LC–MS/MS-based simultaneous quantification of chlorthalidone and cilnidipine in rat plasma: Pharmacokinetic evaluation, green analytical assessment, and DoE-driven optimization

A highly sensitive and reproducible liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was systematically developed and optimized using a Quality by Design (QbD) framework for the simultaneous quantification of chlorthalidone and cilnidipine in rat plasma. Critical method variables—organic phase composition, flow rate, and mobile phase pH—were identified through risk assessment and subsequently optimized via Box–Behnken Design to ensure analytical robustness. Optimal chromatographic conditions comprised 20 % organic content, a flow rate of 1.0 mL/min, and a mobile phase pH of 3.0, facilitating efficient resolution of both analytes. Detection was achieved in positive electrospray ionization mode using multiple reaction monitoring, with transitions of m/z 339.8909 → 85.0951 for chlorthalidone, m/z 493.5237 → 300.1587 for cilnidipine, and m/z 515.6423 → 342.6158 for telmisartan, employed as the internal standard. Method validation, performed in accordance with European Medicines Agency (EMA) guidelines, demonstrated excellent linearity (r² > 0.998), accuracy, and precision, with coefficient of variation consistently <10 %. The method exhibited strong analyte stability and was successfully applied to the pharmacokinetic evaluation of both drugs in Wistar rats. This DoE-optimized LC–MS/MS platform offers a selective, reliable, and environmentally conscious analytical solution for the preclinical assessment of chlorthalidone and cilnidipine.