Simultaneous determination of Enalapril, Enalaprilat and Hydrochlorothiazid in human plasma by LC-MS/MS method

Abstract

A high and specific method using ultra performance liquid chromatography tandem mass spectrometry (LC/MS-MS) for simultaneous determination of enalapril (EPR), enalaprilat (EPL) and hydrochlorothiazide (HCT) in human plasma was developed. Enalapril-d5 (EPR-d5), enalaprilat-d5 (EPL-d5) and hydrochlorothiazide-13C,d2 (HCT-13C,d2) were utilized as internal standards. Analytes were extracted from plasma via protein precipitation using acetonitrile combined with sample acidification with phosphoric acid and sample freezing. Liquid chromatography was performed on Luna C18 (2) - HST; (50 x 3 mm; 2.5 µm) column with gradient program of 0.1% formic acid and methanol as mobile phase at a flow rate of 0.3 mL/min. The Thermo vantage UPLC-MS/MS was operated under the multiple-reaction monitoring (MRM) mode using the ESI+ mode with transitions at (m/z) 377.2 → 234.2 for EPR, 382.2 → 239.2 for EPR-d5, 349.1 → 206.1 for EPL, 354.1 → 211.1 for EPL-d5 and the ESI- mode with transitions at (m/z) 295.9 → 205.1 for HCT and 298.9 → 206.1 for HCT-13C,d2. The method has a rapid analysis time, low LLOQ value and standard curves were found to be linear in the wide range 1.25 to 250 ng/mL for EPR; 0.75 to 150 ng/mL for EPL and 1.0 to 200 ng/mL for HCT. The method’s precision and accurary are within acceptable limit. The validated method has been successfully used to determine enalapril, enalaprilat and hydrochlorothiazide concentrations in healthy adult volunteers and demonstrate its applicability to bioavailability/bioequivalence studies of enalapril maleate and hydrochlorothiazide combination preparations.