Improved enzymatic assay for methotrexate: shape of standard curve, stability of reagents, sensitivity.

An improved enzymatic assay for methotrexate (MXT) is presented. A linear calibration curve for MTX could only be obtained, when a pre-incubation period of MTX with the enzyme preceded the measurement of dihydrofolate reductase (DHFR) activity. Stability for 24 h was achieved by addition of NaN3 to a working solution containing DHFR, albumin, NADPH, and buffer. The detection limit of a rapid assay was 4 X 10(-9) mol/l MTX in plasma. A second but slow assay with increased detection limit (1 X 10(-9) mol/l) is reported.