Background: A new analytical MS method using isotope dilution combined with MALDI-triple quadrupole MS/MS has been developed and validated for the determination of methotrexate and 7-hydroxymethotrexate in plasma. Methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate-d3 were monitored by selected reaction monitoring using the transitions m/z 455.2→308.2, 458.2→311.2, 471.2→324.2 and 474.2→327.2 for methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate- d3, respectively. Results: The LLOQ was 1 nmol/l for methotrexate and 7-hydroxymethotrexate while the limit of detection was 0.3 nmol/l for both analytes. The new developed method was cross-validated by a fluorescence polarization immunoassay and tested for its clinical feasibility by measuring plasma samples from patients suffering from acute lymphoblastic leukemia. Plasma methotrexate concentrations ranged between 66.0 and 954 nmol/l and observed 7-hydroxymethotrexate/methotrexate ratios ranged between 0.1 and 32.4, respectively. Conclusion: The new method showed comparable analytical performances as the fluorescence polarization immunoassay, but analyte specificity and sensitivity of the newly developed method were significantly better.

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Journal Bioanalysis
Meesters, R.J.W, den Boer, E, Mathot, R.A.A, de Jonge, R, van Klaveren, R.J, Lindemans, J, & Luider, T.M. (2011). Ultrafast selective quantification of methotrexate in human plasma by high-throughput MALDI-isotope dilution mass spectrometry. Bioanalysis, 3(12), 1369–1378. doi:10.4155/bio.11.113