A single, selective and simple validated method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma by liquid chromatography-tandem mass spectrometry.

A single, simple and selective method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma was validated using triamterine and hydrochlorothiazide (13)C,d2 as internal standard. The compounds were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mm ammonium acetate pH 3.0 and acetonitrile (30:70, v/v) and detected by tandem mass spectrometry with positive/negative ion mode. The analytes and internal standards were extracted from plasma using simple solid phase extraction. The ion transitions recorded in multiple reaction monitoring mode were m/z 230.1 → 116.0 for amiloride, m/z 254.1 → 237.1 for internal standard, triamterine in positive mode and m/z 296.1 → 204.9 for hydrochlorothiazide, m/z 299.2 → 205.8 for internal standard, hydrochlorothiazide (13)C,d2 in negative ion mode. Linearity in plasma was observed over the concentration range 0.1-10 ng/mL for amiloride and 5.0-500.0 ng/mL for hydrochlorothiazide. The mean recovery was 41.1 and 81.5% for amiloride and hydrochlorothiazide respectively. The coefficient of variation of the assay was less than 11.2 and 5.2% for amiloride and hydrochlorothiazide, respectively, and the accuracy was 89.0-98.1 and 96.6-102.9% for amiloride and hydrochlorothiazide, respectively. The validated method can be applied to the pharmacokinetic study of amiloride and hydrochlorothiazide.