Background/Purpose

Methotrexate (MTX) is a recommended first-line therapy in 2013 EULAR guidelines for active rheumatoid arthritis (RA). Despite this, up to 54% do not adequately respond to MTX. Non-adherence rates vary depending on the method used to measure adherence. Currently there is no gold standard for measurement of MTX adherence. Whilst an ELISA method exists to detect MTX levels at the doses used for chemotherapy regimes, this is not sensitive enough for the low dose MTX used to treat patients with rheumatological conditions. Detection of MTX and its major metabolite 7-OH-MTX in urine may be an improved method to detect adherence in routine practice, when used in the lower dose range (7.5 – 30 mg/wk). The aim, therefore, was to develop a liquid chromatography-selected reaction monitoring mass spectrometry (HPLC-SRM-MS) method to determine the presence of low concentrations of MTX and 7-OH-MTX in urine.

Methods

Donated drug-free samples from RA patients were frozen at -80ᵒC after collection. Samples were thawed at room temperature and then prepared by spiking purchased MTX, 7-OH-MTX and the internal standard MTX-d₃. Samples were diluted and protein removed by precipitation. Supernatent was subsequently analysed using HPLC with a reversed-phase column on line to a triple quadrupole mass spectrometer operated in positive mode. Analytes were measured in selected reaction monitoring mode for the following mass transitions: 455.1>308.1 m/z for MTX 471.1>324.1 m/z for 7-OH-MTX and 458.1>311.1 m/z for MTX-d₃. Method validation consisted of accuracy, lower limit of quantification, recovery, linearity, precision and stability at room temperature and -80°C. All samples were measured in triplicate.

Results

For MTX and 7-OH-MTX respectively, average recovery of analyte following sample preparations was 118%±8.9% and 86%±18.6%. The lower limit of quantitation (LLOQ) was 2.5 and 5nM. The coefficient of variance for intraday run was 3.0% and 2.5% respectively. The method was linear from the LLOQ up to 1000nM (r²=0.99, 1.00 respectively). Stability testing revealed no loss at 7 days when samples were stored at -80°C, although storage at room temperature produced an average loss of 27%±6% and 10%±39% respectively within 24 hours.

Conclusion

We have developed a rapid, simple and cost effective HPLC-SRM-MS method to measure MTX and 7-OH-MTX concentrations in urine, which is sensitive to the low doses used to treat RA and other musculoskeletal diseases. The method requires limited sample preparation and may be a novel biochemical assay for measurement of adherence to MTX therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-hplc-srm-ms-based-method-for-the-detection-and-quantification-of-methotrexate-used-at-doses-in-clinical-practice-for-patients-with-rheumatological-disease-in-urine/