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Thierry Dervieux, Diana Orentas Lein, John Marcelletti, Ken Pischel, Katie Smith, Michael Walsh, Russell Richerson, HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis, Clinical Chemistry, Volume 49, Issue 10, 1 October 2003, Pages 1632–1641, https://doi.org/10.1373/49.10.1632
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Abstract
Background: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential γ-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 γ-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.
