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 m m 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. Copyright © 2011 John Wiley & Sons, Ltd.