Melamine is an organic base and a trimer of cyanamide, which have been implicated as contaminants or biomarkers in Chinese protein adulterations. Melamine is described as "Harmful if swallowed, inhaled or absorbed through the skin. Chronic exposure may cause cancer or reproductive damage, Eye, skin and respiratory irritant". When melamine is absorbed into the bloodstream, they concentrate and interact in the urine-filled renal tubules, then crystallize and form large numbers of round, yellow crystals, which in turn block and damage the renal cells that line the tubes, causing the kidneys to malfunction. As limit of melamine content in Iguratimod is not more than 2.5 ppm per day, hence it needs to quantify melamine content with accuracy in Iguratimod. Hence it’s requirement for development and validation of analytical method for accurate and precise estimation of melamine content in Iguratimod. Analytical method for melamine content in Iguratimod has been developed and validated for the determination of melamine in Iguratimod using liquid chromatography with ultraviolet-visible (UV-Vis) detector. The method has been validated for melamine content in Iguratimod. Melamine is analyzed separately using LC column, Unisphere Extend C18 (250 x 4.6mm, 5µ). Commercially available, standard for Melamine was used for development and validation study. In this methodology, gradient mobile phase is used. For mobile phase-A, prepared 0.1N Tetrabutyl Ammonium hydroxide in Isopropanol and transferred 1ml of this solution to 1000ml water. HPLC grade Acetonitrile is used as mobile phase-B. Flow rate is 1.0ml/minute with 100% mobile phase-A for first 10 minutes, then gradually increased the ratio of mobile phase-B to elute Iguratimod and its possible impurities. Analytical column Unisphere C18 with length 250mm, internal diameter 4.6mm and particle size 5µ is used with wavelength 220nm. Limit of quantitation (LOQ) for melamine was 0.24ppm and LOQ for melamine is 0.72ppm. Analytical method validated as per ICH guideline Q2A, Validation of Analytical Methods, with specificity, LOD & LOQ, precision, linearity, accuracy, etc. Precision and accuracy was determined at lower and higher concentration with respect to specification level. This analytical method is user friendly, ease of use and found very much accurate to determine Melamine content in Iguratimod. By using this methodology to check quality, safe drug can be provided to the patient. Performance of this method may change when different equipment used or when different sample matrices are encountered.
