Development, Optimization, and Validation of a Stability-Indicating Reverse Phase HPLC Method for Simultaneous Estimation of Sitagliptin and Metformin in Drug Substance and Combination Formulations

Abstract

The quantification of impurities and their degradants in the active pharmaceutical ingredients (APIs) of sitagliptin and metformin, as well as their combinations, is essential for quality control analysis and stability indication for product approval. The method was developed and validated in accordance with the International Conference on Harmonisation (ICH) Q2 R2 guidelines. This sensitive method was employed to detect and quantify impurities in both APIs and finished dosage forms for 50 mg/500 mg, 50 mg/850 mg, and 50 mg/1000 mg tablet formulations. The same samples of the reference formulations were compared with the generic compositions. The analytical method developed for sitagliptin quantification utilized Mobile Phase A, consisting of 0.1% orthophosphoric acid (OPA) as mobile phase-A, and Mobile Phase B, comprising 100% acetonitrile in a gradient composition. This was conducted using a Waters µBondapak C18 250 × 4.6 mm, 5 µm column with a column oven temperature of 50°C, an injection volume of 50 µL, and a flow rate of 1.0 mL/min at 210 nm, employing diluent-1 (acetonitrile and 0.1% OPA 90:10% v/v) and diluent-2 (0.1% OPA solution). Metformin HCL quantification was achieved using a C18 column stationary phase with a mobile phase of potassium dihydrogen phosphate and n-hexane sulfonic acid at pH 3.5 ±, and methanol in a ratio of 95:5% v/v as Mobile Phase A, and 100% acetonitrile as Mobile Phase B, with a flow rate of 0.8 mL/min and an injection volume of 10 µL at a column oven temperature of 30°C, utilizing dual wavelengths of 218 nm and 200 nm. The limit of detection was established at 0.05% relative to the test concentrations, ensuring the method's capability to detect all known and unknown impurities in both analytes within the APIs and the finished dosage forms.