Eco-friendly HPTLC method for Trifluridine and Tipiracil determination: Quality-by-design meets green analytical chemistry

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, necessitating efficient treatment strategies and accurate analytical methods. This study presents a green analytical approach using a Quality by Design (QbD)-assisted high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantification of trifluridine (TRI) and tipiracil (TIP) in pharmaceutical formulations. Chromatographic parameters were optimized using a Central Composite Design (CCD) under Response Surface Methodology (RSM), with solvent volume and chamber saturation time identified as critical factors. The optimized method yielded reliable Rf values of 0.64 for TIP and 0.91 for TRI. Validation per ICH Q2 (R1) guidelines confirmed excellent linearity (R² = 0.9944 for TIP and R² = 0.9988 for TRI), low detection limits (0.0011 µg/mL for TIP and 0.0022 µg/mL for TRI), and high precision (intra-day %RSD <0.74, inter-day %RSD <0.92). Robustness testing demonstrated minimal variability in Rf values (%RSD <0.28). Environmental sustainability was assessed using ComplexGAPI, AGREE, and BAGI tools. The developed method achieved an AGREE score of 0.81, an Eco-Scale score of 86, and a BAGI score of 80, highlighting its eco-friendliness and practical applicability. This study demonstrates an efficient, precise, and environmentally sustainable analytical method for TRI and TIP quantification, aligning with green chemistry principles and ensuring minimal environmental impact.