Validated RP-HPLC Bioanalytical Method for Determination of Torsemide in Rabbit Plasma

Abstract

Torsemide, a loop diuretic used for the primary treatment for hypertension, heart failure and pulmonary oedema, requires a reliable and validated bioanalytical method for accurate quantification in biological matrices during pharmacokinetic and preclinical studies. While reversed phase high performance liquid chromatography (RP-HPLC) bioanalytical methods have been reported earlier in human plasma for the quantification of torsemide, no RP-HPLC method has been reported for quantification of torsemide in rabbit plasma, a matrix essential for the preclinical pharmacokinetics and bioavailability studies of new dosage form, human plasma validated methods cannot assumed to transfer directly to species-specific differences in plasma matrix composition. while LC-MS method in rabbit plasma has been reported earlier, they require complex and sophisticated operations. In this work, a simple, sensitive, and reproducible RP-HPLC method was developed and validated for the estimation of torsemide in rabbit plasma using azosemide as the internal standard. Chromatographic separation was achieved using a Intersil ODS (250 × 4.6 mm,5 µm), with a mobile phase composed of acetonitrile and 0.1% trifluoroacetic acid buffer in a ratio of 35:65 v/v, delivered at a flow rate of 1.0 mL/min. The detection wavelength was set at 272.4 nm, with a retention time of 5.198 minutes and 3.415 minutes for torsemide and azosemide respectively. The method exhibited linearity over the concentration range of 0.15-9 µg/mL (R² = 0.9999) in accordance with ICH Q2 (R1) guidelines. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.1582 µg/mL (15.82 ng/mL) and 0.4794 µg/mL (47.94 ng/mL), respectively, confirming the sensitivity of the assay making it suitable for quantification of torsemide in rabbit plasma. The validated bioanalytical method was developed for preclinical pharmacokinetic evaluation and bioavailability studies including chronotherapeutic drug delivery systems..