Development and Application of a UPLC–MRM–MS Method for Quantifying Trimethylamine, Trimethylamine-N-Oxide, and Related Metabolites in Individuals with and Without Metabolic Syndrome

Trimethylamine-N-oxide (TMAO) is associated with various chronic diseases. TMAO is a downstream oxidative metabolite of trimethylamine (TMA) that is generated by the gut microbiota from dietary choline, carnitine, and betaine. Current analytical methods predominantly target TMAO only, due to the challenge of efficiently extracting and quantifying TMA. The present study demonstrates a simple and rapid UPLC–MRM–MS method for concurrent quantification of TMAO, TMA, and related precursors (choline, betaine, and various carnitines) following a methanol extraction from plasma and derivatization using iodoacetonitrile (IACN). Pure methanol resulted in a higher extractability of TMA (up to two-fold) compared to both pure acetonitrile and various methanol/acetonitrile mixtures. The quantification method showed high linearity within the tested range of 0.0625–100 μmol/L (determination coefficient > 0.999) and an intra- (n = 3) and inter-day (n = 9) precision of 2–8% along with an average recovery of above 94% for all metabolites (TMAO, TMA, choline, betaine, L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine). The method’s applicability was confirmed through a comparison of TMAO and its precursor concentrations in plasma samples of overnight-fasted subjects with (n = 12) and without (n = 21) metabolic syndrome.