Clotrimazole

Molecular formula: C22H17CIN2 Molecular weight: 344.8 CAS Registry No.: 23593-75-1

SAMPLE Matrix: blood Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 2 mL 95% EtOH and 2 mL MeCN: water 15:85. 200 jxL Plasma or whole blood + 50 |JLL 100 |xM testosterone propionate in MeOH + 3 mL MeCN: water 15:85, vortex for 30 s, add to the SPE cartridge, wash with 9 mL MeCN: water 30:70, dry, elute with 200 jxL 95% EtOH, inject a 10 JJLL aliquot of the eluate. HPLCVARIABLES Column: 50 X 4.6 5 |xm Supelcosil LC-8DB Mobile phase: MeOH: buffer 72.5:27.5 (Buffer was 25 mM K2HPO4 adjusted to pH 3 with 670 mM phosphoric acid.) Flow rate: 1 Injection volume: 10 Detector: UV 210 CHROMATOGRAM Retention time: 2.60 Internal standard: testosterone propionate (3.60) OTHER SUBSTANCES Extracted: metabolites, doxepin Noninterfering: acetaminophen, N-acetylprocainamide, amitriptyline, aspirin, barbituric acid, brompheniramine, caffeine, carbamazepine, chloramphenicol, chlorpheniramine, clonazepam, desipramine, desmethyldoxepin, digitoxin, digoxin, disopyramide, ethosuximide, felbamate, gentamicin, ibuprofen, imipramine, lidocaine, maprotiline, mephenytoin, mephobarbital, metharbital, methsuximide, methylsuccinimide, nortriptyline, paramethadione, phenacemide, phenobarbital, phensuximide, phenylpropanolamine, phenytoin, primidone, procainamide, protriptyline, quinidine, theophylline, tobramycin, trimethadione, valproic acid, vancomycin Interfering: itraconazole KEYWORDS plasma; SPE; whole blood; pharmacokinetics REFERENCE
Rifai, N.; Sakamoto, M.; Law, T.; Platt, O.; Mikati, M.; Armsby, C C ; Brugnara, C HPLC measurement, blood distribution, and pharmacokinetics of oral clotrimazole, potentially useful antisickling agent. Clin.Chem., 1995, 41, 387-391

SAMPLE Matrix: blood Sample preparation: Prepare a C18 SPE cartridge (Analytichem part 607303) by washing with 2 mL MeOH then 5 mL water (J. Chromatogr. 1986, 377, 287). 1 mL Serum + 200 jiL 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 jxL aliquot.

HPLCVARIABLES

Column: 150 X 3.9 Novopak C18 Mobile phase: MeOH: MeCN: 20 mM KH2PO4 30:30:35, adjusted to pH 6.8 Flow rate: 2 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Retention time: 7.0 Internal standard: clotrimazole

OTHER SUBSTANCES Extracted: ketoconazole KEYWORDS

serum; SPE; cotrimazole is IS

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, tissue Sample preparation: Prepare a SPICE reversed-phase SPE cartridge by washing with 2 mL MeOH then 5 mL water. Tissue. 0.5 g Tissue + 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 |JLL 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 |JLL mobile phase, inject 50 fxL aliquot. Plasma. 1 mL Plasma + 200 |JLL 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 fxL aliquot.
HPLCVARIABLES

Column: 150 X 3.9 5 p,m Novapak C18 Mobile phase: MeCN: MeOH: 20 mM pH 6.8 KH2POVNaOH 30:35:35 Flow rate: 2 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Retention time: 6.8 Internal standard: clotrimazole

OTHER SUBSTANCES Extracted: ketoconazole KEYWORDS

plasma; SPE; clotrimazole is IS; 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: Dissolve in MeCN: THF 94.3:5.7, inject an aliquot. HPLCVARIABLES Column: 250 X 4.6 5 jxm Suplex pKb-100 (Supelco) Mobile phase: MeCN: THF: 15 mM pH 4.1 acetate buffer 41.5:2.5:56 Flow rate: 2 Detector: UV 254 CHROMATOGRAM Retention time: 8 OTHER SUBSTANCES Simultaneous: degradation products, impurities, benzaldehyde, benzyl alcohol, mometasone furoate, orthochlorophenyl-diphenylmethanol KEYWORDS creams REFERENCE
Spangler, M. Isocratic reversed phase HPLC analysis of a pharmaceutical cream. Supelco Reporter, 1994, 13(2), 12-13

SAMPLE Matrix: formulations Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to about 30 mg, add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of filtrate to 5 mL of 100 jJLg/mL ketoconazole in MeOH, make up to 25 mL with MeOH, inject 20 jxL 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 2 mL dichloromethane, elute with 3 mL MeOH: buffer 85:15. Add eluate to 0.5 mL 100 |xg/mL ketoconazole in MeOH, make up to 5 mL with MeOH, inject 20 JULL aliquot. (Buffer was 50 mM triethylamine adjusted to pH 7.0 with phosphoric acid.) HPLC VARIABLES Column: 250 X 4.6 5 imm 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: 9.5 Internal standard: ketoconazole (7) OTHER SUBSTANCES Simultaneous: bifonazole, econazole, fenticonazole, isoconazole, miconazole, tioconazole KEYWORDS tablets; creams

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: formulations Sample preparation: 5 g Ointment containing 1% clotrimazole H - 70 mL MeOH, sonicate for 15 min, make up to 100 mL with MeOH, filter (0.22 \m PTFE), mix an aliquot of the filtrate with an equal volume of 6 |xg/mL chrysene in MeOH, inject a 20 |JLL aliquot.
HPLCVARIABLES

Column: 150 X 4 5 |xm Nucleosil C18 Mobile phase: MeOH: water 9:1 Flow rate: 1 Injection volume: 20 Detector: UV 258
CHROMATOGRAM

Retention time: 5.23 Internal standard: chrysene (9.54)

OTHER SUBSTANCES Interfering: octanol KEYWORDS

ointment
REFERENCE
Valenta, C; Lexer, A.; Spiegl, R Analysis of clotrimazole in ointments by high-performance liquid chromatography Pharmazie, 1992, 47, 641-642

SAMPLE

Matrix: formulations Sample preparation: 0.5 g Ointment containing 1% clotrimazole + 7 mL octanol, sonicate for 15 min, make up to 10 mL with octanol, filter (0.22 |xm PTFE), inject a 20 |xL aliquot.
HPLCVARIABLES

Column: 150 X 4 5 |mm Nucleosil C18 Mobile phase: MeOH:4.25% phosphoric acid 9:1 Flow rate: 1 Injection volume: 20 Detector: UV 258
CHROMATOGRAM Retention time: 2.33 KEYWORDS

ointment
REFERENCE
Valenta, C; Lexer, A.; Spiegl, P. Analysis of clotrimazole in ointments by high-performance liquid chromatography. Pharmazie, 1992, 47, 641-642


ANNOTATED BIBLIOGRAPHY

Stuber, B.; Muller, K.H. [High pressure liquid ehromatography of combined clotrimazole-containing hydrocreams and hydrocream pastes]. Pharm.Acta HeIv., 1984, 59, 210-212 Hoogerheide, J.G.; Strusiak, S.H.; Taddei, CR.; Townley, E.R.; Wyka, B.E. High performance liquid chromatographic determination of clotrimazole in pharmaceutical formulations. J.Assoc. Off.Anal.Chem., 1981, 64, 864-869