Methotrexate determination in pharmaceuticals by enantioselective HPLC

A simple, sensitive and selective high performance liquid chromatographic method with UV detection for the chiral separation of racemic methotrexate (rac-Mtx) and enantiomeric purity of l-methotrexate in pharmaceutical formulations was developed and validated. The chiral separation was optimized studying both the nature of the stationary phase by using Chirobiotic T™, Chiracel OJ and human serum albumin columns and the effect of the mobile phase composition. The best results in terms of enantioresolution and enantioselectivity were achieved with a polar organic mobile phase on Chirobiotic T™ stationary phase. Essential steps in method validation such as precision, accuracy, suitability and stability were studied according to ICH guidelines. At wavelength 303 nm, the limit of detection (S/N = 3) was found to be 0.9 μg/ml for rac-Mtx. The separation of d-Mtx at 0.2% (w/w) level (as limit of quantitation) from the main drug l-Mtx was successfully obtained with 1.72 enantioresolution value. Enantiomeric purity of l-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter- and intra-days relative standard deviation ≤ 1.6%. Under the validated stereoselective HPLC conditions for methotrexate, folic acid was also analysed.