DOI: 10.21276/ajptr
Fri, 22 Mar 2019

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

Syed N. Alvi1, Muhammad M. Hammami*1

1. Clinical Studies and Empirical Ethics Department, King Faisal Specialist Hospital and Research Center, MBC-03, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia.


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 < 3.7% and < 12.0%, and < 10.2% and < 7.8%, respectively. Mean extraction recovery of HCT and the IS from plasma samples were 70% and 90%, respectively. The method was applied to assess the stability of HCT under various conditions generally encountered in the clinical laboratory.

Keywords: Hydrochlorothiazide, Hydroflumethiazide, Human plasma, HPLC.

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