Determination of Hydrochlorothiazide in Plasma by High Performance Liquid Chromatography (HPLC)

Retention times for hydrochlorothiazide (HCTZ) and chlorothiazide (CTZ, internal standard) are highly temperature- (20°-26°) as well as pH-dependent (3.5-6.3). Separation of both drugs is maximized in the pH range 6.3 to 6.9. The 10:90 v/v acetonitrile: 0.01 M sodium acetate ratio was necessary to resolve endogenous interferences. Resolution with this mobile phase was sufficient to permit quantitation at 225 nm, where absorbance is almost twice that obtained at the absorption maximum (270 nm). Calibration curves of peak height ratio versus concentration were linear over the concentration range of 20-700 ng/ml, and the intercept was essentially zero.