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 (7.5-25 mg/week). However, similar to 40% of patients are non-adherent which may explain why some do not respond and need to start more expensive biological 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 using high-performance liquid chromatography-selected reaction monitoring-mass spectrometry (HPLC-SRM-MS) based biochemical analysis of drug levels. Methods 20 patients with RA underwent MTX pharmacokinetic assessment using HPLC-SRM-MS MTX plasma concentration analysis over a 6-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 two-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-SRMMS plasma analysis accurately detects MTX adherence. The validated objective test could easily be used in clinic to identify patients requiring adherence support.