HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis
| Autor: | Katie Smith, Ken D. Pischel, Michael Walsh, Russell Richerson, Diana Orentas Lein, John Marcelletti, Thierry Dervieux |
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| Rok vydání: | 2003 |
| Předmět: |
Analyte
Erythrocytes Clinical Biochemistry Arthritis Sensitivity and Specificity High-performance liquid chromatography Arthritis Rheumatoid Matrix (chemical analysis) chemistry.chemical_compound medicine Humans Chromatography High Pressure Liquid Monitoring Physiologic Detection limit Chromatography Biochemistry (medical) Reproducibility of Results medicine.disease Methotrexate Polyglutamic Acid chemistry Antirheumatic Agents Rheumatoid arthritis Calibration Ammonium acetate medicine.drug |
| Zdroj: | Clinical Chemistry. 49:1632-1641 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1373/49.10.1632 |
| Popis: | 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 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. |
| Databáze: | OpenAIRE |
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