Skip to main content

Quantification of Methotrexate in Human Serum and Plasma by Liquid Chromatography Tandem Mass Spectrometry

  • Protocol
  • First Online:
LC-MS in Drug Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1872))

Abstract

Mass spectrometry (MS) is a highly specific and sensitive technique that is used for the detection of many different analytes with diverse chemical characteristics. It has been adopted by clinical laboratories for the quantification of small molecules and, by extension, has been widely used for therapeutic drug monitoring. It is an attractive alternative to immunoassay methods, because it is not subject to the same interferences. A limitation of MS (relative to immunoassays) is the turnaround time. However, this can be addressed by workflow parallelization with other assays. Herein we describe a tandem LC-MS/MS method for the detection and quantification of methotrexate in human plasma with a lower limit of quantification of 0.01 μM and within-assay and between-assay coefficients of variation of less than 15%. This method lacks interference from high-abundance metabolites and utilizes kindred chromatography to improve turnaround time in the therapeutic drug monitoring laboratory.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Jannetto PJ, Fitzgerald RL (2016) Effective use of mass spectrometry in the clinical laboratory. Clin Chem 62:92–98. https://doi.org/10.1373/clinchem.2015.248146

    Article  CAS  PubMed  Google Scholar 

  2. Strathmann FG, Hoofnagle AN (2011) Current and future applications of mass spectrometry to the clinical laboratory. Am J Clin Pathol 136:609–616. https://doi.org/10.1309/AJCPW0TA8OBBNGCK

    Article  CAS  PubMed  Google Scholar 

  3. Garg U, Zhang YV (2016) Mass spectrometry in clinical laboratory: applications in therapeutic drug monitoring and toxicology. In: Clinical applications of mass spectrometry in drug analysis. Springer, New York, pp 1–10

    Chapter  Google Scholar 

  4. Al-Turkmani MR, Law T, Narla A, Kellogg MD (2010) Difficulty measuring methotrexate in a patient with high-dose methotrexate-induced nephrotoxicity. Clin Chem 56:1792. https://doi.org/10.1373/clinchem.2010.144824

    Article  CAS  PubMed  Google Scholar 

  5. Albertioni F, Rask C, Eksborg S et al (1996) Evaluation of clinical assays for measuring high-dose methotrexate in plasma. Clin Chem 42(39):39–44

    CAS  PubMed  Google Scholar 

  6. Bath RK, Brar NK, Forouhar FA, Wu GY (2014) A review of methotrexate-associated hepatotoxicity. J Dig Dis 15:517–524. https://doi.org/10.1111/1751-2980.12184

    Article  CAS  PubMed  Google Scholar 

  7. Widemann BC, Adamson PC (2006) Understanding and managing methotrexate nephrotoxicity. Oncologist 11:694–703. https://doi.org/10.1634/theoncologist.11-6-694

    Article  CAS  PubMed  Google Scholar 

  8. Cavone JL, Yang D, Wang A (2014) Glucarpidase intervention for delayed methotrexate clearance. Ann Pharmacother 48:897–907. https://doi.org/10.1177/1060028014526159

    Article  CAS  PubMed  Google Scholar 

  9. Green JM (2012) Glucarpidase to combat toxic levels of methotrexate in patients. Ther Clin Risk Manag 8:403–413. https://doi.org/10.2147/TCRM.S30135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ahmed YAAR, Hasan Y (2013) Prevention and management of high dose methotrexate toxicity. J Cancer Sci Ther 5:106–112. https://doi.org/10.4172/1948-5956.1000193

    Article  CAS  Google Scholar 

  11. Evans WE, Crom WR, Abromowitch M et al (1986) Clinical pharmacodynamics of high-dose methotrexate in acute lymphocytic leukemia. N Engl J Med 314:471–477. https://doi.org/10.1056/NEJM198602203140803

    Article  CAS  PubMed  Google Scholar 

  12. Aumente D, Buelga DS, Lukas JC et al (2006) Population pharmacokinetics of high-dose methotrexate in children with acute lymphoblastic leukaemia. Clin Pharmacokinet 45:1227–1238. https://doi.org/10.2165/00003088-200645120-00007

    Article  CAS  PubMed  Google Scholar 

  13. Przybylski M, Preiss J, Dennebaum R, Fischer J (1982) Identification and quantitation of methotrexate and methotrexate metabolites in clinical high-dose therapy by high pressure liquid chromatography and field desorption mass spectrometry. Biomed Mass Spectrom 9:22–32. https://doi.org/10.1002/bms.1200090106

    Article  CAS  PubMed  Google Scholar 

  14. Wu D, Wang Y, Sun Y et al (2015) A simple, rapid and reliable liquid chromatography-mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring. Biomed Chromatogr BMC 29:1197–1202. https://doi.org/10.1002/bmc.3408

    Article  CAS  PubMed  Google Scholar 

  15. Schofield RC, Ramanathan LV, Murata K et al (2015) Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites. J Chromatogr B Analyt Technol Biomed Life Sci 1002:169–175. https://doi.org/10.1016/j.jchromb.2015.08.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gunther V, Mueller D, von Eckardstein A, Saleh L (2016) Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay. Clin Chem Lab Med 54:823–831. https://doi.org/10.1515/cclm-2015-0578

    Article  CAS  PubMed  Google Scholar 

  17. Steinborner S, Henion J (1999) Liquid-liquid extraction in the 96-well plate format with SRM LC/MS quantitative determination of methotrexate and its major metabolite in human plasma. Anal Chem 71:2340–2345

    Article  CAS  Google Scholar 

  18. Nair H, Lawrence L, Hoofnagle AN (2012) Liquid chromatography–tandem mass spectrometry work flow for parallel quantification of methotrexate and other immunosuppressants. Clin Chem 58:943. https://doi.org/10.1373/clinchem.2011.175067

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Andrew N. Hoofnagle .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Winston-McPherson, G.N., Schmeling, M., Hoofnagle, A.N. (2019). Quantification of Methotrexate in Human Serum and Plasma by Liquid Chromatography Tandem Mass Spectrometry. In: Langman, L., Snozek, C. (eds) LC-MS in Drug Analysis. Methods in Molecular Biology, vol 1872. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8823-5_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8823-5_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8822-8

  • Online ISBN: 978-1-4939-8823-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics