Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.

Abstract

Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.