Methotrexate from various commercial sources has been found to contain 0.5 to 48% (w/w) of the enantiomer d-methotrexate. The two methotrexate enantiomers were separated by using chiral high-performance liquid chromatography with an octadecyl silica column and a mobile phase containing l-proline and cupric nitrate. For the assay of d-methotrexate impurity in commercial methotrexate, l-methotrexate was hydrolyzed with carboxypeptidase G1, and the remaining d-methotrexate was quantitated by high-performance liquid chromatography. The biological effects of d-methotrexate were investigated and compared to that of l-methotrexate. d-Methotrexate was found to be a good inhibitor of dihydrofolate reductase from both murine and human tumor cells, but was a poor inhibitor of L1210 and CCRF-CEM cell growth. In animal experiments with dogs and mice, d-methotrexate was rapidly absorbed from the intestine and excreted by the kidneys.

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Supported by Grants CA-21948, GM-20993, and in part by Grants CA-08341 and CA-08010 from NIH, United States Department of Health and Human Resources.

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©1984 American Association for Cancer Research.
1984
Cancer Research, Inc.