Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry
Open accessAbstract
Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5μM), mid (5μM), and high (100μM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at -80°C for 5years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) show a range of 0.73-126μM, median (interquartile range) levels of 3.45 (2.25-5.79)μM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.
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Peak plasma TMAO levels from acute TMAO injection were similar to levels in mice chronically exposed to choline-supplemented diet and in some human clinical studies.
“Of note, the peak plasma TMAO levels with this protocol were similar to levels observed in mice chronically exposed to the choline‐supplemented diet (Figure 1C) and in some human clinical studies.9, 10, 17”
From Trimethylamine N‐Oxide Promotes Vascular Inflammation Through Signaling of Mitogen‐Activated Protein Kinase and Nuclear Factor‐κB (2016) · cites this paper as [17]
TMAO was measured by stable isotope dilution HPLC with online electrospray ionization tandem mass spectrometry using d9-labeled internal standards.
“Trimethylamine-N-oxide (TMAO) levels were determined by stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/MS/MS) on an AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX, Framingham MA) using d9-(trimethyl)-labeled internal standards as previously described10, 21.”
Plasma TMAO levels increase with age in humans and rats.
“Some studies performed in human and rats have revealed that plasma TMAO levels show an age related fashion, i.e., levels increase with age [32,33].”
From Implication of Trimethylamine N-Oxide (TMAO) in Disease: Potential Biomarker or New Therapeutic Target (2018) · cites this paper as [33]
In 349 apparently healthy subjects, fasting plasma TMAO median concentration was 3.45 μM (IQR 2.25-5.79), did not differ by sex, but increased with advancing age.
“Wang et al. measured fasting plasma levels of TMAO in 349 apparently healthy subjects using stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) and reported a median concentration of 3.45 μM (Interquartile Range 2.25–5.79), which did not differ by sex, but the levels increased with advancing age [21].”
From Trimethylamine N-oxide: the good, the bad and the unknown. (2016) · cites this paper as [21]
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References 13 · Cited by 320
Research works printed but not linked: 1
- [11]Wang Z Tang WH Cho L Brennan DM Hazen SL Targeted metabolomic evaluation of arginine methylation and cardiovascular risks: potential mechanisms beyond nitric oxide synthase inhibition Arterioscler. Thromb. Vasc. Biol 2009 29 1383 1391 19542023 PMC2755199
- Authors
- Wang, Z., Levison, B. S., Hazen, J. E., Donahue, L., Li, X.-M., & Hazen, S. L.
- Affiliations
- Cleveland Clinic (US)
- Corresponding author
- Wang, Zeneng
- Venue
- Analytical Biochemistry
- Publisher
- Elsevier BV
- Published
- 2014-06
- Volume
- 455
- Pages
- 35-40
- ISSN
- 0003-2697 (ISSN-L)
- PMC
- PMC4167037
- OpenAlex
- W2016019060
- Cited by
- 320
- References
- 13
- Citations by year
- 2026: 17 · 2025: 14 · 2024: 31 · 2023: 27 · 2022: 36 · 2021: 33 · 2020: 40 · 2019: 31 · 2018: 34 · 2017: 10 · 2016: 30 · 2015: 13 · 2014: 3
- FWCI
- 7.66
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Office of Dietary Supplements (P20 HL113452, R01 HL103866)
- American Heart Association (12SDG12050473)
- National Institutes of Health
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Keywords
- trimethylamine N-oxide, stable isotope dilution, plasma biomarkers, cardiovascular risk markers, assay validation, sample stability
- MeSH terms
- Adult, Aged, Chromatography, Liquid, Deuterium, Fasting, Female, Humans, Indicator Dilution Techniques, Male, Methylamines, Middle Aged, Reference Values, Reproducibility of Results, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry, Limit of Detection
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