Liquid chromatography–tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5  ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO4 followed by liquid–liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC–MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC–MS/MS method for indapamide was linear over the range of 0.5–80.0 ng/ml with correlation coefficient, r ≥ 0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of ±20% at 0.5 ng/ml and ±15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5 mg sustained-release (SR) formulations.