Capillary electrophoretic drug monitoring of methotrexate and leucovorin and their metabolites.

High-dose methotrexate is an important element of treatment protocols in childhood acute lymphocytic leukemia or osteosarcoma. A capillary electrophoretic method has been developed to measure peak levels and the metabolite pattern in patients with delayed methotrexate elimination. It serves to determine plasma levels of methotrexate, leucovorin and their metabolites, 7-hydroxymethotrexate, 2,4-diamino-N10 -methylpteroic acid and 5-methyltetrahydrofolic acid. For the determination of high concentrations (>10 microM) protein precipitation by acetonitrile will suffice for sample preparation. All other samples undergo solid-phase extraction and upgrading on C18 columns. Aminopterin, a therapeutic antecedent of methotrexate, serves as internal standard. Detection is done on a UV detector with a 300 nm filter. Five hundred microl of serum are needed to determine 0.2 microM of a specified substance (0.5 microM 5-methylterahydrofolic acid) with good precision and accuracy. For peak levels, 20 microl of capillary serum are sufficient.