ArticleJournal of chromatography. A2026
Simultaneous quantification of cytidine, methylcytidine, and hydroxymethylcytidine by isotope-dilution LC-MS/MS with application to mouse liver samples.
Article in Journal of chromatography. A, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
DNA methylation and hydroxymethylation are important epigenetic modifications that play key roles in cancer development and aging processes by regulating gene expression and genome stability. Traditionally, bisulfite conversion-based or antibody-based enzyme-linked immunosorbent assays are used to find DNA methylation. These tests are non specific, tedious, and not able to differentiate the difference between methylation and hydroxymethylation. To address these issues, we developed a sensitive, reproducible, and specific LC-MS/MS method for simultaneous quantification of two major DNA methylation products, 5-methyl-2'-deoxycytidine (5-mdC) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), as well as 2'-deoxycytidine (2-dC), using corresponding stable isotope-labeled internal standards: 5-methyl-2'-deoxycytidine-d₃, 5-(hydroxymethyl)-2'-deoxycytidine-d₃, and 2'-deoxycytidine-¹³C,¹⁵N₂. We purified DNA samples from mouse liver tissue, broke them down with enzymes, filtered them, added internal standards, and then run them through a SCIEX 6500+ Triple Quad LC-MS/MS system with an Atlantis T3 C18 column under a binary gradient. The method showed great chromatographic separation and specificity, with MRM transitions of m/z 228.154 to 112.1 for 2-dC, 242.143 to 126.2 for 5-mdC, and 258.135 to 142.1 for 5-hmdC.Peak area ratio of analyte to internal standard exhibited linearity across calibration ranges of 5-5000 ng /mL for 2-dC, 0.5-500 ng/ mL for 5-mdC, and 0.05-10 ng/mL for 5-hmdC (R² > 0.999), using 2 µL injection and a total runtime of 9 min. The 5-hmdC level in female mouse liver significantly increased with aging from two to sixteen months old (0.0958 % to 0.1984 %; P<0.001), whereas 5-mdC remained unchanged (3.47 % to 3.56 %; n.s.). These data confirm the accurate and reproducible quantification of DNA methylation and hydroxymethylation in tissue samples using the developed LC-MS/MS assay and indicate a broad application to cell culture and clinical biomarker studies.
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