hplc analysis of alprazolam

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  • 7/28/2019 HPLC Analysis of Alprazolam


    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:

  • 7/28/2019 HPLC Analysis of Alprazolam


    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

  • 7/28/2019 HPLC Analysis of Alprazolam


    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

  • 7/28/2019 HPLC Analysis of Alprazolam


    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

  • 7/28/2019 HPLC Analysis of Alprazolam


    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: