Greenness evaluation and quality by design-driven LC-MS/MS method for simultaneous estimation of Irbesartan and Cilnidipine in rat plasma

To facilitate the concurrent determination of irbesartan and cilnidipine—two antihypertensive agents in rat plasma, a LC-MS/MS method was systematically developed and validated using a Quality by Design (QbD) framework. Key method variables, including the organic solvent proportion, flow rate, and mobile phase pH, were identified through an Ishikawa diagram-supported risk assessment combined with Risk Priority Number (RPN) analysis. These parameters were subsequently optimized using Box-Behnken Design (BBD) to achieve reliable and robust chromatographic separation. Pharmacokinetic studies were conducted in rat plasma, and the method’s environmental impact was assessed using green analytical chemistry tools. Optimal conditions, 30% organic phase, 1.0 mL/min flow rate, and pH 3.2-enabled effective separation. Response surface methodology validated model predictability, with strong agreement in adjusted and predicted R2 values. Positive ion electrospray ionization (ESI) confirmed transitions for irbesartan (m/z 429.5346 → 293.1042), cilnidipine (m/z 493.5237 → 300.1587), and azilsartan (internal standard, m/z 457.4696 → 121.0643). The method demonstrated high accuracy, precision (%CV < 10%), and linearity (r2 > 0.998), complying with European Commission guidelines. Stability studies confirmed robustness. The QbD-optimized LC-MS/MS method demonstrated excellent precision, reliability, and environmental sustainability for the simultaneous estimation of irbesartan and cilnidipine in rat plasma.