Method Development and Validation using RP-HPLC for estimation of Genotoxic impurity i.e. Melamine mainly present as contaminant in diversified categories of API
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A simple and precise gradient Reversed Phase High Performance Liquid Chromatography (RP-HPLC) common method was developed for determination and quantification of genotoxic impurity i.e. Melamine. The gradient mobile phase consisted of A = 0.1%v/v Tetra butyl Ammonium Hydroxide 0.1N solution in Isopropyl Alcohol in water and B = 100% Acetonitrile, the flow rate was kept 1 mL/min using Unisphere Extend C18 column from Agela Technologies (250 mm x 4.6 mm x 5μm). Detection was performed at 220 nm using PDA detector. The common method was validated for LOD, LOQ, Linearity and Accuracy as per ICH guidelines. This method is suitable for detecting and quantifying melamine in various categories of API's such as for cardiovascular problems, Anti psychotic, API's for stomach and esophageal problems, Anti-diabetic, Antibiotic, Anti fungal, Anti gout, Antimalarial, Anti-Alzheimer and Antiadrenergic API's. Precision and accuracy was performed for all the above mentioned categories of API's on individual basis at three concentrations in three replicates i.e. at 80%, 100% and 150% of the limit concentration. Also LOQ level was shown to be precise and accurate by spiking the individual API's at LOQ level.
The developed and validated common method was found to be simple, precise, repeatable and accurate for the estimation of Melamine genotoxic impurity in diversified categories of API's.
RP-HPLC, Development, Validation, Melamine, Genotoxic Impurity.