European Journal of Biomedical and Pharmaceutical Sciences

STABILITY-INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS QUANTITATION OF THE ANTIDIABETIC AGENTS IN A COMBINED SYNTHETIC MIXTURE

Alisha Banafar*

ABSTRACT

Background: Fixed-dose combination therapy containing a biguanide, a sulfonylurea, and a thiazolidinedione has emerged as a highly effective strategy for managing type 2 diabetes mellitus, particularly in patients with inadequate glycaemic control on dual therapy. Despite widespread clinical use, a single stability-indicating assay capable of resolving all three actives from their degradation products in a combined synthetic matrix remains scarcely reported. Objective: The present study was aimed at developing and validating a novel, stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous quantitation of Metformin Hydrochloride (MET), Glimepiride (GLI), and Pioglitazone Hydrochloride (PIO) in a combined synthetic mixture. Methods: Chromatographic separation was achieved on a Waters Symmetry C18 column (250 × 4.6 mm, 5 µm) using a gradient mobile phase consisting of 0.05 M ammonium acetate (pH 3.5 ± 0.05, glacial acetic acid) and acetonitrile, at a flow rate of 1.0 mL/min and column temperature of 35°C. Photodiode array detection was employed in the range 200–400 nm, with quantitation extracted at 232 nm (MET), 228 nm (GLI), and 269 nm (PIO). The method was validated in accordance with ICH Q2(R1) guidelines. Forced degradation studies were conducted under hydrolytic (acidic, alkaline), oxidative, thermal, photolytic, and humidity stress conditions per ICH Q1A(R2). Results: Base-line separation was attained within 20 min with resolution (Rs) > 2.5 between all analytes and degradation products. Calibration curves were linear over the ranges of 250–1500, 0.5–3.0, and 7.5–45 µg/mL for MET, GLI, and PIO, respectively (r² > 0.999). Accuracy (% recovery) ranged between 98.5% and 101.2%, while intra- and inter-day precisions showed RSD < 1.5%. Peak purity indices derived from PDA data (purity angle < purity threshold) confirmed the homogeneity of each analyte peak under all stress conditions. Significant degradation was observed under alkaline and oxidative stress, while the drugs remained relatively stable under thermal and photolytic exposure. Conclusion: The proposed RP-HPLC method is specific, accurate, precise, and stability indicating. It is suitable for routine quality control, stability testing, and dissolution profiling of triple-combination antidiabetic formulations.

Keywords: Metformin Hydrochloride; Glimepiride; Pioglitazone Hydrochloride; Stability-indicating; RP-HPLC; Forced degradation; Method validation; ICH guidelines.