HPLC Analysis of Alprazolam

7/28/2019 HPLC Analysis of Alprazolam

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SAMPLEMatrix: bloodSample preparat ion: 2 mL Whole blood or pla sm a + 2 mL buffer + 5 mL chloroform:isopropanol: n-h epta ne 60 :1 4:2 6, sh ake gently horizontally for 10 min, centrifuge a t 2800g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuumat 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min,inject a 50 jxL aliquot of th e su pe rn ata nt. (Buffer w as sa tur ate d am mo nium chloridesolution 25% diluted w ith water, adjusted to pH 9.5 with 25% amm onia solution.)

HPLCVARIABLESColumn: 300 X 3.9 4 |jim NovaPack C18Mobile phase: M eOH :TH F:buffer 65 :5 :3 0 (Buffer wa s 0.68 g/L (10 mM (sic)) KH 2PO 4adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each sessionwash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)Colum n tem pera ture: 30Flow rate: 0 .8Inject ion volum e: 50Detector: UV 222CHROMATOGRAMRetention t ime: 4 .17Limit of detect ion:


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phine, naloxone, naltrexone, naproxen, nialamide, nicardipine, niflumic acid, nimodipine,nitrazepam, nitrendipine, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol,oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phen obar bital,phenol, phenylbutazon e, pimozide, pindolol, pipam perone , prazepa m, prazo sin, prilocaine,procainamide, procarbazine, proguanil, promethazine, propafenone, propranolol, protrip-tyline, pyrimetham ine, quinidine, quinine, quinupram ine, ramipril , ranitidine, reserpine,ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulpride, sultopride, suriclone,temazepam , tenoxicam, terbu taline, terfenadine, tetrac aine, tetrazepam , thiopental, thio-properazin e, thio ridazine, tiane ptin e, tiaprid e, tiaprofenic acid, ticlopidine, timolol, tioclo-marol, tofisopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, triflu-peridol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vinblastine,vincristine, vindes ine, wa rfarin, yohim bine, zolpidem, zopiclone, zorubicineInterfering: cycloguanil, debrisoquine, ketamine, lorazepam, methaqualone, metoprolol,nifedipine, piroxicam, sulindac

KEYWORDSwhole blood; plasmaREFERENCETracqui, A.; Kintz, R; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic Sci.,1995, 40, 254-262SAMPLEMatrix: bloodSample preparat ion: 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 at1100 g for 5 min. Remove ether layer and evaporate it at 40 under nitrogen. Take upresidue in 50 JJLL mobile phase, inject an aliquot.HPLCVARIABLESColumn: 150 X 2 5jxm Ultrasphere C18Mobile phase: M eCN : M eOH : 43 mM pH 2.4 sodium aceta te buffer 8:45:47Flo w rate: 0 .3Inject ion volum e: 20Detector: UV 230CHROMATOGRAMRetention time: 7Internal s tandard: demoxepam (4)Limit of detect ion: 5 ng/mLOTHER SUBSTANCESExtracted: metabolites, triazolamSimul taneous: chlorpromazine, clonazepam, diazepam, flurazepam, hexobarbital, oxaze-pam, phenobarbital, temazepamNoninterfering: amphetamine, buspirone, chlordiazepoxide, cocaine, cocathylene, fluma-zenil, midazolam, norcocaineKEYWORDSrat; serumREFERENCEJin, L.; Lau, CE. Determination of alprazolam and its major metabolites in serum microsamples byhigh-performance liquid chromatography and its application to pharmacokinetics in rats.J.Chromatogr.B, 1994, 654, 7 7 - 8 3


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SAMPLEMatrix: bloodSample preparat ion: Inject 100-200 |xL pla sm a o nto column A w ith m obile ph as e A andelute to w aste, after 5 min backflush th e co ntents of column A onto column B with mobileph ase B , after 5 min remove column A from th e circuit, elute column B with m obile pha seB, mon itor the effluent from column B. W ash column A with M eCN : wa ter 60:4 0 at 1mL/min for 6 min then re-equilibrate with pH 7.5 buffer for 10 min.HPLCVARIABLESColumn: A 45 X 4 12 [xm TSK-gel G 3 PW (Tosohass); B 75 X 4.6 U ltras ph ere ODS C18 3ixmMobi le phase: A 50 mM pH 7.5 phos pha te buffer; B G radien t. X wa s MeCN. Y was 65 mMKH 2PO 4 + 1% diethy lam ine adjusted to pH 5.4 with phosphoric acid. X:Y 22 :78 for 5min, to 25:75 over 10 min, to 60:40 over 15 min.Flow rate: 1Inject ion vo lum e: 100-200Detector: UV 230CHROMATOGRAMRetention time: 23OTHER SUBSTANCESExtracted: bromazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clotiaze-pam, desmethylclobazam, desmethyldiazepam, diazepam, estazolam, flunitrazepam, lof-lazepate, lorazepam, medazepam, nitrazepam, oxazepam, prazepam, temazepam, tetra-zepam, tofisopam, triazolamNoninterfer ing : carbamazepine, phenytoin, ethosuximide, phenobarbital, primidone, val-proic acidKEYWORDSplasma; column-switchingREFERENCELacroix, C ; W ojciechowski, R; Danger, P. Mo nitoring of benzodiaze pines (clobazam, diazepam and the irmain active metabolites) in human plasma by column-switching high-performance liquid chromatog-raphy. J.Chromatogn, 1993, 617, 285-290SAMPLEMatrix: bloodSample preparat ion: 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 andallow to soak in for 10 min, elute with 20 mL dichloromethane. Evaporate eluant at 30und er reduced press ure, tak e up residue in 1 mL MeC N: wate r 5:9 5, stan d for 15 min,centrifuge at 14000 g for 2 min, remove supernatant. Inject a 250 jxL aliquot of thesu pe rn ata nt onto column A with m obile phas e A and elute to w aste. After 7 min forward

flush th e c ontents of column A onto column B w ith m obile ph ase B. After 0.47 m in removecolumn A from th e circuit and elute column B with mobile ph ase B , mo nitor the effluentfrom column B. When not in u se, flush column A with mobile ph ase A. Between injectionsclean column A with two injections of 250 |xL MeCN.HPLCVARIABLESColumn: A 30 X 2.1 10 ^m MPL C cartridg e PR P-I (Kontron); B 100 X 4.6 MPLC cartridg e5 jxm RP-8 Spheri-5 (Kontron)Mobi le phase: A l L wa ter + 20 mL MeCN + 50 |xL phosphoric acid (pH 3.2); B MeCN:buffer 40 :60 (Buffer wa s 1 L w ater + 20 mL MeCN + 50 \xL phosphoric acid (pH 3.2).)Flo w rate: A 0.3; B 1


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Injection volume: 250Detector: UV 230CHROMATOGRAMRetention time: 7.6Internal standard: triazolam (7.0)Limit of quantitation: 1 ng/mLOTHER SUBSTANCESSimultaneous: metabolites, bromazepam, clobazam, diazepam, lorazepam, oxazepamKEYWORDSplasma; SPE; column-switching; pharmacokineticsREFERENCERieck, W.; Platt, D. High-performance liquid chromatographic method for the determination of alpra-zolam in plasm a u sing the column-switching technique. J.Chromatogr., 1992, 578, 2 5 9 - 2 6 3SAMPLEMatrix: bloodSample 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, shake15 min, centrifuge at 177 g for 10 min. Remove aqueous layer and freeze residual aqueouslayer in dry ice-acetone for 30 s. Decant organic layer, dry under nitrogen at 50, vortexresidue with 200 |xL mobile phase, inject a 125 |xL aliquot.HPLCVARIABLESColumn: 150 X 4.6 5 \xm C18 (Supelco)Mobile phase: MeOH: buffer 40:60 (Buffer was 1 mM phosphate and 3 mM hexyltriethy-lammonium phosphate in water at pH 7.4.)Column temperature: 35Flow rate: 2Injection volume: 125Detector: UV 221CHROMATOGRAMRetention time: 26Internal standard: triazolam (30)Limit of detection: 1 ng/mLOTHER SUBSTANCESExtracted: metabolitesKEYWORDSserumREFERENCESchmith, V.D.; Cox, S.R.; Zemaitis, M.A.; Kroboth, RD. New high-performance liquid chromatographicmethod for the determination of alprazolam and its metabolites in serum: instability of 4-hydroxy-alprazolam. J.Chromatogr., 1991, 568, 253-260SAMPLEMatrix: bloodSample 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 aceta te: heptane 85:15, shake


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10 min, centrifuge at 220 g for 10 min. Remove organic layer and evaporate it to drynessunder a stream of nitrogen, reconstitute in 100 |xL mobile phase, inject an aliquot.HPLCVARIABLESCo lum n: 100 X 4.6 5 \xm C18 (IBM)M obile 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: 1.5Detec tor: UV 214C H R O M A TO G R A MRetention time: 12Internal standard: triazolam (13.2)Lim it of qu an tita tio n: 2 ng/mLOTHER SUBSTANCESExtracted: metabolitesSimultaneous: nitrazepamKEYWORDSplasma; pharmacokineticsREFERENCEMiller, R.L.; DeVane, C L . Alprazolam, a-hydroxy- a nd 4-hydroxyalprazolam analy sis in plasm a by high-performance liquid chromatography. J.Chromatogr., 1988, 430, 180-186SAMPLEMatrix: bloodSample preparation: 2 mL Serum or plasma + 100 (JLL 1 \g/mL IS in water + 0.5 mLwater, vortex, extract w ith 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 nitrogenat 37, take up in 150 jxL mobile phase, vortex for 2 min, add 0.5 mL hexane, vortexbriefly, centrifuge for 5 min, discard upper hexane layer, inject a 100 |JLL aliquot of thelower layer.HPLCVARIABLESColumn: 250 x 4 Bio-Sil ODS-10 (Bio-Rad)M obile phase: MeCN :pH 4.5 50 mM phosphate buffer 30:70 (Buffer was 6.9 g KH2PO4 in1 L water adjusted to pH 4.5 with orthophosphoric acid.)Column temperature: 45Flow rate: 2.5Injection volume: 100Detecto r: UV 202C H R O M A TO G R A MRetention time: 8.4In te rn al s ta nd ar d: U-31485 (6.9)Lim it of dete ctio n: 1 ng/mLOTHER SUBSTANCESExtracted: metabolites, desipramine, protriptylineNoninterfering: N-acetylprocainamide, amitriptyline, caffeine, chlordiazepoxide, chlor-promazine, diazepam, flurazepam, lorazepam, oxazepam, prazepam, procainamide, pro-pranolol, thioridazineIn te rf er in g: imipramine, nortriptyline, triazolam


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KEYWORDSserum; plasmaREFERENCEMcCormick, S.R.; Nielsen, J.; Jatlow, P. Quantification of alprazolam in serum or plasma by liquidchromatography. Clin.Chem., 1984, 30, 1652-1655SAMPLEMatrix: solutionsHPLCVARIABLESColumn: 250 X 4.6 5 jmm Supelcos il L C-DP (A) or 250 X 4 5 \xm LiC hrospher 100 RP-8 (B)Mobile phase: M eCN : 0.025% phosph oric acid-.buffer 25 :1 0 :5 (A) or 60 :2 5: 1 5 (B) (Bufferwas 9 mL concentrated phosphoric acid and 10 mL triethy lam ine in 900 mL water, adjustpH to 3.4 with dilute phosphoric acid, make up to 1 L.)Flow rate: 0 .6Inject ion volume: 25Detector: UV 229CHROMATOGRAMRetent ion t ime: 6.40 (A), 6.44 (B)OTHER SUBSTANCESAlso analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,albuterol, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azata-

dine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine,buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlor-propamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomi-pramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclo-benzaprine, 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, fu-rosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine,hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine,ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ie-vorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamicacid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyl-dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen,nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazo-line, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine,pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, pra-zosin, probenecid, procainam ide, p rocaine, pro chlorperazine, procyclidine, prom azine, pro-methazine, propafenone, propantheline, propiomazine, propofol, propranolol, protripty-line, quazepam, quinidine, quinine, racem ethorpha n, ranitidine, remoxipride, risperidone,salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyra-zone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethyl-perazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, tri-methoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

KEYWORDSsome details of plasma extraction in paper


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REFERENCEKoves, E.M. Use ofhigh-performance liquid chromatography-diode array detection in forensic toxicology.J.Chromatogr.A, 1995,692, 103-119

SAMPLEMatrix: urineSample preparation: Heat 5 mL urine + 1 mL temazepam in MeOH with 1 mL (3-glu-curonidase at 37 for 2.5 h, cool, adjust to pH 8.5 with saturated Na2CO3, extract with 10mL dichloromethane. Evaporate, take up the residue in 200 |xL mobile phase, inject an

aliquot.

HPLCVARIABLESColumn: 250 X 4.6 Brownlee 5 |xm RP-8Mobile phase: MeCN: 10 mM KH2PO4: n-nonylamine 450:550:0.6 adjusted to pH 3.2 with

phosphoric acidFlow rate: 1.6Detector: UV 225

CHROMATOGRAMRetention time: 8Internal standard: temazepam (7)

OTHER SUBSTANCESExtracted: metabolitesInterfering: triazolam

REFERENCEFraser, 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 BIBLIOGRAPHYHall, M.A.; Robinson, CA.; Brissie, R.M. High-performance liquid chromatography of alprazolam inpostmortem blood using solid-phase extraction. J.Anal.ToxicoL, 1995,19,51 1-5 13 [SPE; blood]Goldnik, A.; Gajewska, M. Determination of estazolam and alprazolam in serum by HPLC. AdaPol.Pharm., 1994,51, 311-312Bogusz, M.; Erkens, M.; Franke, J.P.; Wijskbeek, J.; de Zeeuw, R.A. Interlaboratory applicability of aretention index library of drugs for screening by reversed phase HPLC in systematic toxicologicalanalysis. J.Liq.Chromatogr., 1993,16, 1341-1354 [gradient; also acebutolol, acetazolamide, amino-phenazone, amphetamine, aspirin, atenolol, caffeine, carbromal, clomipramine, codeine, cyclobarbi-tal, diamorphine, diazepam, dibenzepine, diclofenac, droperidol, flunarizine, fluphenazine, fluraze-pam, hydrochlorothiazide, ibuprofen, lidocaine, lormetazepam, methadone, mianserin, mianserine,

normethadone, oxycodone, perphenazine, phenacetin, phenazone, phenobarbital, phenylbutazone,promazine, propranolol, propyphenazone, salicylamide, strychnine, tetrazepam, theobromine, thio-pental, tilidine, tolbutamide, trifluoperazine, trifluoropromazine, triflupromazine, vinylbital,warfarin]Atta-Politou, J.; Parissi-Poulou, M.; Dona, A.; Koutselinis, A. A simple and rapid reversed phase highperformance liquid chromatographic method for quantification of alprazolam and a- hydroxyalpra-zolam in plasma. J.Liq.Chromatogr., 1991, 14,3531-3546 [plasma; LOD 1ng/mL; extracted hydrox-yalprazolam ; flunitrazepam (IS)]Noggle, F.T., Jr.; Clark, CR.; DeRuiter, J. Liquid chromatographic separation of some common benzo-

diazepines and their metabolites. J.Liq.Chromatogr., 1990,13,40 05 -40 21 [also bromazepam, chlor-


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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 al-prazolam in human serum. Anal.Chem., 1984, 56, 1590-1594

HPLC Analysis of Alprazolam

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