Development and validation of a methotrexate adherence assay

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Background: The first-line therapy for rheumatoid arthritis (RA) is weekly oral methotrexate (MTX) at low-dosages (7.5-25mg/week). However, ~40% of patients are non-adherent which may explain why some do not respond and need to start more expensive biologic therapies. To monitor adherence more accurately and develop strategies to improve it, a validated objective MTX adherence test is required.
Objective: To develop and validate the diagnostic accuracy of a novel MTX adherence assay utilising High Performance Liquid Chromatography (HPLC) Selected Reaction Monitoring (SRM) Mass Spectrometry (MS) based biochemical analysis of drug levels.
Methods: 20 RA patients underwent MTX pharmacokinetic assessment using HPLC-SRM-MS MTX plasma concentration analysis over a six day period. Directly observed therapy was the reference standard. Pharmacokinetic model validation was performed using independent plasma samples from real-world patients (n=50) with self-reported times of drug administration. Following assay optimisation the sensitivity of the assay to detect adherence was established using samples from an observational cohort study (n=138).
Results: A 2-compartment pharmacokinetic model was developed and validated. Simulations described the sensitivity required for MTX detection over 7 days; subsequent assay optimisation and retesting of samples confirmed that all patients were correctly identified as MTX adherers. Using real-world samples the assays sensitivity was 95%.
Conclusion: Non-adherence to MTX is common and can have a significant effect on disease activity. HPLC-SRM-MS plasma analysis accurately detects MTX adherence. The validated objective test could easily be used in clinic to identify patients requiring adherence support.
Original languageEnglish
JournalAnnals Of Rheumatic Diseases
Early online date5 Jun 2019
Publication statusPublished - 5 Jun 2019


  • Methotrexate
  • assay
  • adherence
  • rheumatoid arthritis
  • pharmacokinetic


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