A U-HPLC-ESI-MS/MS-based stable isotope dilution method for the detection and quantitation of methotrexate in plasma

Ethan Den Boer, Sandra G. Heil, Bertrand D. Van Zelst, Roland J.W. Meesters, Birgit C.P. Koch, Mariël L. Te Winkel, Marry M. Van Den Heuvel-Eibrink, Theo M. Luider, Robert De Jonge

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28 Citations (Scopus)


INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 μM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 x FPIA - 7.3). CONLUSIONS: The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.

Original languageEnglish
Pages (from-to)432-439
Number of pages8
JournalTherapeutic Drug Monitoring
Issue number4
Publication statusPublished - Aug 2012
Externally publishedYes


  • FPIA
  • human plasma
  • mass spectrometry
  • methotrexate
  • stable isotope
  • therapeutic drug monitoring


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