Ketoconazole

Molecular formula: C26H28CI2N4O4 Molecular weight: 531.4 CAS Registry No.: 65277-42-1

SAMPLE Matrix: blood Sample preparation: 500 (JLL Plasma + 2 jxg clotrimazole + hexane: isoamyl alcohol 98.5:1.5, vortex, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 250 |xL MeCN, inject an aliquot. HPLCVARIABLES Column: 150 X 3.9 NovaPak C18 Mobile phase: MeCN:MeOH:50 mM phosphate buffer 40:5:55 Detector: UV 220 CHROMATOGRAM Limit of detection: 100-200 ng/mL KEYWORDS plasma; pharmacokinetics REFERENCE
von Moltke, L.L.; Greenblatt, D.J.; Harmatz, J.S.; Duan, S.X.; Harrel, L.M.; Cotreau-Bibbo, M.M.; Pritchard, G.A.; Wright, CE.; Shader, R.I. Triazolam biotransformation by human liver microsomes in vitro: Effects of metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole. J.Pharmacol.Exp.Then, 1996, 276, 370-379

SAMPLE Matrix: blood

Sample preparation: 200 JULL Serum + 500 JULL MeCN, mix, centrifuge, inject a 200 JJLL

aliquot of the supernatant. HPLCVARIABLES Guard column: 15 mm long C18 Column: 250 X 4.6 5 \xm C18 (Beckman) Mobile phase: MeCN: 50 mM pH 2.2 phosphoric acid 40:60 Flow rate: 2 Injection volume: 200 Detector: UV 207 CHROMATOGRAM Limit of quantitation: 20 ng/mL KEYWORDS serum; pharmacokinetics REFERENCE
Chin, T.W.F.; Loeb, M.; Fong, LW. Effects of an acidic beverage (Coca-Cola) on absorption of ketoconazole. Antimicrob.Agents Chemother., 1995, 39, 1671-1675

SAMPLE Matrix: blood

Sample preparation: 200 jxL Serum + 300 |xL 5 jxg/mL terconazole in MeCN, vortex, centrifuge, inject 75 |JLL supernatant. HPLCVARIABLES Column: 300 X 4.5 jxBondapak C18 Mobile phase: MeOH: buffer 60:40 (Buffer was 25 mM KH2PO4 + 4 mM heptanesulfonic acid, adjusted to pH 8.0 with 1 M NaOH.) Flow rate: 1.8 Injection volume: 75 Detector: UV 226 CHROMATOGRAM Internal standard: terconazole Limit of quantitation: 10 ng/mL KEYWORDS serum REFERENCE
Carver, P.L.; Berardi, R.R.; Knapp, M.J.; Rider, J.M.; Kauffman, CA.; Bradley, S.F.; Atassi, M. In vivo interaction of ketoconazole and sucralfate in healthy volunteers. Antimicrob.Agents Chemother., 1994, 38, 326-329

SAMPLE Matrix: blood Sample preparation: Condition a C18 SPE cartridge (Analytichem part 607303) by washing with 2 mL MeOH then 5 mL water. 1 mL Serum + 100 JJLL 3 mg/mL clotrimazole in MeOH + 200 |xL ammonium hydroxide, add to cartridge, wash with 6 mL water, elute with 3 mL MeOH. Evaporate the residue to dryness under a stream of nitrogen at 45, reconstitute in 1 mL mobile phase, inject 50 JJLL aliquot. (SPE preparation from J. Chromatogr. 1986, 377, 287.) HPLC VARIABLES Column: 150 X 3.9 Novopak C18 Mobile phase: MeOH: MeCN: 20 mM KH2PO4 30:30:35, adjusted to pH 6.S Flow rate: 2 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 4.3 Internal standard: clotrimazole (7.0) Limit of quantitation: 200 ng/mL KEYWORDS serum; SPE REFERENCE
Piscitelli, S.C.; Goss, T.F.; Wilton, J.H.; D'Andrea, D.T.; Goldstein, H.; Schentag, J.J. Effects of ranitidine and sucralfate on ketoconazole bioavailability. Antimicrob.Agents Chemother., 1991, 35, 1765-1771

SAMPLE Matrix: blood Sample preparation: Condition a 3 mL Bond-Elut C18 cartridge with 6 mL MeOH and 6 mL water. 1 mL Serum + 110 jxL 50 mg/L terconazole in water + 250 jxL 100 mM NaOH + 3 mL water, add to cartridge, wash with 9 mL water, wash with 200 |JLL MeOH, elute

with 1 mL MeOH. Evaporate eluent at 60, resuspend in 200 \iL mobile phase, centrifuge at 13000 g for 2 min, inject 20-40 jiL. HPLC VARIABLES Guard column: Chrompack C18 Column: 100 X 3 Hypersil ODS in a Chrompack glass cartridge Mobile phase: MeCN: water 45:55 containing 500 |xL/L diethylamine, pH adjusted to 8.0 with orthophosphoric acid Flow rate: 0.6 Injection volume: 20-40 Detector: UV 254 CHROMATOGRAM Retention time: 5.0 Internal standard: terconazole (11.4) Limit of quant it ation: 50 ng/mL OTHER SUBSTANCES Noninterfering: acetaminophen, acyclovir, allopurinol, amoxicillin, amphotericin B, ampicillin, aspirin, azlocillin, bendrofluazide, bumetanide, buprenorphine, carbenicillin, cefazolin, cefotaxime, cefoxitin, ceftazidime, cefuroxime, cephalexin, chlorambucil, chloramphenicol, chlordiazepoxide, chlorpheniramine, chlorpropamide, cyclophosphamide, cyclosporin, cytarabine, daunorubicin, dextropropoxyphene, dihydrocodeine, domperidone, flucytosine, furosemide, gentamicin, griseofulvin, melphalan, methotrexate, metochlopramide, metronidazole, miconazole, nabilone, netilmicin, nicotinamide, nitrazepam, penicillin G, piperacillin, prednisolone, procarbazine, prochlorperazine, riboflavin, rifampin, sulfamethoxazole, thioguanine, tobramycin, tolbutamide, trimethoprim Interfering: diazepam KEYWORDS serum REFERENCE
Turner, CA.; Turner, A.; Warnock, D.W. High performance liquid chromatographic determination of ketoconazole in human serum. J.Antimicrob.Chemother., 1986, 18, 757-763

SAMPLE Matrix: blood, tissue Sample preparation: Condition a SPICE reversed-phase SPE cartridge by washing with 2 mL MeOH then 5 mL water. Tissue. 0.5 g Tissue + 100 |xL 3 mg/mL clotrimazole in MeOH + 4 mL MeCN, homogenize for 2 min using a PTFE pestle in a tissue grinder, centrifuge at 1500 g for 15 min. Remove supernatant and evaporate it under a stream of nitrogen at 45. Reconstitute residue in 1 mL 10 mM HCl, add 200 jxL ammonium hydroxide to adjust pH to about 10.5, add to cartridge, wash with 6 mL water, elute with 3 mL MeOH. Evaporate the residue to dryness under a stream of nitrogen at 45, reconstitute in 200 |xL mobile phase, inject 50 |JLL aliquot. Plasma. 1 mL Plasma + 100 |xL 3 mg/mL clotrimazole in MeOH + 200 jxL ammonium hydroxide, add to cartridge, wash with 6 mL water, elute with 3 mL MeOH. Evaporate the residue to dryness under a stream of nitrogen at 45, reconstitute in 1 mL mobile phase, inject 50 |xL aliquot. HPLCVARIABLES Column: 150 X 3.9 5 \m Novapak C18 Mobile phase: MeCN: MeOH: 20 mM pH 6.8 KH2PO4ZNaOH 30:35:35 Flow rate: 2 Injection volume: 50 Detector: UV 254

CHROMATOGRAM Retention time: 4.5 Internal standard: clotrimazole (&8) Limit of detection: 200 ng/mL (plasma), 400 ng/g (tissue) KEYWORDS plasma; SPE; lung; liver; adrenal REFERENCE
Riley, CM.; James, M.O. Determination of ketoconazole in the plasma, liver, lung and adrenal of the rat by high-performance liquid chromatography. J.Chromatogr., 1986, 377, 287-294

SAMPLE Matrix: formulations Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to about 30 mg ketoconazole, add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of filtrate to 5 mL of 200 |xg/mL clotrimazole in MeOH, make up to 25 mL with MeOH, inject 20 JULL aliquot. Cream. Condition a 500 mg Bond-Elut diol cartridge with 6 mL dichloromethane. Weigh out cream equivalent to about 5 mg of drug, add 30 mL dichloromethane, sonicate for 3 min, make up to 100 mL with dichloromethane, filter. Add a 2 mL aliquot to the cartridge, wash with 2 mL dichloromethane: methanol 4:1, wash with 1 mL MeOH, elute with 3 mL MeOH-.buffer 85:15. Add eluate to 1 mL 200 \xg/mL clotrimazole in MeOH, make up to 5 mL with MeOH, inject 20 |xL aliquot. (Buffer was 50 mM triethylamine adjusted to pH 7.0 with phosphoric acid.) HPLC VARIABLES Column: 250 X 4.6 5 |xm Spherisorb CN Mobile phase: THF: buffer 30:70 (Buffer was 50 mM triethylamine adjusted to pH 3.0 with phosphoric acid.)

Flow rate: 1 Injection volume: 20

Detector: UV 230 [Enhanced sensitivity with photoreactor (Beam Boost model C6808 with 10 m X 0.3 mm reaction coil) followed by UV detection at 270 nm.] CHROMATOGRAM Retention time: 7 Internal standard: clotrimazole (9.5) OTHER SUBSTANCES Simultaneous: bifonazole, econazole, fenticonazole, isoconazole, miconazole, tioconazole KEYWORDS tablets; creams; post-column reaction REFERENCE
Di Pietra, A.M.; Cavrini, V.; Andrisano, V.; Gatti, R. HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations. J.Pharm.Biomed.AnaL, 1992, 10, 873-879

SAMPLE Matrix: solutions HPLCVARIABLES Column: 5 |xm Deltabond CN (Keystone) Mobile phase: Carbon dioxide: MeOH

Flow rate: 0.5 (CO2), 0.05 to 0.12 in 7 min (MeOH) Detector: UV 254
CHROMATOGRAM

Retention time: 7 OTHER SUBSTANCES Simultaneous: impurities KEYWORDS outlet pressure 3600 psi; SFC; back-pressure regulator heated to 60 REFERENCE
Ashraf-Khorassani, M.; Levy, J.M. Addition of modifier in supercritical fluid chromatography using a microbore reciprocating pump. Chromatographia, 1995, 40, 78-84

SAMPLE Matrix: solutions HPLC VARIABLES Column: 250 X 4.6 5 fjtm Supelcosil LC-DP (A) or 250 X 4 5 fxm 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: 11.30 (A), 5.92 (B) OTHER SUBSTANCES Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, 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, ketoprofen, ketorolac, labetalol, levorphanol, 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, protripty-

line, 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

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: tissue Sample preparation: Skin sample extracted with 250 |xL mobile phase, vortex 1 min, centrifuge at 8000 rpm for 10 min, inject 40 J U L L aliquot.
HPLCVARIABLES

Column: 125 X 4.5 Whatman 5 |xm reverse-phase C18 Mobile phase: MeCN: 10 mM pH 6.0 K2HPO4 65:35 Flow rate: 0.7 Injection volume: 40 Detector: UV 254
CHROMATOGRAM

Retention time: 8.6 Limit of detection: 50 ng/mL

KEYWORDS

skin
REFERENCE
Pershing, L.K.; Corlett, J.; Jorgensen, C. In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole and miconazole in human stratum corneum. Antimicrob.Agents Chemother., 1994, 38, 90-95

ANNOTATED BIBLIOGRAPHY

Hoffman, D.W.; Jones-King, K.L.; Ravaris, CL.; Edkins, R.D. Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva. Anal.Biochem., 1988, 172, 495-498 Badcock, N.R. Micro-determination of ketoconazole in plasma or serum by high-performance liquid chromatography. J.Chromatogr., 1984, 306, 436-440 Pascucci, V.L.; Bennett, J.; Narang, P.K.; Chatterji, D.C. Quantitation of ketoconazole in biological fluids using high-performance liquid chromatography. J.Pharm.ScL, 1983, 72, 1467-1469

Ketoprofen
Molecular formula: C16H14O3 Molecular weight: 254.3 CAS Registry No.: 22071 -15-4

SAMPLE Matrix: bile, blood, perfusate Sample preparation: Dilute bile with saline. Inject 200 fxL plasma, 100 JULL perfusate, or 100 JULL diluted bile onto column A and elute to waste with mobile phase A, after 15 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 and monitor the effluent from column B. Re-equilibrate column A with mobile phase A for 5 min before the next injection. (See also J.Pharm.Sci. 1995, 84, 1327.) HPLCVARIABLES Column: A 30 X 4.6 L-column (porous silica gel with internal surfaces coated with octadecyl groups and external surfaces coated with glycerylpropyl groups) (Chemical Inspection and Testing Institute, Tokyo); B 250 X 4.6 Sumichiral OA-2500S ((R)-N-(3,5-dinitrobenzoyl)1-naphthylglycine bonded to aminopropyl silica) (Sumika, Osaka) Mobile phase: A 20 mM pH 6.8 ammonium acetate buffer; B MeOH: buffer 95:5, pH 6.2 (Buffer was 1 M acetic acid:l M ammonium acetate 20:80, pH 4.0. Dilute to 600 mM acetate before use.) Flow rate: 1 Injection volume: 100-200 Detector: UV 262 CHROMATOGRAM Retention time: 27 (-), 29 (+) Limit of detection: 20 ng/mL KEYWORDS chiral; rat; column-switching; plasma REFERENCE
Yagi, M.; Shibukawa, A.; Nakagawa, T. Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system. Chem.Pharm.BulL, 1990, 38, 2513-2517

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 100 |xL tolmetin solution + 500 jxL pH 1.8 phosphate buffer, extract with 1-butanol/MTBE. Remove the organic layer and add it to 500 JULL pH 6.1 ammonium acetate buffer, mix, inject an aliquot of the aqueous layer. HPLCVARIABLES Column: 150 X 4.6 5 jmrn Cosmosil C18 Mobile phase: MeCN: 250 mM pH 5.0 ammonium acetate buffer 20:80 Flow rate: 1.8 Detector: UV 350 CHROMATOGRAM Internal standard: tolmetin (UV 258) Limit of quantitation: 5 ng/mL