Development and validation of an LC-MS/MS bioanalytical method for simultaneous determination of five main metabolites from the folate-methionine cycle in biological samples : implications for anti-folate treatment

Rotman Primec, Jaka (author), Geršak, Ksenija (author), Mlinarič-Raščan, Irena (author), Urbančič, Dunja (author), Roškar, Robert (author)

The folate-methionine cycle metabolites are essential for methylation reactions and cell growth, with their imbalances implicated in cardiovascular disease, neural tube defects, cancer, and psychiatric disorders. Reliable quantification of these metabolites is important for disease prevention and monitoring of antifolate therapies. However, existing analytical methods are often limited by a low number of analytes, lengthy sample preparation, high reagent consumption, derivatisation requirements or lack of efficiency for simultaneous analysis. In this study, we report the development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of five key metabolites: 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, folic acid, and homocysteine in biological samples utilising simple and fast sample preparation by protein precipitation. Method validation followed the guideline M10 on bioanalytical method validation, demonstrating selectivity, concentration-response relationship, precision, accuracy and stability across short- and long-term storage. The method covered wide dynamic ranges between the lower and upper limits of quantification, namely 50–4000 μg/L for homocysteine, 500–10,000 μg/L for S-adenosylmethionine, 2.5–50 μg/L for S-adenosylhomocysteine, 2.5–100 μg/L for folic acid and 2.5–50 μg/L for 5-methyltetrahydrofolate. In addition, the method's robustness was shown in endogenous matrices of six different cell lines. Applicability was demonstrated on human lymphoblastoid cells treated with methotrexate, a known antifolate, revealing expected metabolic changes; increase in intracellular levels of folic acid and homocysteine, and decreased levels of 5-methyltetrahydrofolate and S-adenosylmethionine. These results highlight the ability of the method to detect metabolic changes and its potential as a robust tool for assessing methylation and folate status in biomedical research.

• Language: English
• Keywords: LC-MS/MS, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cell lysates
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 10 str.
• Numbering: Vol. 220, art. 116399
• PID: 20.500.12556/RUL-182833
• UDC: 543.2/.9
• ISSN on article: 1095-9149
• DOI: 10.1016/j.microc.2025.116399
• COBISS.SI-ID: 260447235
• Publication date in RUL: 25.05.2026

Record is a part of a journal

• Title: Microchemical journal
• Publisher: Elsevier
• ISSN: 1095-9149
• COBISS.SI-ID: 23088645

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: LC-MS/MS, folna kislina, 5-metiltetrahidrofolat, S-adenozilmetionin, S-adenozilhomocistein, homocistein, celični lizati, tekočinska kromatografija, masna spektrometrija