Quantification of trimethylamine-N-oxide (TMAO) and its main related trimethylammonium-containing compounds in human plasma by LC-MS/MS

Background: Trimethylammonium-containing compounds, including choline (CHOL), carnitine (CAR), trimethylglycine (TMG), ergothioneine (ERT), Nε,Nε,Nε-trimethyllysine (TML), γ-butyrobetaine (gBB), and dimethylglycine (DMG) contribute to trimethylamine N-oxide (TMAO) production, a metabolite linked to cardiovascular, renal, and metabolic diseases. An LC-MS/MS method has been established for their simultaneous measurement in human plasma, as an accurate quantification of TMAO and its precursors is crucial for understanding its clinical relevance. Methods: Blood samples from ten healthy male volunteers were processed using acetonitrile protein precipitation. Analysis was performed using a HILIC column and an isocratic methanol-formic acid mobile phase, achieving a total run time of less than 6 min. Linearity was adequate for all analytes (R2 > 0.995), with mean intra- and inter-assay variation coefficients of 2.88 % and 4.23 %, respectively. Recoveries ranged from 95 % to 101 %, limits of detection from 0.009 to 0.068 µmol/L, and limits of quantification from 0.031 to 0.187 µmol/L. Plasma mean concentrations were 3.18 ± 0.73 µmol/L for TMAO, 3.99 ± 0.65 µmol/L for DMG, 9.84 ± 2.08 µmol/L for CHOL, 24.22 ± 6.19 µmol/L for TMG, 0.54 ± 0.22 µmol/L for gBB, 57.29 ± 8.89 µmol/L for CAR, 1.10 ± 0.42 µmol/L for ERT, and 0.40 ± 0.11 µmol/L for TML. Significant inter-individual variability (mean RSD% of 26 %) was observed. Conclusion: The developed LC-MS/MS method enables rapid, sensitive, and selective quantification of TMAO and its precursors in human plasma. The analytical performance supports its application in clinical and metabolomic studies, contributing to a better understanding the role of TMAO in disease states.