Liquid chromatography–mass spectrometric determination of losartan and its active metabolite on dried blood spots

Abstract

A simple and rapid quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for simultaneous determination of losartan and its active metabolite, losartan carboxylic acid on rat dried blood spots was developed and validated as per regulatory guidelines. Losartan and its metabolite were extracted from dried blood spots using 50% aqueous methanol and separated on Waters XTerra® RP18 (250mm×4.6mm, 5μm) column using mobile phase composed of 40% acetonitrile and 60% aqueous ammonium acetate (10mM). The eluents were monitored using ESI tandem mass spectrometric detection with negative polarity in MRM mode using ion transitions m/z 421.2→179.0, m/z 435.3→157.0 and m/z 427.3→193.0 for losartan, losartan carboxylic acid and Irbesartan (internal standard), respectively. The method was validated over the linear range of 1–200ng/mL and 5–1000ng/mL with lower limits of quantification of 1.0ng/mL and 5.0ng/mL for losartan and losartan carboxylic acid, respectively. Inter and intra-day precision and accuracy (Bias) were below 5.96% and between −2.8 and 1.5%, respectively. The mean recoveries of the analytes from dried blood spots were between 89% and 97%. No significant carry over and matrix effects were observed. The stability of stock solution, whole blood, dried blood spot and processed samples were tested under different conditions and the results were found to be well within the acceptable limits. Additional validation parameters such as influence of hematocrit and spot volume were also evaluated and found to be well within the acceptable limits.