Simultaneous Determination of Hydrochlorothiazide, Cilazapril and Its Active Metabolite Cilazaprilat in Urine by Gradient RP-LC

A rapid and simple liquid chromatographic method with UV detection has been developed for the determination of hydrochlorothiazide (HCTZ), cilazapril (CL) and its active metabolite cilazaprilat (CLT) in urine. Sample preparation for urine consisted of solid-phase extraction using styrene-divinylbenzene (SDB-2) cartridges. The chromatographic system was a Zorbax Eclipse XDB-C-18 column with a mixture of methanol and 10 mM phosphate buffer, pH 2.3 with gradient (20 to 60% of methanol) as mobile phase at a flow rate of 1.0 mL min(-1). The detection was performed at the wavelength of 206 nm. Enalapril maleat was used as an internal standard. The detector response was linear in the range of 2.4-30.0, 1.6-15.0 and 1.8-20.0 mu g mL(-1) for HCTZ, CL and CLT, respectively. LOQ was determined to be 2.4, 1.6 and 1.8 mu g mL(-1) for HCTZ, CL and CLT, respectively. Both intra- and inter-day precision were within acceptable limits. The method has been applied to urine samples obtained from three hypertensive patients after intake of HCTZ and CL therapeutic dose.