title: A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity.
abstract: This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.
authors.affiliation: Department of Clinical Pharmacology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Shimizu
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Uno
authors.forename: Hiro-omi
authors.initials: HO
authors.lastname: Tamura
authors.forename: Hideko
authors.initials: H
authors.lastname: Kanazawa
authors.forename: Isao
authors.initials: I
authors.lastname: Murakami
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Sugawara
authors.forename: Tomonori
authors.initials: T
authors.lastname: Tateishi
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D015394:Molecular Structure; D015203:Reproducibility of Results
chemical_list: C040081:1-hydroxymethylmidazolam; D051544:Cytochrome P-450 CYP3A; D008874:Midazolam