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A simple liquid chromatography mass spectrometric method was developed for quantitative determination of Ramipril (RAM) and Hydrochlorothiazide (HCTZ) simultaneously in human plasma. RAM, HCTZ and the internal standards (RAM-D5, HCTZ-13C, D2) were extracted from human plasma by direct protein precipitation. The chromatographic separation was achieved using ACE 5 C8, (50 mm x 4.6 mm, 5µm) column with gradient elution mobile phase of 0.25 mM Ammonium chloride and methanol. The method validation intends to investigate sensitivity, linearity, precision and accuracy, recovery, matrix effect, specificity and stability according to the European Medicines Agency (EMA) guideline. Standard calibration levels were prepared in pooled human plasma to attain final dynamic range of 0.2 - 20.0 ng/mL for RAM and 1.0 - 150 ng/mL for HCTZ. Clinical bioequivalence study was successfully investigated by application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products for 5mg RAM/ 25mg Hydrochlorothiazide single dose.
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Author(s): Mahmoud AbdusSalam Alawi