbital. benzocaine. brotizolam. fentanyl. cetirizine. labetalol. Ievorphanol. lovastatin. phenol. clozapine. loxapine. encainide. metaproterenol. Jatlow. phenylbutazone. thiopental. timolol. fluphenazine. Clin. hydrochlorothiazide. albuterol. lidocaine. flavoxate. zopiclone KEYWORDS some details of plasma extraction in paper . chlorzoxazone. pimozide. dipyridamole. flurbiprofen. salicylic acid. 1652-1655 SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4. chlorprothixene. acetaminophen. indomethacin. procainamide. chlorthalidone. codeine. fenoprofen. paroxetine.. quinidine. nizatidine. terfenadine. cocaine. chlormezanone. thioridazine. prazepam. triflupromazine. triamterene. oxymetazoline. methadone. mepivacaine.6 Injection volume: 25 Detector: UV 229 CHROMATOGRAM Retention time: 6. ketorolac. tolbutamide. naphazoline. S. propafenone. antipyrine. propranolol. lorazepam. acepromazine. methdilazine. sotalol. dobutamine. morphine.KEYWORDS serum. procyclidine. methylphenidate. warfarin..) Flow rate: 0. yohimbine. phenyltoloxamine. trifluoperazine. ketoprofen. dantrolene. bromocriptine. triazolam. acetazolamide. meperidine. perphenazine. tolmetin. methotrimeprazine.6 5 jmm Supelcosil LC-DP (A) or 250 X 4 5 \xm LiChrospher 100 RP-8 (B) Mobile phase: MeCN: 0. propantheline. clomipramine. doxapram.R. trimipramine. glyburide. flurazepam. probenecid. pemoline. norepinephrine. quinine. procaine. colchicine. terbutaline. glutethimide. oxazepam.025% phosphoric acid-. hydroxychloroquine. cyclizine. metoprolol.

Interlaboratory applicability of a retention index library of drugs for screening by reversed phase HPLC in systematic toxicological analysis.P.. Wijskbeek. Dona. diamorphine.. J. 11.Pharm. Use of high-performance liquid chromatography-diode array detection in forensic toxicology. theobromine. 3531-3546 [plasma. J.M. Jr. codeine..Chromatogr.. blood] Goldnik. R. Ada Pol. Urinary screening for alprazolam. dibenzepine. triflupromazine. A simple and rapid reversed phase high performance liquid chromatographic method for quantification of alprazolam and a. A. strychnine. warfarin] Atta-Politou.T. perphenazine. M.5 h.Anal. propranolol. 1987. M. 13. diclofenac. cyclobarbital.REFERENCE Koves. diazepam. A. flurazepam. 692. chlor- . benzodiazepine metabolite assay. vinylbital. Liquid chromatographic separation of some common benzodiazepines and their metabolites. carbromal. tolbutamide. mianserin. flunitrazepam (IS)] Noggle.5 with saturated Na2CO3. trifluoperazine. M.. tetrazepam. DeRuiter.. Franke. salicylamide. caffeine.Chromatogr. Brissie. Clark. J.2 with phosphoric acid Flow rate: 1.Chromatogr. 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. Gajewska.. fluphenazine. 4005-4021 [also bromazepam. 16. High-performance liquid chromatography of alprazolam in postmortem blood using solid-phase extraction. Robinson..a. phenylbutazone. 1995. de Zeeuw. J.A. J. propyphenazone. droperidol.M. atenolol. lormetazepam. phenazone. acetazolamide. J.Liq.6 adjusted to pH 3. 311-312 Bogusz. E. 263-266 * + + ANNOTATED BIBLIOGRAPHY Hall. extracted hydroxyalprazolam. 1341-1354 [gradient.. 1994. A.. J. take up the residue in 200 |xL mobile phase.Anal.. CR. triazolam. Erkens.D. 511-513 [SPE. F. R. LOD 1 ng/mL. extract with 10 mL dichloromethane. 14.A. phenacetin. J. oxycodone.. normethadone. ibuprofen. 1990. hydrochlorothiazide. methadone. cool.Liq. trifluoropromazine.. J.. 1991.6 Brownlee 5 |xm RP-8 Mobile phase: MeCN: 10 mM KH2PO4: n-nonylamine 450:550:0..Chromatogr. lidocaine. adjust to pH 8. mianserine.. 51. aspirin. Parissi-Poulou. and their metabolites with the EMIT d. phenobarbital. 1995. promazine.u. 19. tilidine. inject an aliquot. Evaporate. clomipramine. amphetamine. also acebutolol. 1993. aminophenazone. J.Liq. M. Determination of estazolam and alprazolam in serum by HPLC.6 Detector: UV 225 CHROMATOGRAM Retention time: 8 Internal standard: temazepam (7) OTHER SUBSTANCES Extracted: metabolites Interfering: triazolam REFERENCE Fraser. thiopental. M. A.ToxicoL. CA.hydroxyalprazolam in plasma. Koutselinis. flunarizine.A..ToxicoL. HPLCVARIABLES Column: 250 X 4.

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