Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution.

A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of captopril and hydrochlorothiazide in human plasma samples was developed. Captopril was derivatized with 2,4'-dibromoacetophenone (pBPB) to form a captopril-pBPB adduct. From acidified serum plasma samples, the hydrochlorothiazide and derivatized captopril was extracted with 5 ml ether, then with 5 ml dichloromethane. Effective chromatographic separation was achieved using a C(18) column (DIAMONSIL 150 mmx4 mm i.d., 5 microm) based on an acetonitrile-trifluoroacetic acid-water gradient elution at a flow rate of 1.2 ml/min. The internal standard (IS), derivatized captopril and hydrochlorothiazide were detected at 263 nm and were eluted at 4.2, 6.8 and 16.9 min, respectively. No endogenous substances were found to interfere. The limit of quantification for hydrochlorothiazide and derivatized captopril in plasma were 3.3 and 7 ng/ml. The calibration curve for derivatized captopril showed linearity in the range 20-4000 ng/ml, with a regression coefficient corresponding to 0.9993 and the coefficient of the variation of the points of the calibration curve being lower than 10%. The calibration curve for hydrochlorothiazide showed linearity in the range 10-1200 ng/ml, with a regression coefficient corresponding to 0.9999 and the coefficient of the variation of the points of the calibration curve being lower than 10%. The method was suitably validated and successfully applied to the determination of captopril and hydrochlorothiazide in human plasma samples.