Determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, using capillary zone electrophoresis and laser-induced fluorescence detection

High-dosage methotrexate therapy requires careful monitoring of the drug in serum to ensure minimal toxic effects. A simple, rapid and sensitive method for the separation and quantitation of methotrexate and its major metabolite, 7-hydroxymethotrexate, using high-voltage capillary zone electrophoresis combined with laser-induced fluorescence detection is described. The detection limit for methotrexate is as low as 5.10(-10) M (signal-to-noise ratio = 3), while that for 7-hydroxymethotrexate is 2.10(-9) M. The linearity of the system extends over nearly four orders of magnitude for both methotrexate and 7-hydroxymethotrexate. The extraction efficiency for the drug and its metabolite from serum is 80-85% using a Sep-Pak C18 cartridge. Quantitation of methotrexate in serum was possible in the 10(-10) M range, nearly two orders of magnitude lower than that currently obtainable by existing methods. Good correlation (r = 0.99) for serum methotrexate concentrations was obtained with an enzyme-multiplied immunoassay technique. Comparison with an enzyme inhibition assay also provided similar results.