LC-MS/MS-based simultaneous quantification of chlorthalidone and cilnidipine in rat plasma: Pharmacokinetic...

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.