Development and Validation of Reverse Phase HPLC Method for the Determination of Midazolam in Human Plasma

Abstract: A simple, rapid, sensitive and specific high performance liquid chromatograpic (HPLC-UV) method was developed and fully validated for the quantification of midazolam in human plasma. Alprazolam was used as an internal standard. The analyte was extracted from human plasma samples by liquid-phase extraction technique. Analyte was chromatographed on a Zorbax XDB C18 column (3.5 µm, 4.6 x 100 mm i.d. from Agilent Technologies) by using a mixture of 0.05 M Na3PO4 (pH-4): toluene: acetonitrile (40:40:20, v/v/v) as the mobile phase at a flow-rate of 0.9 ml min−1 and the chromatogram was monitored with UV detection at 220 nm. The calibration curve obtained was linear (r2=0.99) over the concentration range of 2-200 ng ml−1. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Extraction recoveries of MDZ from plasma were 85.73, 91.93 and 93.68 % at 5, 84 and 148 ng ml−1 respectively. The mean recovery of the internal standard was found to be 102.8 % at the spiked concentration of 1 µg ml−1. The method was validated and can be successfully applied for routine analyses. © 2014, Har Krishan Bhalla & Sons.