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

Abstract

Background: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential ␥-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 L of packed erythrocytes were converted to MTX in the presence of plasma ␥-glutamyl hydrolase and mercaptoethanol at 37°C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 L 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 C 18 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

Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis

“…The proportion of individual RBC MTXGlu concentrations at steady state in this study is similar to that in other studies, with MTXGlu 3 accounting for the largest portion (30% of MTXGlu total ) and MTXGlu 5 accounting for the smallest proportion (ϳ5% of MTXGlu total ) (23,26,32). Interestingly, some patients starting MTX had no detectable RBC MTXGlu 5 at any time during the study.…”

“…These proportions are similar to those reported in other studies described in the literature (23,26). RBC MTXGlu n elimination in patients stopping MTX treatment.…”

“…The washed RBCs were counted to normalize MTXGlu 1-5 concentrations to 8 ϫ 10 12 RBCs, so that results were comparable and not confounded by changes in the RBC count between patients and between visits. The concentrations of MTXGlu n in RBCs were analyzed by HPLC with fluorescence detection, using a modification of a previously described method (26,27). All samples were analyzed in duplicate, and the mean concentration of each RBC MTXGlu n concentration from each sample was used in the analysis.…”

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

“…Detection of each MTXGlu was carried out using a micromass Ultima triplequadrupole mass spectrometer, operating in positive ion mode. The lower limits of detection were found to be in the 0.1-0.5 nM range for each MTXGlu, which is an order of magnitude lower than the 5 nM limit of detection presented by Dervieux et al (24).…”

Risk genotypes in folate‐dependent enzymes and their association with methotrexate‐related side effects in rheumatoid arthritis

“…MTHFR C677T (alanine-to-valine substitution at codon 622) was measured using a standard real-time TaqMan allelic discrimination method with fluorogenic 3Ј-minor groove binding probes (Assay By Design; Applied Biosystems, Foster City, CA). Concentrations of MTX polyglutamate (MTXPG) in red blood cells (RBCs) (expressed as nmoles/liter RBCs) were measured using a high-performance liquid chromatography-fluorometry procedure (13).…”