Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate

A fluorescence polarization immunoassay (FPIA) procedure for measuring methotrexate was evaluated. The dynamic range of the assay is from 0.05 to 810 microM, and the calibration curve can be stored for at least 2 weeks. The FPIA procedure is automated and rapid; one result can be obtained in 18 min and five results in 25 min. There was no interference from hemoglobin (800 mg/dl), triglycerides (500 mg/dl), bilirubin (20 mg/dl), and protein (12.1 g/dl). Cross-reactivity with 7-hydroxy methotrexate and 2,4-diamino-N-methylpteroic acid was 0.6 and 44%, respectively. The coefficient of variation for the within-run and between-run precision was less than 5.0%. For the comparison studies, the samples were divided into four groups. The methotrexate concentrations in group 1 were 0.05-2.1 microM; in group 2, 2.2-9.3 microM; in group 3, 10-80 microM; and in group 4, greater than 80 microM. Linear regression analysis of the results obtained with the FPIA procedure and the enzyme multiplied immunoassay gave a correlation coefficient of at least 0.95 for all groups.