A Validated Reversed-Phase HPLC Method for the Determination of Hydrochlorothiazide in Human Plasma

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of hydrochlorothiazide (HCT) in human plasma was developed and validated. Using hydroflumethiazide as an internal standard (IS), separation was achieved on Atlantis dC 18 column. The mobile phase, 10 mM monobasic potassium phosphate and acetonitrile (80:20, v: v), was delivered at a flow rate of 1.2 ml/min. The eluent was monitored by photodiode array detector, with the wavelength set at 272 nm. Plasma samples containing HCT and IS were extracted with methyl tert butyl ether and reconstituted in mobile phase. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of HCT in plasma and peak area ratio of HCT to the IS was linear over the range of 5-300 ng/ml. The intra-day and inter inter-day coefficient of variation and bias were