Quality by design-assisted development and validation of a sustainable LC–MS/MS method for simultaneous quantification of berahyaluronidase alfa and pembrolizumab in rat plasma: Pharmacokinetic application and comprehensive greenness assessment

A sensitive, robust, and environmentally conscious liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantification of berahyaluronidase alfa and pembrolizumab in rat plasma. Method development was performed using a Quality by Design-based strategy involving risk assessment, experimental design, and multivariate optimization to establish a reliable analytical design space. Critical chromatographic parameters, including organic phase composition, mobile phase pH, and flow rate, were systematically optimized using Taguchi screening and Box–Behnken design methodologies. Chromatographic separation was achieved using a phenyl-hexyl column coupled to an AB SCIEX QTRAP® 5500 mass spectrometer operated in positive electrospray ionization mode. Quantitative analysis was performed in selected reaction monitoring mode using the transitions m/z 49.52→36.00 for berahyaluronidase alfa and m/z 146.68→70.01 for pembrolizumab, with amivantamab employed as the internal standard. The method exhibited excellent linearity over concentration ranges of 5–200 ng/mL for berahyaluronidase alfa and 8.25–330 ng/mL for pembrolizumab, with correlation coefficients greater than 0.999. The limits of detection and quantification were 1.35 and 4.10 ng/mL for berahyaluronidase alfa and 2.58 and 7.80 ng/mL for pembrolizumab, respectively. Validation studies demonstrated satisfactory selectivity, accuracy, precision, recovery, matrix tolerance, carryover control, and stability in accordance with international council for harmonisation (ICH) M10 requirements. The validated method was successfully applied to a pharmacokinetic study in Wistar rats, yielding Cmax values of 96.075 and 155.211 ng/mL and elimination half-lives of 15.0 and 20.0 h for berahyaluronidase alfa and pembrolizumab, respectively. Sustainability assessment using Analytical GREEnness (AGREE), AGREEprep, Analytical Eco-Scale, Carbon Footprint Reduction Index (CaFRI), Click Analytical Chemistry Index (CACI), Complex Green Analytical Procedure Index (ComplexGAPI), and the Complex modified Green Analytical Procedure Index (ComplexMOGAPI), and Blue Applicability Grade Index (BAGI) confirmed the favorable environmental and practical attributes of the proposed analytical procedure.