Development and validation of a practical LC–MS/MS method for the quantification of finasteride in rat plasma using liquid–liquid extraction from 50 μL plasma samples

Reliable quantification of finasteride (FNS) in small-volume plasma samples is essential for preclinical pharmacokinetic studies but requires analytical methods that combine adequate sensitivity with practical sample preparation. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method coupled with liquid–liquid extraction (LLE) was developed and validated for the determination of FNS in rat plasma. Sample preparation was systematically optimized by evaluating plasma volume and extraction solvent composition. The optimized procedure required only 50 μL of plasma and employed methyl tert- butyl ether/dichloromethane (70:30, v/v) as the extraction solvent with dutasteride as a structurally related surrogate internal standard. The method exhibited excellent linearity over the validated concentration range of 1–500 ng/mL (R 2 = 0.99963 ± 0.00075), with acceptable accuracy and precision, consistent recovery, and limited matrix effects. The validated method was successfully applied to the determination of FNS in rat plasma samples collected during a pharmacokinetic study, demonstrating its suitability for routine bioanalysis. Compared with a previously reported LC–MS/MS method for FNS in rat plasma, the proposed assay requires substantially less plasma while providing a lower limit of quantification, a broader validated working range, and a simple LLE procedure. Overall, the proposed method provides a practical balance between analytical performance, minimal sample consumption, and operational simplicity, making it a useful analytical tool for routine determination of FNS in rat plasma and future preclinical pharmacokinetic studies.