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.
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 0.05 M 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 proposed method was adequately sensitive to measure midazolam hydroxylase activity as a marker for CYP3A activity, and was applied to measure midazolam pharmacokinetics in human plasma.
Elbarbry
Fawzy
F
School of Pharmacy, Pacific University, Hillsboro, OR 97123, USA.
Attia
Ahmed
A
Shoker
Ahmed
A
D000328:Adult; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008874:Midazolam / Q000032:analysis* / Q000493:pharmacokinetics*; D010100:Oxygen / Q000737:chemistry; D011786:Quality Control; D011817:Rabbits; D015203:Reproducibility of Results
D051544:Cytochrome P-450 CYP3A; D008874:Midazolam; D010100:Oxygen