A stability-indicating HPLC method has been developed and validated for the determination of Related Substances by HPLC method for Levonorgestrel was performed to demonstrate that the method is suitable for its intended purpose i.e. to determine the Related Substances in Levonorgestrel drug substances in-house. Simple HPLC chromatographic separation was achieved on a Waters Kromasil C18 (250mmx4.6mm, 5m) with mobile phase containing water in gradient combination with acetonitrile (ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm. In the developed method, the resolution of Levonorgestrel from any of impurities was found to be greater than 1.5. The test solution and Standard solutions were found to be stable in the diluent for 24 h. The developed method resolved the drug from its impurities.
Regression analyses indicate correlation coefficient value greater than 0.999 for Levonorgestrel. The LOD & LOQ for Levonorgestrel were observed 0.013% and 0.039%. The high recoveries and low standard deviations confirm the suitability of the method.The method has shown good, consistent recoveries for Levonorgestrel and also for its impurities w.r.t. variation in column temperature and flow. The method was found to be accurate, precise, linear, specific, sensitive, rugged, robust, and stability-indicating.