Abstract
Trimethylamine-N-oxide (TMAO), a microbiome-derived metabolite from the metabolism of choline, betaine, and carnitines, is associated to adverse cardiovascular outcomes. A method suitable for routine quantification of TMAO and its precursors (trimethylamine (TMA), choline, betaine, creatinine, and propionyl-, acetyl-, and l-carnitine) in clinical and food samples has been developed based on LC-MS. TMA was successfully derivatized using iodoacetonitrile, and no cross-reactions with TMAO or the other methylamines were detected. Extraction from clinical samples (plasma and urine) was performed after protein precipitation using acetonitrile:methanol. For food samples (meatballs and eggs), water extraction was shown to be sufficient, but acid hydrolysis was required to release bound choline before extraction. Baseline separation of the methylamines was achieved using a neutral HILIC column and a mobile phase consisting of 25 mmol/L ammonium formate in water:ACN (30:70). Quantification was performed by MS using external calibration and isotopic labelled internal standards. The assay proved suitable for both clinical and food samples and was linear from ≈ 0.1 up to 200 μmol/L for all methylamines except for TMA and TMAO, which were linear up to 100 μmol/L. Recoveries were 91–107% in clinical samples and 76–98% in food samples. The interday (n=8, four duplicate analysis) CVs were below 9% for all metabolites in clinical and food samples. The method was applied successfully to determine the methylamine concentrations in plasma and urine from the subjects participating in an intervention trial (n=10) to determine the effect of animal food ingestion on methylamine concentrations.Graphical abstract
Highlights
The methylamines trimethylamine N-oxide (TMAO) and its precursors, trimethylamine (TMA), choline, betaine, and carnitines, have received increasing attention over the past decade owing to their potential link to adverse cardiovascular outcomes [1,2,3,4]
Available methods are based on nuclear magnetic resonance (NMR) spectroscopy [12, 13], liquid chromatography–tandem mass spectrometry (LC-MS/MS), or isotope dilution liquid chromatography-mass spectrometry [15,16,17,18,19,20,21,22]
The high polarity of methylamines makes the use of reversed phase separation difficult due to the poor retention, which can be improved by using hydrophilic liquid interaction chromatography (HILIC)
Summary
The methylamines trimethylamine N-oxide (TMAO) and its precursors, trimethylamine (TMA), choline, betaine, and carnitines, have received increasing attention over the past decade owing to their potential link to adverse cardiovascular outcomes [1,2,3,4]. While TMAO, choline, betaine, and carnitines can be measured directly using LC-MS/MS [12, 13], TMA, which does not allow fragmentation, needs to be derivatized. Most of the present methods for the quantification of TMAO and its related metabolites have been restricted to small numbers of metabolites, focusing mainly on clinical samples. The high polarity of methylamines makes the use of reversed phase separation difficult due to the poor retention, which can be improved by using hydrophilic liquid interaction chromatography (HILIC). The aim of the present study was to develop and internally validate a robust method using a HILIC separation mode and the widely available single quadrupole MS equipment for the simultaneous quantification of the most dominant methylamines in clinical and food samples (TMAO, TMA, betaine, choline, L-carnitine, acetyl-carnitine, and propionyl-carnitine). The usefulness of the method was demonstrated using a subsample of plasma and urine from subjects participating in an intervention trial to determine the effects of ingesting animal foods on methylamine concentrations in plasma and urine
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