HPLC method for simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in human plasma

ABSTRACT

A simple, sensitive and rapid chromatographic method was developed and validated for the quantification of a binary mixture namely; [amiloride hydrochloride and hydrochlorothiazide] in human plasma using chlorthalidone as internal standard [IS]. The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyI column with isocratic elution with 10 mM KH[2]PO[4] [pH 4.50]-methanol [7030 v/v] at ambient temperature with flow rate 1 ml/min and UV detection. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range 0.1-10 micro g ml[-1]. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The within-and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze-thaw cycles [deviation <15%]. The proposed method is specific for determination of the mixture in human plasma where there is no interference from endogenous biological substances