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Clinical Chemistry 49: 1632-1641, 2003; 10.1373/49.10.1632
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(Clinical Chemistry. 2003;49:1632-1641.)
© 2003 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis

Thierry Dervieux1,a, Diana Orentas Lein1, John Marcelletti1, Ken Pischel2, Katie Smith1, Michael Walsh1 and Russell Richerson1

1 Research and Development, Prometheus Laboratories, 5739 Pacific Center Blvd., San Diego, CA 92121.
2 Division of Rheumatology, Scripps Clinic, San Diego, CA 92037.

aAuthor for correspondence. Fax 858-410-0298; e-mail tdervieux{at}prometheuslabs.com.

Background: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential {gamma}-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes.

Methods: Two alternative approaches were developed. In the first approach, MTXPGs from 50 µL of packed erythrocytes were converted to MTX in the presence of plasma {gamma}-glutamyl hydrolase and mercaptoethanol at 37 °C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 µL packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C18 reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes.

Results: Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2–8 °C.

Conclusion: The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.




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


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 Am J Health Syst PharmHome page
A. Gaffo, K. G. Saag, and J. R. Curtis
Treatment of rheumatoid arthritis
Am. J. Health Syst. Pharm., December 15, 2006; 63(24): 2451 - 2465.
[Abstract] [Full Text] [PDF]

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 Ann Rheum DisHome page
T Dervieux, D Furst, D O Lein, R Capps, K Smith, J Caldwell, and J Kremer
Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study
Ann Rheum Dis, August 1, 2005; 64(8): 1180 - 1185.
[Abstract] [Full Text] [PDF]

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 Pharmacol. Rev.Home page
B. N. Cronstein
Low-Dose Methotrexate: A Mainstay in the Treatment of Rheumatoid Arthritis
Pharmacol. Rev., June 1, 2005; 57(2): 163 - 172.
[Abstract] [Full Text] [PDF]


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