Development and validation of a methotrexate adherence assay

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.