Methotrexate is an antineoplastic drug used solely or in combination with other drugs for the treatment of a variety of diseases. High dose methotrexate (usually >20 mg/kg body weight) is used with citrovorum factor (folinic acid) "rescue" for the treatment of some sarcomas and occasionally other malignancies. In this treatment, methotrexate levels must be very carefully monitored to avoid serious toxicity.

Methotrexate is measured by a competitive binding immunoassay using FPIA (fluorescence polarization immunoassay) on the Abbott TDX analyzer. Samples may be serum (preferred), plasma, urine or CSF.​​

Methotrexate requests are processed STAT. Samples are to be protected from the light and sent immediately to the TML. The requisition must include the diagnosis, sample date & time, methotrexate dose and time last administered.

All methotrexate results will be phoned as soon as they are available. Turnaround time is 90 minutes from time of receipt in the TML. Please provide a contact number on the requisition, so the results may be called to the appropriate physician or location.

A safe level for methotrexate depends on duration of exposure and aggressiveness of "rescue." For many protocols values <0.02 umol/L are considered safe, but this must be interpreted in relation to the specific administration protocol.

Interferences: most compounds tested showed a cross-reactivity of less than 1%, including doxorubicin, cyclophosphamide, dihydrofolic acid, folinic acid, 5FU, prednisone, and vinblastine. The principle known metabolite of methotrexate, 7-hydroxy-methotrexate, shows a low cross-reactivity of 1.5%.