HPLC-MS/MS Method for the Simultaneous Quantification of SGLT2 Inhibitor YWS22026 and Its M3 Metabolite in Rat Plasma, With Application to Toxicokinetic Study.

A sensitive and robust HPLC-MS/MS method was developed and validated for the simultaneous determination of YWS22026 and its major metabolite M3 in rat plasma using protein precipitation. Chromatographic separation was achieved on a Shim-pack Scepter C18 column (4.6 × 50 mm, 3 μm) with a gradient elution program (mobile phase A: 0.2% formic acid in water; mobile phase B: methanol) at a flow rate of 0.9 mL/min. Detection was performed via positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The method demonstrated excellent specificity, accuracy (relative error, %RE < 9.1%), precision (relative standard deviation, %RSD < 9.9%), and linearity (5-2000 ng/mL for both analytes). The lower limit of quantification (LLOQ) was 5 ng/mL. Stability studies confirmed that plasma samples remained stable under various storage conditions (at room temperature for 10 h, -20°C for 22 days, three freeze-thaw cycles, and 4°C autosampler storage for 73 h). This validated method was successfully applied to a 26-week concomitant toxicokinetic study in Sprague-Dawley rats, which revealed significant sex-related differences in exposure for both YWS22026 and M3. These findings provide valuable insights for informing the safety assessment of YWS22026 in subsequent clinical trials.