STABILITY-INDICATING RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION OF REMOGLIFLOZIN ETABONATE, TENELIGLIPTIN, AND METFORMIN HYDROCHLORIDE IN SYNTHETIC MIXTURE

Abstract

A simple, accurate, precise, specific, robust, and stability-indicating Reverse Phase High-
Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the
simultaneous determination of remogliflozin etabonate (REM), teneligliptin (TEN), and
metformin hydrochloride (MET) in a synthetic mixture. Chromatographic separation was
achieved on a Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 μm) using an isocratic
mobile phase consisting of phosphate buffer (pH 4.0) and acetonitrile (60:40 v/v) at a flow rate
of 1.0 mL/min, with UV detection at 226 nm. Under optimized conditions, baseline-resolved
peaks were obtained at retention times of 4.23 min (REM), 7.89 min (TEN), and 11.45 min
(MET). The method was validated according to ICH Q2(R1) guidelines for system suitability,
specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ),
robustness, and solution stability. Excellent linearity was observed over the ranges 2–12 μg/mL
(REM), 2–12 μg/mL (TEN), and 10–60 μg/mL (MET), with correlation coefficients (r²) ≥
0.9997. Mean percentage recoveries were 100.05 ± 0.49%, 99.96 ± 0.53%, and 100.13 ± 0.48%
for REM, TEN, and MET respectively. Percentage relative standard deviation (%RSD) for both
intraday and interday precision was below 0.6%. Forced degradation studies under acidic,
alkaline, oxidative, thermal, and photolytic conditions confirmed the stability-indicating
capability of the method, with all degradation products completely resolved from parent drug
peaks. Mass balance ranged from 97.8% to 99.8% across all stress conditions. Assay of the
synthetic mixture yielded results within 98–102% of label claim. The validated method is
suitable for routine quality control, stability studies, and regulatory submissions for this
clinically important triple antidiabetic combination.