K.Venkataramana

  A simple, fast, and precise reverse phase, gradient HPLC method was developed for the separation and quantification of metformin hydrochloride and its related compounds Cyanoguanidine impurity, Melamine impurity, 1- methylbiguanidine impurity, Monoguanylmelamine impurity, N,N Dimethylmelamine impurity in tablet formulations. Liquid chromatography with using Partisil SCX, 250 X 4.6 mm, 10µm and mobile phase is 17 gms of ammonium Dihydrogen phosphate in 1000 ml water and adjust the pH to 3.0 with phosphoric acid and degassed under sonication. The flow rate was 1.0 ml/min and the effluent was monitored at 218 nm. This new method was validated in accordance with USP requirements for new methods for assay determination, which include accuracy, precision, linearity, range and robustness. The current method demonstrates good linearity over the range of 0.01µg/mL to 10µg/mL for Impurity A and metformin HCl. Remaining all impurities with concentration from 0.02 µg/mL to 15 µg/mL for six levels. The accuracy is carried out with concentrations ranging from 50% to 200% of Target concentration the Mean % recovery for each impurity at each level should be between 85.0 % and 115.0.The precision of this method reflected by relative standard deviation of replicates all metformin Related Substances is NMT 10%. Validation of the same method was also performed according to USP requirements for quantitative determination of impurities which include robustness and limit of quantification (LOQ) and Limit of detection LOD. No significant variation in RRT of Metformin and its substances at flow rate (0.8 to 1.2mL/min.), at pH (2.8 to 3.2), column temperature (23°C to 27°C), hence the method is robust. Key words: Metformin Hydrochloride, Metformin related substances, HPLC, method validation.