The antifolate methotrexate is a widely used drug for the treatment of various malignant tumors. Enzyme and fluorescence-based immunoassay tests are used to analyze methotrexate concentrations, but are known for their cross reactivity to the inactive metabolite 2,4-Diamino-N¹⁰methylpteroic acid (DAMPA).

Research on high-dose methotrexate (>500 mg/m²) for central nervous system (CNS) lymphoma is usually accompanied by the administration of glucarpidase or folinic acid rescue therapy to reduce methotrexate concentrations to below 1 mmol/L. Immunoassay techniques can overestimate methotrexate concentrations post-glucarpidase administration due to elevated levels of DAMPA, emphasizing the importance of using a selective detection method.

UPLC-MS/MS analysis with the ACQUITY UPLC I-Class PLUS and Xevo TQD provides the selectivity needed to achieve an accurate measurement of methotrexate.