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.
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