DEVELOPMENT AND VALIDATION OF AN ANALYTICAL QUALITY BY DESIGN-DRIVEN REVERSED PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF METFORMIN AND ITS IMPURITY

Objectives: The occurrence of impurities such as 1-Methylbiguanide Hydrochloride (Impurity E) is a major safety and regulatory issue of the widely used antidiabetic drug metformin (MTF) hydrochloride. The objective of the current study was to design and develop a rapid, sensitive, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) methodology to simultaneously estimate MTF and its impurity E in an analytical quality by design (AQbD) methodology, as required by International Conference on Harmonization (ICH) Q2(R1), Q3A, Q3B, and Q14 requirements. Methods: The method was developed with Design-Expert software (version 13) by implementing a central composite design to investigate how key method parameters (including mobile phase composition and pH) affect chromatographic responses. The separation was performed by using a HiQ Sil C18 column (250 × 4.6 mm, 5 μm) and the mobile phase with orthophosphoric acid (0.1 M) to adjust the pH (3.6) and a 1.0 mL/min flow rate. The method was validated as specified in ICH Q2(R2) under linearity, accuracy, precision, robustness, specificity, Limit of detection (LOD), and limit of quantification (LOQ). Results: The developed technique in the concentration range of 25–150 μg/mL of MTF and 0.2–1.2 μg/mL of Impurity E was found to be very linear with a correlation coefficient (r2=0.999). Accuracy studies revealed mean percentage recoveries ranging from 99.81 to 100.02% for MTF and 98.20–100.00% for Impurity E, with % relative standard deviation values below 2%. Both analytes demonstrated intra-day and inter-day precision with a <2.0% of RSD, indicating good repeatability and intermediate precision. MTF was determined to have LOD and LOQ of 4.08 μg/mL and 12.35 μg/mL, respectively, and Impurity E LOD and LOQ of 0.0305 μg/mL and 0.0925 μg/mL, respectively. Robustness studies showed that there was no great impact of small intentional changes in the chromatographic conditions. Conclusion: A straightforward, sensitive, precise, and robust AQbD-based RP-HPLC procedure was effectively developed and confirmed to determine MTF and its impurity E simultaneously. The procedure meets the existing regulatory requirements and can be used in daily quality control and impurity profiling of MTF in pharmaceutical dosage forms.