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.