Simultaneous HPLC quantification of diltiazem and N-demethyldiltiazem in plasma

ABSTRACT

A simple and sensitive reversed phase high-performance liquid chromatographic method was developed for simultaneous quantification of diltiazem and its major metabolite N-demethyldiltiazem in human plasma. The method involves one-step solvent extraction of diltiazem, N-demethyldiltiazem and the internal standard, verapamil with n-hexane and diethyl ether [5050 v/v]. The mobile phase comprised 0.1M ammonium dihydrogen phosphate-acetonitrile [6238 v/v] and triethylamine [0.08%] was added before the pH was ajusted to 5.9 with 85% phosphoric acid. Analysis was run at a flow rate of 1.0 ml/mm at a detection wavelength of 238 nm. The completion time for assay was not more than 10 minutes and lower limit of quantification was 5 ng/ml. The calibration curve for diltiazem and its metabolite was linear over a concentration range of 5-200 ng/ml and average recovery was about 90%. The coefficient of variation and percent error values of the assay method within and between day were all less than 10%