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RATIONALE Methotrexate (MTX) is an antineoplastic therapeutic medicine that acts as an antimetabolite of folic acid. In this study we identified the impurities in MTX drug substances produced by different manufacturers and in different batches using high-performance liquid chromatography coupled with a photodiode array detector and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-PDA/FTICR-MS). METHODS MTX and its impurities were separated on a Restek Pinnacle II C18 column (250 × 4.6 mm, 5 µm) with a gradient elution system composed of 0.2% formic acid and acetonitrile at a flow rate of 1.0 mL/min. Ultraviolet (UV) detection was set at 305 nm. Mass detection was carried out using FTICR-MS with full-scan mass analysis at a resolving power of 100 000 coupled with multiple-stage mass analysis using a parent list of compounds. RESULTS Fifteen impurities were detected in MTX drug substances, and their structures were predicted from using HPLC-PDA/FTICR-MS data, including their UV spectra, high-resolution mass spectrometry (HRMS), fragmentation patterns, and MS n spectra. Ten of the impurities detected in the MTX drug substances are reported for the first time. There was a high abundance of esterified impurities in some batches of MTX drug substances, over the identification threshold of International Conference on Harmonization (ICH) guidelines, which requires particular attention. CONCLUSIONS This paper describes a HPLC-PDA/FTICR-MS method to profile and identify impurities in MTX drug substances. The results suggest that HPLC-PDA/FTICR-MS is a valuable analytical technique for the rapid identification of impurities. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]