Simultaneous Determination and Pharmacokinetic Study of Losartan, Losartan Carboxylic Acid, Ramipril, Ramiprilat, and Hydrochlorothiazide in Rat Plasma by a Liquid Chromatography/Tandem Mass Spectrometry Method

Abstract

The monitoring of the plasmatic concentrations of cardiovascular drugs is crucial for understanding their pharmacokinetics and pharmacodynamics. A simple, sensitive, specific, and high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous estimation and pharmacokinetic study of losartan (LOS), losartan carboxylic acid (LCA), ramipril (RAM), ramiprilate (RPT), and hydrochlorothiazide (HCZ) in rat plasma using irbesartan (IBS) and metolazone (MET) as internal standards (ISs). After solid phase extraction (SPE), analytes and ISs were separated on an Agilent Poroshell 120, EC-C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column with a mobile phase consisting of methanol/water (85:15, v/v) containing 5 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.4 mL/min. The precursor → product ion transitions for the analytes and ISs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode and switching the electrospray ionization (ESI) mode during chromatography from positive (to detect LOS, LCA, RAM, RPT, and IBS) to negative (to detect HCZ and MET). The method was validated as per the FDA guidelines and it exhibited sufficient specificity, accuracy, and precision. The method was found to be linear in the range of 3-3000 ng/mL for LOS and LCA, 0.1-200 ng/mL for RAM and RPT, and 1-1500 ng/mL for HCZ. The described method was successfully applied to the preclinical pharmacokinetic study of analytes after oral administration of a mixture of LOS (10 mg/kg), RAM (1 mg/kg), and HCZ (2.5 mg/kg) in rats.

Keywords: LC-MS/MS; Pharmacokinetic; Plasma; Simultaneous estimation; Validation.

Fig. 1

Chemical structures and product ion mass spectra of [Losartan (LOS), (A)], [Losartan carboxylic acid (LCA), (B)], [Ramipril (RAM), (C)], [Ramiprilat (RPT), (D)], [Hydrochlorothiazide (HCZ), (E)], [Irbesartan (ISB, IS), (F)], and [Metolazone (MET, IS), (G)

Fig. 2

Representative MRM chromatograms of blank rat plasma samples: LOS (A1), LCA (A2), RAM (A3), RPT (A4), HCZ (A5), IS (IBS, A6), and IS (MET, A7). Representative MRM chromatograms of a blank rat plasma sample spiked with: LOS at the LLOQ of 3 ng/mL (B1), LCA at the LLOQ of 3 ng/mL (B2), RAM at the LLOQ of 0.1 ng/mL (B3), RPT at the LLOQ of 0.1 ng/mL (B4), HCZ at the LLOQ of 1 ng/mL (B5), IS (IBS) at 100 ng/mL (B6), and IS at 100 ng/mL (B7). Representative MRM chromatograms of a plasma sample of LOS (C1), LCA (C2), RAM (C3), RPT (C4), and HCZ (C5), obtained from a rat at 1.5 hr after oral administration of a mixture of LOS (10 mg/kg), RAM (1 mg/kg), HCZ (2.5 mg/kg), IS (ISB) at 100 ng/mL (C6), and IS (MET) at 100 ng/mL

Fig. 3

Mean plasma concentration vs. time curve of LOS (A), LCA (B), RAM (C), RPT (D), and HCZ (E) after oral administration of a mixture of LOS (10 mg/kg), RAM (1 mg/kg), and HCZ (2.5 mg/kg) in rats