TITLE:
Simultaneous Determination of Hydrochlorothiazide, Ramipril and Its Active Metabolite Ramiprilat in Human Plasma by Using Liquid Chromatography-Tandem Mass Spectrometry
AUTHORS:
Nihal Saraner, Yelda Karacan, Berrak Guney, Onursal Saglam
KEYWORDS:
Hydrochlorothiazide (HCT), Ramipril, Ramiprilat, Human Plasma, LC-MS/MS
JOURNAL NAME:
Open Journal of Applied Sciences,
Vol.16 No.4,
April
2,
2026
ABSTRACT: This study describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of hydrochlorothiazide (HCT), ramipril and its active metabolite ramiprilat in human plasma. Plasma samples were prepared using protein precipitation followed by chromatographic separation on a reversed-phase C18 analytical column with a mobile phase consisting of methanol and 0.2% formic acid in 10 mM ammonium formate solution. Stable isotope-labeled internal standards ramipril D5, [2H5] ramiprilat and HCT 15N2 13C D2 were employed to ensure accurate quantification. Detection was performed using electrospray ionization in both positive and negative ion modes under multiple reaction monitoring (MRM) conditions; ramipril and ramiprilat were analyzed in positive ion mode, whereas HCT was monitored in negative ion mode. The method demonstrated linear calibration ranges of 1 - 400 ng/mL for HCT, 0.1 - 70 ng/mL for ramipril, 0.05 - 35 ng/mL for ramiprilat, with correlation coefficients (r2) exceeding 0.99 for all analytes. Intra- and inter-day precision and accuracy met the accepted bioanalytical validation criteria. No significant matrix effects were observed and the analytes remained stable under various storage and processing conditions. The total chromatographic run time was 5 minutes per sample. The validated method was successfully applied to the analysis of human plasma samples.