A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Telmisartan and Chlorthalidone in Rat Plasma and its Application to a Pharmacokinetic Study

Abstract

A selective, sensitive, rapid, and stable liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed for the simultaneous quantification of chlorthalidone (CLT) and telmisartan (TEL) in rat plasma. Sample preparation involved liquid-liquid extraction by ethyl acetate. Analytes and internal standard (IS) were separated on Gemini C18 (50 x 4.6 mm, 5 μm) using a mixture of methanol-2 mM ammonium acetate (80:20 v/v) as the mobile phase at a flow rate of 0.4 mL/min. Hydrochlorothiazide (HCTZ) was used as an internal standard. Detection was carried out using Electrospray positive and negative ionization mass spectrometry using multiple reaction monitoring of the transition at m/z 515.90→276 for TEL and m/z 337.1→190.05 for CLT, m/z 296.1→205.3 for HCTZ respectively. For TEL and CLT, the method was linear (R2 ≥ 0.995) within the range of 6-1200 ng/mL and 3-600 ng/mL respectively. The retention time for TEL, CLT, and IS was found to be 3.65±0.03 min, 1.83±0.02 min, and 1.76±0.01 min. The mean recovery for both analytes and IS was greater than 70 %. The developed method was validated according to the USFDA guideline. The quantitative method was validated for specificity, linearity, accuracy, precision, recovery, dilution integrity, matrix effect, and analyte stability. The method was successfully applied to a pharmacokinetic study in rat plasma following oral administration.