Development of an AQbD-guided sustainable RP-HPLC method for simultaneous quantification of saracatinib and rapamycin in lipid–polymer hybrid nanocarriers

Abstract

Saracatinib (SRC) and Rapamycin (RAP) are promising targeted anticancer agents that act through dual Src/Abl kinase inhibition and mTOR pathway suppression, respectively, demonstrating synergistic efficacy in head and neck cancer models. To overcome limitations such as poor tumor selectivity, low solubility, and systemic toxicity, both drugs were co-encapsulated into lipid–polymer hybrid nanoparticles (LPHNPs) to enhance bioavailability and therapeutic performance. However, no validated analytical approach has been reported for their simultaneous quantification in such nanocarrier systems. This research presents the development and optimization of a robust, sustainable and green reverse-phase high-performance liquid chromatography (RP-HPLC) method using an Analytical Quality by Design (AQbD) strategy. A Box–Behnken design was utilized to systematically evaluate critical method parameters (CMPs) and establish a design space, ascertaining consistent performance. Chromatographic separation was achieved under optimized isocratic RP-HPLC conditions with baseline resolution of both analytes. SRC and RAP were eluted at 3.787 and 9.462 minutes, respectively. The validated method showed outstanding linearity over the studied concentration, with an r² of 0.9999 for SRC and 1 for RAP. The % RSD below 2% indicated high precision, robustness and complied with international regulatory standards, specifically the ICH Q2(R2) validation requirements. Furthermore, the method's applicability was confirmed by determining the drug entrapment efficiency (DEE) and monitoring in vitro drug release profiles of LPHNPs. Environmental sustainability was assessed using complementary green analytical metrics, including AGREE (0.68), AGREEprep (0.63), and ComplexMoGAPI (81), indicating a favourable sustainability profile with moderate-to-high environmental compatibility. Overall, the study delivers a validated, regulatory-compliant, and moderately green RP-HPLC method for the simultaneous quantification of dual-drugs, supporting the advancement of nanocarrier-based combination therapies in HNC management.