Methotrexate assay by enzymatic inhibition, with use of centrifugal analyzer

We present a method for measuring methotrexate by enzymatic inhibition, with use of the centrifugal analyzer. In the presence of dihydrofolate reductase (EC 1.5.1.3), dihydrofolic acid is converted to tetrahydrofolate. NADPH acts as a coenzyme. Methotrexate inhibits the action of dihydrofolate reductase. Increasing concentrations of methotrexate depress the conversion of NADPH to NADP+, and the reaction may be followed at 340 nm. Precision, accuracy, and sensitivity are satisfactory. A comparison to the radioimmunoassay method (x) showed the following least-squares regression: y = 0.9813x + 5.6 microgram/liter; r = .9812; Sy = 6.3 microgram/liter. Some interference was noted from bilirubin, lipermia, and hemoglobin. Up to 60 patients' samples per hour may be estimated by this technique.