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Development of an Assay for Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS

  • Ryan C. Schofield
  • Lakshmi V. Ramanathan
  • Kazunori Murata
  • Martin Fleisher
  • Melissa S. Pessin
  • Dean C. CarlowEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1383)

Abstract

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to1.0 × 106 nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.

Key words

Methotrexate Carboxypeptidase-G2 Therapeutic drug monitoring Mass spectrometry Turbulent flow liquid chromatography 

References

  1. 1.
    LaCasce AS (2014) Therapeutic use and toxicity of high-dose methotrexate. In: Post TW (ed) UpToDate. Waltham, MA. Accessed 1 Oct 2014Google Scholar
  2. 2.
    Lennard L (1999) Therapeutic drug monitoring of antimetabolic cytotoxic drugs. Br J Clin Pharmacol 47:131–143PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Widemann BC, Adamson PC (2006) Understanding and managing methotrexate nephrotoxicity. Oncologist 11:694–703CrossRefPubMedGoogle Scholar
  4. 4.
    DeAngellis LM, Tong WP, Lin S, Fleisher M et al (1996) Carboxypeptidase G2 rescue after high-dose methotrexate. J Clin Oncol 14:2145–2149Google Scholar
  5. 5.
    Widemann BC, Sung E, Anderson L et al (2000) Pharmacokinetics and metabolism of the methotrexate metabolite 2,4-diamino-N-methylpteroic acid. J Pharmacol Exp Ther 294:894–901PubMedGoogle Scholar
  6. 6.
    Albertioni F, Rask C, Eksborg S et al (1996) Evaluation of clinical assays for measuring high-dose methotrexate in plasma. Clin Chem 42:39–44PubMedGoogle Scholar
  7. 7.
    Kumar VS, Law T, Kellogg M (2010) Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for monitoring methotrexate in the setting of carboxypeptidase-G2 therapy. In: Garg U, Hammett-Stabler CA (eds) Clinical applications of mass spectrometry: methods in molecular biology 603. Humana Press, New YorkGoogle Scholar
  8. 8.
    Guo P, Wang X, Liu L et al (2007) Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 43:1789–1795PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Nair H, Lawrence L, Hoofnagle AN (2012) Liquid chromatography-tandem mass spectrometry work flow for parallel quantification of methotrexate and other immunosuppressants. Clin Chem 58:943–945CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ryan C. Schofield
    • 1
  • Lakshmi V. Ramanathan
    • 1
  • Kazunori Murata
    • 1
  • Martin Fleisher
    • 1
  • Melissa S. Pessin
    • 1
  • Dean C. Carlow
    • 1
    Email author
  1. 1.Department of Laboratory MedicineMemorial Sloan Kettering Cancer CenterNew YorkUSA

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