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Occurrence and Significance of D-Methotrexate as a Contaminant of Commercial Methotrexate¹

Department of Chemical Engineering [S. M. C., K. K. K., C. H.], and Departments of Medicine and Pharmacology [J. H. S., J. R. B.], Yale University, New Haven, Connecticut 06520

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.

¹ 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.

² American Cancer Society Professor of Medicine and Pharmacology.

³ To whom requests for reprints should be addressed.

Received 10/26/83. Accepted 1/31/84.