A simple preparation of the methotrexate metabolites 7-hydroxymethotrexate and 4-deoxy-4-amino-N10-methylpteroic acid.

Simple and convenient preparations of two metabolites of the antineoplastic drug methotrexate (MTX) are described. MTX is incubated in a Tris-HCl rabbit liver homogenate to produce 7-hydroxymethotrexate. Filtration, lyophilization, and recrystallization from water gives a chromatographically pure product. Analytical amounts of 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA) are prepared by 0.05 N NaOH hydrolysis of an amide bond within the MTX molecule. Preparative high-performance liquid chromatography was used for purification of DAMPA. Nominal molecular weights of these metabolites have been confirmed by fast atom bombardment mass spectrometry. These synthetic methods provide a simple and efficient source of these compounds for use as standards in analytical methods, drug metabolism and cytotoxicity studies, and therapeutic drug monitoring.