Development of an assay for methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum by LC-MS/MS Journal Article


Authors: Schofield, R. C.; Ramanathan, L. V.; Murata, K.; Fleisher, M.; Pessin, M. S.; Carlow, D. C.
Article Title: Development of an assay for methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum by LC-MS/MS
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-N-10-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 mu L) after protein precipitation with a methanolic solution containing internal standard (MTX-D-3) 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 to 1.0 x 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.
Keywords: methotrexate; plasma; tandem mass-spectrometry; high-dose methotrexate; mass; spectrometry; therapeutic drug monitoring; carboxypeptidase-g2; turbulent flow liquid chromatography
Journal Title: Methods in Molecular Biology
Volume: 1383
ISSN: 1064-3745
Publisher: Humana Press Inc  
Publication Place: New York, NY
Date Published: 2016-01-01
Start Page: 213
End Page: 222
Language: English
ACCESSION: WOS:000368837100024
PROVIDER: wos
DOI: 10.1007/978-1-4939-3252-8_23
PUBMED: 26660190
Notes: Article; Book Chapter 23 in "Clinical Applications of Mass Spectrometry in Drug Analysis: Methods and Protocols" (ISBN: 978-1-4939-3251-1)-- 999 RIVERVIEW DR, STE 208, TOTOWA, NJ 07512-1165 USA -- Source: Wos
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MSK Authors
  1. Kazunori Murata
    12 Murata
  2. Martin Fleisher
    237 Fleisher
  3. Dean Christian Carlow
    20 Carlow