A simultaneous LC–MS/MS assay for the quantification of xanomeline and trospium chloride in rat plasma: Method validation as per FDA guidelines and evaluation using green analytical chemistry principles and the blue tool

The fixed-dose combination of xanomeline and trospium chloride has emerged as a promising therapeutic option for schizophrenia; however, no validated bioanalytical method is available for their simultaneous quantification in biological matrices, limiting pharmacokinetic and translational studies. A green, Quality by Design (QbD)–driven LC–MS/MS method was developed and optimized using a Box–Behnken design. Chromatographic separation was achieved using 50% acetonitrile (pH 3.0) at a flow rate of 1.0 mL/min. Detection was performed in positive electrospray ionization mode with selective reaction monitoring transitions of m/z 282→173 for xanomeline and m/z 428→220 for trospium chloride. The method was validated in accordance with regulatory guidelines. Both analytes were baseline-resolved within 7 min, exhibiting excellent linearity (R² > 0.999), high extraction recovery (>95%), and low detection limits (1.351 ng/mL for xanomeline and 0.452 ng/mL for trospium chloride). The method demonstrated robust stability under all tested conditions and was successfully applied to a rat pharmacokinetic study, revealing rapid elimination of xanomeline and sustained exposure of trospium chloride. This first simultaneous LC–MS/MS method for xanomeline and trospium chloride offers superior sensitivity, reduced solvent consumption, and high analytical throughput, making it well-suited for routine pharmacokinetic and drug development applications.