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

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.