Separation, determination of six impurities in methotrexate drug substance using ultra-performance liquid chromatography

Methotrexate (MTX) is an antineoplastic therapeutic medicine as antimetabolite of folic acid. In this paper, a sensitive and rapid ultra-performance liquid chromatographic (UPLC) method was developed and validated for the separation and determination of impurities in MTX drug substances. The UPLC method was accomplished on an Agilent Zorbax Extend C-18 (50 mm × 4.6 mm, 1.8 μm) with a gradient elution system composed of sodium dihydrogen phosphate in water (20 mmol/L, pH 3.0) and acetonitrile. The flow rate was 2.2 mL/min. The method was validated. The calibration curves displayed good linearity (r > 0.999) within the tested concentration ranges. The limit of detection (LOD) and limit of quantification (LOQ) of the six analytes were all less than 0.774 μg/mL and 1.03 μg/mL. The relative standard deviation (RSD) for intra- and inter-day precision of the six analytes was less than 9.8%, including at the LOQ. The average recovery ranged from 95.2% to 103% except at the LOQ, where recovery ranged from 82.7% to 117%. The validated method was successfully used to determine the relative abundance of six impurities in the MTX drug substances.

Graphical abstractA good and rapid separation of the six impurities in MTX drug substance was achieved under the Agilent Zorbax Extend-C18 (4.6 mm × 50 mm, 1.8 μm) column with a gradient elution system composed of sodium dihydrogen phosphate in water (20 mmol/L, pH 3.0) and acetonitrile. Additionally, this was the first report to detect the contents of methotrexate 5-ethyl ester (termed impurity N) in MTX drug substances.Figure optionsDownload full-size imageDownload as PowerPoint slide