ABSTRACT A robust, sustainable, and stability‐indicating RP‐HPLC method was developed and validated for the simultaneous quantification of pazopanib and piperine in bovine serum albumin–based nanoparticle formulations intended for hepatocellular carcinoma therapy. Method development was guided by a quality by design framework, incorporating risk assessment, Taguchi orthogonal array–based factor screening, and response surface methodology using a Box–Behnken design to ensure analytical robustness and repeatability. Chromatographic separation was achieved on a C18 column using an optimized mobile phase of acetonitrile and 0.1% formic acid (70:30, v/v) at a flow rate of 1.0 mL/min, with the column temperature maintained at 30°C, and detection at an isosbestic wavelength of 290 nm. The method was fully validated in accordance with International Council for Harmonisation Q2 (R2) guidelines. Excellent linearity was observed over the concentration range of 2–12 µg/mL for both analytes ( R 2 > 0.999). The method demonstrated high sensitivity, precision, and accuracy, with LOD of 0.531 µg/mL for pazopanib and 0.432 µg/mL for piperine, and LOQ of 1.578 µg/mL for pazopanib and 1.337 µg/mL for piperine, %RSD values below 2%, and recoveries ranging from 98.14% to 101.05%. Forced degradation studies confirmed the stability‐indicating capability of the method under acidic, basic, oxidative, and photolytic stress conditions. The validated method was successfully applied to quantify pazopanib and piperine in bovine serum albumin nanoparticles, which exhibited nanoscale size, narrow polydispersity, high encapsulation efficiency, and good colloidal stability. Environmental sustainability and applicability were evaluated using Complementary Green Analytical Procedure Index, Analytical GREEnness metric, AGREEprep, and Analytical Eco‐Scale, and Blue Applicability Grade Index tools, confirming the method's green and white analytical credentials. Overall, the developed RP‐HPLC method is reliable, eco‐conscious, and well‐suited for routine analysis of pazopanib and piperine in nanoparticulate drug delivery systems.
Raikar et al. (Sun,) studied this question.