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Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method

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Abstract

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5–100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1–1,000 nM for all MTXPGn (R 2 > 0.99). The coefficient of variation ranged from 1–4% for intraday precision and 6–15% for interday precision. Samples were stable for at least 1 month at −80 °C. Recovery ranged from 98–100%, and the relative matrix-effect varied from 95–99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

The seperation of methotrexate polyglutamates using UPLC.

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Abbreviations

MTX:

Methotrexate

MTXPGn:

Methotrexate polyglutamate

MTXPGn(M+6):

Methotrexate polyglutamate (13C5, 15N)-stable isotope labeled analogue

RBC:

Red blood cell

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Authors and Affiliations

  1. Department of Clinical Chemistry, Erasmus University Medical Centre Rotterdam, ‘s Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands

    E. den Boer, B. D. van Zelst, S. G. Heil & R. de Jonge

  2. Faculty of Sciences, Department of Chemistry, Universidad de los Andes, Cra. 1 No. 18A-10, Bogota D.C., Colombia

    R. J. W. Meesters

  3. Department of Neurology, Erasmus University Medical Centre Rotterdam, ‘s Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands

    T. M. Luider

  4. Department of Rheumatology, Erasmus University Medical Centre Rotterdam, ‘s Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands

    J. M. W. Hazes

  5. Department of Clinical Chemistry, Erasmus MC, ‘s Gravendijkwal 230, room L-137, 3015 CE, Rotterdam, The Netherlands

    R. de Jonge

Authors
  1. E. den Boer
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  2. R. J. W. Meesters
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  3. B. D. van Zelst
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  4. T. M. Luider
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  5. J. M. W. Hazes
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  6. S. G. Heil
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  7. R. de Jonge
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Correspondence to R. de Jonge.

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den Boer, E., Meesters, R.J.W., van Zelst, B.D. et al. Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method. Anal Bioanal Chem 405, 1673–1681 (2013). https://doi.org/10.1007/s00216-012-6581-7

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  • DOI: https://doi.org/10.1007/s00216-012-6581-7

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