Validation of a new HPLC method for determination of midazolam and its metabolites: Application to determine its pharmacokinetics in human and measure hepatic CYP3A activity in rabbits
HPLC, CYP3A, Therapeutic drug monitoring, Midazolam, Pharmacokinetics
Journal of Pharmaceutical and Biomedical Analysis
Pharmacy and Pharmaceutical Sciences
Midazolam (MDZ) is a commonly used benzodiazepine in clinical practice. In addition, its metabolic oxidation is used as a surrogate marker for Cytochrome P450 (CYP) 3A enzyme activity as well. Thus, a new simpler method to measure MDZ and its metabolites is welcomed. Herein we report a new and simple HPLC method with ultraviolet detection for the simultaneous determination of midazolam and its hydroxyl metabolites using lorazepam as an internal standard. A liquid–liquid extraction was used to extract the compounds from rabbit hepatic microsomes and human plasma. The separation was performed on a Zorbax Eclipse XDB C18 column using a mobile phase composed of 0.05M Na2PO4 (pH 4.5) and acetonitrile mixture (67:33) pumped at 1.2 mL/min. The calibration curves showed good linearity with correlation coefficient higher than 0.999 for all analytes in the range 10–500 ng/mL. Accuracy in the measurement of quality control (QC) samples was in the range 95–106% of the nominal values. The intra-day and inter-day precisions in the measurement of QC samples were less than 11% coefficient of variation. Although less sensitive than GC–MS, the proposedmethodwas adequately sensitive to measure midazolam hydroxylase activity as a marker for CYP3A activity, and was applied to measure midazolam pharmacokinetics in human plasma.
Elbarbry, F., Attia, A., & Shoker, A. (2009). Validation of a new HPLC method for determination of midazolam and its metabolites: Application to determine its pharmacokinetics in human and measure hepatic CYP3A activity in rabbits. Journal of Pharmaceutical and Biomedical Analysis, 50(5), 987-993. doi:DOI: 10.1016/j.jpba.2009.07.004