Work

Quantitation of methotrexate polyglutamates in red blood cells and application in patients with Crohn’s disease

Year: 2025

Type: article

Abstract: Methotrexate (MTX), a folate antagonist, is commonly administered at low doses for the treatment of Crohn's disease (CD).Anti-inflammatory effects of MTX are facilitated by its intracellular conversion to MTX polyglutamates (MTX-PGs).Because plasma-based monitoring of therapeutic response does not accurately reflect the therapeutic efficacy of MTX, quantifying intracellular MTX-PGs, potential biomarkers of the MTX response, is crucial.However, it is challenging to routinely monitor intracellular MTX metabolites in patients with CD due to the low concentrations of MTX-PGs.Therefore, quantitating MTX-PGs in clinical samples with a high-sensitivity method is necessary.We established a high-sensitivity method to quantify three MTX-PGs using perchloric acid deproteinization followed by high-performance liquid chromatography-tandem mass spectrometry.Calibration curves were generated using human red blood cells as biological matrix.This method was applied to analyze MTX-PGs in red blood cells (RBCs) from patients with CD undergoing MTX therapy.The method achieved a lower limit of quantification of 1 ng/mL for individual MTX-PGs.A nine-point calibration curve covering 1-400 ng/mL showed excellent linearity.Precision (relative standard deviation < 15%) and accuracy (93.41-109.37%)were satisfactory in both intra-and inter-day assays.Plasma MTX levels were not significantly correlated with any individual RBC MTX-PG level (p = 0.998, 0.640, and 0.587, respectively).The lack of correlation supports our conclusion that plasma MTX levels may not reliably represent intracellular accumulation.The developed quantitative method provides a useful tool to improve our understanding of MTX metabolism and may facilitate therapeutic drug monitoring in MTX therapy.

Source: Translational and Clinical Pharmacology

DOI: 10.12793/tcp.2025.33.e7