STABILITY-INDICATING RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION OF METFORMIN HYDROCHLORIDE, GLIMEPIRIDE AND PIOGLITAZONE HYDROCHLORIDE IN FIXED-DOSE COMBINATION TABLETS

Abstract

Background: Fixed-dose combination (FDC) tablets containing metformin hydrochloride
(MET), glimepiride (GLP) and pioglitazone hydrochloride (PIO) represent an important
pharmacological approach in the management of Type 2 Diabetes Mellitus (T2DM), addressing
multiple pathophysiological defects through complementary mechanisms. Reliable analytical
methods for simultaneous quantification of all three active ingredients are essential for
pharmaceutical quality control.
Methods: An isocratic RP-HPLC method was developed on a C18 column (250 × 4.6 mm, 5
μm) using methanol and 25 mM phosphate buffer (pH 4.3) in 75:25 v/v ratio as mobile phase,
at a flow rate of 1.0 mL/min with UV detection at 258 nm and an injection volume of 20 μL.
Method development followed a systematic approach involving UV wavelength selection,
mobile phase pH and composition optimization, and chromatographic parameter refinement.
Results: Baseline separation was achieved for all three analytes with retention times of 2.51
min (MET), 4.32 min (PIO) and 6.85 min (GLP), with resolution values of 3.2 (MET–PIO) and
4.1 (PIO–GLP). The method was validated per ICH Q2(R2) guidelines. Linearity was
established over 400–600 μg/mL (MET), 12–18 μg/mL (PIO) and 3.2–4.8 μg/mL (GLP) with
r² ≥ 0.9998. Mean percentage recoveries were 100.14 ± 0.32%, 100.09 ± 0.36% and 100.08 ±
0.41% for MET, PIO and GLP respectively. Repeatability %RSD values were 0.20%, 0.12%
and 0.15% for MET, PIO and GLP respectively. LOD and LOQ values confirmed sub-
microgram sensitivity. The method was robust to deliberate variations in mobile phase
composition. Assay of marketed FDC tablets yielded results within 95–105% of label claim.
Conclusion: The developed RP-HPLC method is simple, economical, accurate, precise,
specific and robust, making it suitable for routine quality control analysis of fixed-dose
combination tablets containing metformin, glimepiride and pioglitazone in pharmaceutical
dosage forms.