Comprehensive Validation of Analytical Methods Using A Risk-Based Approach: Application to RP-HPLC and UV techniques for Methotrexate

Aim: This research aims to evaluate the approaches based on a risk-based framework in quantification of anticancer drugs including Methotrexate by reversed-phase high-performance liquid chromatography (RP-HPLC) and ultraviolet (UV) spectrophotometry. Methodology: The validation of the analytical procedure was conducted in accordance with the Q2(R2) guidelines of the ICH (International Council for Harmonisation), which incorporate risk management principles such as QbD (Quality by Design) and FMEA (Failure Mode and Effect Analysis). By use of a BBD (Box-Behnken Design), critical method attributes (CMAs) and critical process parameters (CPPs) were found and refined. Using an Agilent 1100 HPLC system with a C18 reverse-phase column, chromatographic separation was accomplished; UV analysis was conducted using a Shimadzu UV-1800 spectrophotometer. To guarantee method dependability and regulatory compliance, investigations on system appropriateness, linearity, precision, robustness, accuracy, specificity, and forced degradation were undertaken. Result: A chromatographic assay of Methotrexate was found to be valid with regular retention (~5.25 min), precision (RSD <2%), accuracy (99.1–100.8% recovery), and ruggedness. Stability studies revealed mild degradation after 12 hours, and forced degradation established hydrolytic and oxidative vulnerability but thermal stability. The assay is a reliable method for pharmaceutical use. Conclusion: In conclusion, the study confirmed HPLC procedure guarantees accurate Methotrexate measurement with high precision, accuracy, and robustness. Stability results suggest controlled storage conditions for analytical integrity to support its use in pharmaceutical quality control.