A highly-sensitive and selective LC-MS/MS platform for quantitative determination of diosgenin in mice plasma with comprehensive bioanalytical validation and pharmacokinetic study.

Abstract

A robust and highly sensitive LC-MS/MS method was developed and validated for the quantification of diosgenin in mice plasma in accordance with ICH M10 guidelines. Chromatographic separation was achieved using Kinetex C8 column (50 x 2.1 mm, 5 µm) with 0.1% formic acid in distilled water and methanol as mobile phase at a flow rate of 0.6 mL/min, and the run time was 17 min. Detection was performed in positive ESI+ mode using MRM at m/z 415.10 → 271.20, employing sarsasapogenin as the internal standard. Protein precipitation yielded efficient extraction with minimal matrix interference. The method exhibited excellent linearity over 3.125-1000 ng/mL (r2 = 0.996) with an LLOQ of 3.125 ng/mL. The accuracy during within-day and between-day evaluations ranged from 91.7% to 110.99%, and precision (%CV) remained below 5.14%. Dilution integrity indicated that the analyte can be reliably quantified in biological samples up to 24,000 ng/mL. The stability studies confirmed that the analyte was stable under various conditions. Further, this validated method was applied for pharmacokinetic studies in female mice via oral (10mg/kg) and intramuscular (5mg/kg) administration. Overall, the method is reliable for trace-level quantification and demonstrated its applicability in pharmacokinetic, bioavailability, and formulation studies of diosgenin.