Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays

Abstract Methotrexate (MTX) is a widely used chemotherapeutic agent in pediatric oncology, where high-dose protocols (HDMTX; >500 mg/m²) are standard for treating hematological and central nervous system malignancies. Due to its narrow therapeutic index and potential for severe toxicity, therapeutic drug monitoring (TDM) of plasma MTX concentrations is essential to guide leucovorin rescue therapy and prevent adverse effects. The presented study aimed to compare the analytical performance of two immunoassays—enzyme-multiplied immunoassay technique (EMIT) and enzyme immunoassay (EIA)—against a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS assay demonstrated excellent linearity, sensitivity (LLOQ = 0.01 µmol/L), and precision, meeting ICH M10 regulatory guidelines. Clinical samples from pediatric patients receiving HDMTX were analyzed using all three methods. Results showed strong correlations (r > 0.93) between methods; however, immunoassays exhibited biases related to cross-reactivity with MTX metabolites such as DAMPA (2, 4-diamino-N(10)-methylpteroic acid) and 7-OH-MTX, which may lead to overestimation of MTX levels and unnecessary prolongation of leucovorin rescue. While immunoassays remain practical for routine monitoring due to their accessibility and speed, LC-MS/MS provides superior accuracy and should be the method of choice in critical clinical situations. These findings underscore the importance of selecting the appropriate assay in optimizing HDMTX therapy and ensuring patient safety.