Development and optimization of a RP-HPLC method to Quantify Midazolam in Rat Plasma after Transdermal Administration: Validation and Application in Pharmacokinetic Study

Abstract

A reversed phase high performance liquid chromatographic assay coupled with UV detection (239 nm) has been developed and validated for the determination of midazolam in rat plasma samples after transdermal administration. A liquid-liquid extraction was used to extract the compound from plasma sample. The separation was performed on a Hypersil ODS C18 column using a mobile phase composed of acetonitrile and 0.1% triethylamine aqueous solution (52:48, V/V), pumped at a flow rate 1.0 mL min-1. The calibration curves showed good linearity with correlation coefficient higher than 0.998 for all analytes in the range 0.10-10.0 μg mL-1. Accuracy in the measurement of quality control (QC) samples was in the range 95-107% of the nominal values. The intra-day and inter-day precisions in the measurement of QC samples were less than 10% coefficient of variation. The developed method is suitable for quality control of midazolam in their mixtures and in transdermal delivery system pharmaceutical preparations. The validated assay was found to be suitable for the pharmacokinetic study of midazolam in rats with transdermal administration.