title: Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1'-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry.
abstract: Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 microL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 microL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm x 100 mm, 3.5 microm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100-250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.
authors.affiliation: Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 125 Fogarty Hall, 41 Lower College Road, Kingston, RI 02881, USA.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Dostalek
authors.forename: Joyce S
authors.initials: JS
authors.lastname: Macwan
authors.forename: Shripad D
authors.initials: SD
authors.lastname: Chitnis
authors.forename: Ileana A
authors.initials: IA
authors.lastname: Ionita
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Akhlaghi
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D016018:Least-Squares Analysis; D008862:Microsomes, Liver; D008874:Midazolam / Q000031:analogs & derivatives / Q000032:analysis / Q000097:blood / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C042940:4-hydroxymidazolam; C040081:1-hydroxymethylmidazolam; D008874:Midazolam