BACKGROUND AND AIMS: Therapeutic drug monitoring (TDM) of methotrexate (MTX) is challenging due to its pharmacokinetics and short plasma half-life. Intracellular MTX-polyglutamates (PG1-5), which accumulate over time, have not been assessed in pediatric inflammatory bowel disease (IBD). This study aimed to evaluate erythrocyte MTX-PG as a potential TDM tool in pediatric IBD. METHODS: In this cross-sectional study, MTX-PG concentrations were measured in erythrocytes of children with IBD on stable low-dose MTX for at least 12 weeks using stable-isotope dilution liquid chromatography-tandem mass spectrometry. The influence of administration route, MTX dosage, and anthropometrics on MTX-PG concentrations was examined. RESULTS: Seventy-eight patients were included, showing MTX-PG3 as the predominant subspecies (median 27.0 nmol/L) with a median MTX-PGtotal of 74.8 nmol/L. A higher MTX dose correlated significantly with elevated levels of MTX-PG3, MTX-PG4, MTX-PG5, and MTX-PGtotal (P <
.01). Adjusted for body surface area, MTX dose remained significantly associated with higher MTX-PG concentrations (P <
.01). However, comparison by administration route was limited due to a few patients on subcutaneous MTX (n = 4). CONCLUSIONS: We observed high interindividual variability in the reached erythrocyte MTX-PG concentrations. Body surface adjusted or unadjusted MTX dosage showed a positive linear correlation with erythrocyte MTX-PG concentrations in children with IBD. This is a prerequisite for TDM and provides a strong basis for further research into the relation between TDM of MTX, efficacy, and toxicity.