Clinical Chemistry
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Clinical Chemistry 45: 223-228, 1999;
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(Clinical Chemistry. 1999;45:223-228.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Dihydrofolate Reductase Enzyme Inhibition Assay for Plasma Methotrexate Determination Using a 96-Well Microplate Reader

Brigitte C. Widemanna, Frank M. Balis and Peter C. Adamson

a Address correspondence to this author at: Pediatric Oncology Branch, National Cancer Institute, Bldg. 10, Rm. 13N240, 10 Center Dr., Bethesda, MD 20892-1928. Fax 301-402-0575; e-mail widemanb{at}pbmac.nci.nih.gov.

Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 µmol/L. The within-run CVs at 0.03 µmol/L and 0.08 µmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.




The following articles in journals at HighWire Press have cited this article:


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Understanding and managing methotrexate nephrotoxicity.
Oncologist, June 1, 2006; 11(6): 694 - 703.
[Abstract] [Full Text] [PDF]

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T. Dervieux, D. Orentas Lein, J. Marcelletti, K. Pischel, K. Smith, M. Walsh, and R. Richerson
HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis
Clin. Chem., October 1, 2003; 49(10): 1632 - 1641.
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L. R. Belur, D. Boelk-Galvan, M. D. Diers, R. S. McIvor, and C. L. Zimmerman
Methotrexate Accumulates to Similar Levels in Animals Transplanted with Normal versus Drug-resistant Transgenic Marrow
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B. C. Widemann, E. Sung, L. Anderson, W. L. Salzer, F. M. Balis, K. S. Monitjo, C. McCully, M. Hawkins, and P. C. Adamson
Pharmacokinetics and Metabolism of the Methotrexate Metabolite 2,4-Diamino-N10-methylpteroic Acid
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