STABILITY INDICATING RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF PROCESS IMPURITIES AND DEGRADATION PRODUCTS OF LEVOLEUCOVORIN CALCIUM FOR INJECTION

Levoleucovorin calcium contains amide and aldehyde groups which makes it vulnerable to degradation. Stability indicating method for determination of all seven process-related impurities and degradation impurities in Fusilev® 50 mg/mL injection was developed with the help of RP-HPLC. A Forced degradation study supports method development to ensure stability indicating conditions. Solutions of the lyophilized injection were stress-tested in different conditions like Acidic, Basic, Oxidative, Thermal, Photolytic conditions, and the mass balance was found close to 90-110%. Validation has been performed according to ICH guidelines. The method was developed in ACE C18 column (150 × 4.6 mm, 3 µm) with binary gradient elution consisting of mobile phase A, which consists a mixture of Potassium Phosphate Triabasic and Tetra butyl ammonium hydrogen sulfate in water (pH 6.8 with diluted Ortho Phosphoric Acid, 0.05 M, 1000 ml) and mixed with Methanol in ratio of 90:10 % V/V and mobile phase B consist methanol at a flow rate of 0.5 mL/min and detection performed at 280 nm with column thermostat 40 °C, injection volume was 10 µl. The HPLC method allowed good resolution between peaks of Levoleucovorin Calcium, process impurities, and its degradation products with good linearity, precision, accuracy, specificity, LOD, LOQ. Regression analysis indicates the correlation coefficient value greater than 0.9900 for all process-related impurities and degradants. The LOD of all process impurities was at a level of 0.015%. The method has shown consistent recoveries for all process impurities (98.0%-102%). The method was found to be linear, accurate, precise, specific, sensitive, robust and stability-indicating.