Abstract
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.