Development, Optimization and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Metformin Hydrochloride and Linagliptin in Bulk Drug Using Design of Experiments

Abstract

A systematic reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed for simultaneous estimation of Metformin Hydrochloride and Linagliptin in bulk drug. Preliminary chromatographic trials were followed by a three-factor Design of Experiments (DoE) study in which flow rate, column temperature and mobile-phase pH were investigated as independent variables. Peak area, retention time, theoretical plate count and tailing factor for both analytes were evaluated as responses. The DoE comprised 15 observations and the response models were assessed by analysis of variance (ANOVA) and regression analysis. Column temperature significantly affected Linagliptin retention time (p=0.0044) and theoretical plate count (p=0.0177). Metformin Hydrochloride retention time was significantly affected by flow rate (p=0.0056), column temperature (p=0.0052) and mobile-phase pH (p=0.0048). The Metformin Hydrochloride retention-time model showed R²=0.7671, adjusted R²=0.7035 and predicted R²=0.5002, while the corresponding Linagliptin retention-time model showed R²=0.5886, adjusted R²=0.4764 and predicted R²=0.1982. Numerical optimization predicted a solution at 1.000 mL/min flow rate, 40.0°C column temperature and pH 4.5, with desirability 0.664. The predicted retention times were 5.154 min for Metformin Hydrochloride and 6.751 min for Linagliptin. The available validation dataset demonstrated excellent reported linearity (r=1.000 for both analytes), mean recoveries of 99.7–100.2% for Metformin Hydrochloride and 99.7–99.9% for Linagliptin, and method precision of 0.2% and 0.1% RSD, respectively. Forced-degradation experiments under acidic, alkaline and oxidative conditions produced additional chromatographic responses while retaining identifiable principal analyte peaks. Importantly, the DoE optimum is a model prediction and should be experimentally confirmed at 40°C/pH 4.5 before those conditions are claimed as the final validated method.