Abstract
Objectives
To develop a sensitive liquid chromatography-tandem mass spectrometry method capable of measuring serum methotrexate in patients with rheumatoid arthritis to assess adherence to drug treatment.
Methods
Isotopically labelled internal standard and deionised water were added to sample prior to solid phase extraction using a Waters Oasis Max ion-exchange 96-well plate. Following extraction, samples were analysed by LC-MS/MS on a TQS-micro mass spectrometer.
Results
Mean recovery was 107 % for four different concentrations of methotrexate spiked into seven patient samples, whilst post extraction spiking gave a mean recovery of 100 %. Between-batch and within-batch CVs were ≤6 % at three different concentrations of methotrexate in fresh frozen plasma. Mean bias was <5 % for between-batch and within batch analysis at three different weighed in concentrations of methotrexate certified reference material. The lower limit of quantification of the assay was 0.1 nmol/L with linearity up to approximately 100 nmol/L. Dilution linearity studies were used to validate the dilution of patient samples prior to analysis. There was no significant interference in the method from lipaemia, haemolysis or icterus.
Conclusions
A sensitive LC-MS/MS assay for methotrexate has been developed and validated. The method has been used to measure methotrexate adherence in patient samples from clinical trials and could be used in future research to assess the ability of the assay as a biofeedback intervention to improve adherence to methotrexate therapy.
Funding source: The MIRA study is funded by The Association of Physicians of Great Britain & Ireland and supported by Versus Arthritis (21754) and the NIHR Manchester Biomedical Research Centre (NIHR203308). N/A
Award Identifier / Grant number: N/A
Acknowledgments
We thank laboratory staff in biochemistry at Wythenshawe Hospital for providing a productive working environment allowing this work to be carried out. Additionally we thank UKNEQAS for providing samples free of charge.
-
Research funding: The MIRA study is funded by The Association of Physicians of Great Britain & Ireland and supported by Versus Arthritis (21754) and the NIHR Manchester Biomedical Research Centre (NIHR203308).
-
Author contributions: Professor Brian Keevil developed the method. Dr Malcolm McTaggart performed the other laboratory work including method validation and clinical trial work. Dr Malcolm Mctaggart produced the first draft of the manuscript. Dr James Bluett provided the clinical trial samples. All authors edited the manuscript and contributed to the final version.
-
Competing interests: No conflict of interest.
-
Informed consent: Informed consent was obtained from all individuals included in the MIRA study.
-
Ethical approval: Ethical approval for MIRA was given by the North West – Haydock Research Ethics Committee (19/NW/0047) and all participants provided written informed consent. The method validation did not require ethical approval.
References
1. Smolen, JS, Landewe, RB, Bijlsma, JW, Burmester, GR, Dougados, M, Kerschbaumer, A, et al.. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update. Ann Rheum Dis 2020;79:685–99. https://doi.org/10.1136/annrheumdis-2019-216655.Search in Google Scholar PubMed
2. National Institute for Health and Care Excellence. Rheumatoid arthritis in adults: management. NG100; 2018. https://www.nice.org.uk/guidance/ng100/resources/rheumatoid-arthritis-in-adults-management-pdf-66141531233989 [Accessed 1 Feb 2023].Search in Google Scholar
3. Bluett, J, Riba-Garcia, I, Verstappen, SM, Wendling, T, Ogungbenro, Unwin, RD, et al.. Development and validation of a methotrexate adherence assay. Ann Rheum Dis 2019;78:1192–7. https://doi.org/10.1136/annrheumdis-2019-215446.Search in Google Scholar PubMed PubMed Central
4. Barton, A, Jani, M, Bundy, C, Bluett, J, McDonald, S, Keevil, B, et al.. Translating research into clinical practice: quality improvement to halve non-adherance to methotrexate. Rheumatology 2020;60:125–31. https://doi.org/10.1093/rheumatology/keaa214.10.1093/rheumatology/keaa214Search in Google Scholar PubMed PubMed Central
5. Food and Drug Administration, Department of Health and Health Services. Guidance for industry. Bioanalytical method evaluation. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf [Accessed 7 Jul 2023].Search in Google Scholar
6. McTaggart, MP, Keevil, BG. A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy. Ann Clin Biochem 2021;58:599–604. https://doi.org/10.1177/00045632211031284.Search in Google Scholar PubMed
7. ClinicalTrials.gov. Methotrexate use improvement in rheumatoid arthritis using biomarker feedback. https://clinicaltrials.gov/ct2/show/NCT03913728 [Accessed 7 Jul 2023].Search in Google Scholar
8. Bluett, J, Hyrich, KL, Keevil, B, Doran, P, Barton, A. Feasibility study of methotrexate use improvement in rheumatoid arthritis using biomarker feedback. Rheumatology 2023;62(2 Suppl):P106.10.1093/rheumatology/kead104.147Search in Google Scholar
9. Engel, A, Ruhe, L, Singh, N, Wright, JA, Liesch, F, Bauland, F, et al.. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of methotrexate in human serum and plasma. Clin Chem Lab Med 2023;61:1917–29. https://doi.org/10.1515/cclm-2022-1001.Search in Google Scholar PubMed
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0350).
© 2023 Walter de Gruyter GmbH, Berlin/Boston
