Abstract The main objective of this study is to study the impact of forced degradation on the assay of Methylene Blue by RP-HPLC. A simple, specific, accurate and precise RP-HPLC method has been validated as per ICH guideline for determination of assay of Methylene Blue. Validation parameters like Linearity, Accuracy, Precision, System suitability, Specificity were tested. The Methylene Blue was dissolved in HPLC water at the concentration of 1 mg/mL and appropriate chromatographic condition was kept as per the monograph of Methylene Blue in USP. No interference was observed in the parameter specificity of any blank or other impurities. The Recovery of the Spiked Sample in parameter accuracy was within the acceptance limit of 97 to 103 % at three different concentrations 80 %, 100 % and 120 %. The Method Precision and Intermediate Precision of this method was achieved within the limit of RSD 2 %. The Linearity of the method was achieved through the correlation coefficient of 0.99983.Forced Degradation of Methylene Blue was performed through Acid, Base, Oxidation, and Thermal Degradation to get the % degradation data of methylene blue. The study was performed for 48 hr in which the % degradation was within the acceptance limit of 20 % except Thermal Degradation. This study can be applicable in the profiling of Methylene Blue and Sterile Preparation of Methylene Blue Injection. This study can be used for the Pharmaceutical Industries for the referral of forced degradation studies.
Keywords Methylene Blue, RP-HPLC, Validation, ICH, Forced Degradation.