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An isotope dilution selective and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of hydrochlorothiazide (HCTZ) and ramipril in human plasma through a new concept of periodical polarity switching. Extraction of HCTZ, ramipril and their deuterated analogs as internal standards (ISs) was carried out from 150 μL of human plasma by solid-phase extraction method. Chromatographic separation of analytes was performed on Hypurity C18 (150 mm × 4.6 mm, 5 μ) column under gradient conditions with methanol:0.2% (v/v) formic acid in water as the mobile phase. The method was validated over a concentration range of 0.750–300 ng/mL for HCTZ and 0.125–80.0 ng/mL for ramipril. The mean extraction recovery for analytes and ISs were &gt;(86.0%), consistent across all four QC levels. The challenges to evaluate matrix effect and continuous reproducibility of method during long analytical run was studied and resolved. Processed samples, freeze–thaw, long-term and whole blood stability were evaluated for both the analytes. The method was applied to support a bioequivalence study of 25 mg of HCTZ and 5 mg of ramipril tablet formulation in nine healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 42 incurred samples.

Challenges in Simultaneous Determination of Hydrochlorothiazide and Ramipril in Human Plasma: Application to a Bioequivalence Study