QbD-based, greenness and whiteness-assessed LC-MS/MS method for simultaneous determination of sodium phenylbutyrate and Taurursodiol with pharmacokinetic application.

Sodium phenylbutyrate (NaPB) and taurursodiol (TRS) are clinically important agents frequently investigated in combination for therapeutic applications. To facilitate pharmacokinetic studies, a sensitive, robust, and environmentally sustainable bioanalytical method for their simultaneous determination in plasma was developed. A Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method was systematically optimized using a Box-Behnken design within the Design of Experiments framework. The influence of organic solvent proportion, flow rate, and mobile phase pH was evaluated, yielding optimal conditions of 40% (v/v) organic modifier, a flow rate of 1.0 mL/min, and mobile phase pH 2.6. Detection was achieved via electrospray ionization in positive selected reaction monitoring (SRM) mode with transitions m/z 187.2 → 144.7 for NaPB, m/z 500.7 → 218.9 for TRS, and m/z 516.5 → 440.0 for taurocholic acid (internal standard). Method validation, performed according to EC 2002/657/EC guidelines, confirmed excellent linearity (r2 > 0.999), accuracy, precision within 15% CV, and analyte stability under various storage and handling conditions. The method was successfully applied to a pharmacokinetic study in Wistar rats. Greenness and whiteness assessment further demonstrated strong environmental compatibility. Overall, the developed LC-MS/MS protocol is precise, reproducible, and sustainable, providing a valuable tool for preclinical pharmacokinetic investigations of NaPB and TRS.