title: Sensitive determination of midazolam and 1'-hydroxymidazolam in plasma by liquid-liquid extraction and column-switching liquid chromatography with ultraviolet absorbance detection and its application for measuring CYP3A activity.
abstract: This manuscript described a new sensitive determination of midazolam and its metabolite 1'-hydroxymidazolam by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 2 ml of plasma using chloroform-hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (C18 STR ODS-II analytical column (5 microm, 150 mm x 4.6 mm i.d.) for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (60%) and acetonitrile (57.9:0.1:42, v/v/v) and was delivered at a flow-rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.3-100 ng/ml, and good linearity (r > 0.998) was confirmed. Intra- and inter-day coefficient variations for midazolam and 1'-hydroxymidazolam were less than 8.5 and 6.1%, respectively, at the concentrations of 0.5, 5 and 50 ng/ml for the test compounds. Relative errors ranged from -14 to 6% and mean recoveries were 78-85%. The limit of quantification was 0.5 ng/ml for each compound. This method was sensitive enough for pharmacokinetic studies measuring CYP3A activity in human volunteers following an intravenous (1 mg) and a single-oral administration (2 mg) of a subtherapeutic midazolam dose.
authors.affiliation: Department of Clinical Pharmacology, School of Medicine, Hirosaki University, 5 Zaifu, Hirosaki 036-8562, Japan. yasufuru@cc.hirosaki-u.ac.jp
authors.forename: Norio
authors.initials: N
authors.lastname: Yasui-Furukori
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Inoue
authors.forename: Tomonori
authors.initials: T
authors.lastname: Tateishi
mesh_terms: D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D005260:Female; D006801:Humans; D008297:Male; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: C040081:1-hydroxymethylmidazolam; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam