Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

doi:10.1016/j.ab.2014.03.016

Analytical Biochemistry

Volume 455, 15 June 2014, Pages 35-40

https://doi.org/10.1016/j.ab.2014.03.016 Get rights and content

Abstract

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 >200 μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 μM), mid (5 μM), and high (100 μM) 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 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n = 349) show a range of 0.73–126 μM, 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.

Section snippets

Reagents

Deuterated trimethylamine-N-oxide (d₉-TMAO) was purchased from Cambridge Isotope Laboratories (Cat. No. DLM-4779-1, Andover, MA). All other reagents were purchased from either Sigma-Aldrich (St. Louis, MO) or Fisher Scientific Chemicals (Pittsburg, PA) unless otherwise stated.

Research subjects

Samples and associated clinical data were collected from fasting subjects undergoing community health screens. All subjects gave written informed consent and the Institutional Review Board of the Cleveland Clinic approved

Optimization of parameters for mass spectrometric assay of TMAO and d₉-TMAO

In order to determine the TMAO concentration in human blood, we first optimized the parameters for TMAO and its isotope labeled internal standard, d₉-TMAO, by direct infusion of these two standards. Shown in Fig. 1A and B are the collision-induced dissociation (CID) mass spectra of TMAO and d₉-TMAO, which are fragmented with collision energy of 25 eV. The product ions are 58 and 59 amu for TMAO, which are due to loss of water and hydroxyl group, respectively, as are the product ions for d₉-TMAO

Discussion

Herein we present an LC/MS/MS method for the measurement of TMAO levels in biological matrices with high accuracy, precision, and throughput that is amenable to the demands of clinical diagnostic practice. Using multiplexed HPLC with column switching, shorter column, and further optimization of the column gradients, we have adapted the stable isotope dilution LC/MS/MS assay described to permit a between sample acquisition time of 5 min (averaged) without affecting assay results or the column

Competing financial interests

Drs. Hazen, Wang, and Levison report being listed as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics. Dr. Hazen reports having been paid as a consultant for the following companies: AstraZeneca Pharmaceuticals LP, BG Medicine, Inc., Cleveland Heart Lab, Esperion, Lilly, Liposcience, Inc., Merck & Co., Inc., Pfizer, Inc., and Proctor & Gamble, and Takeda. Dr. Hazen reports receiving research funds from Cleveland Heart

Acknowledgments

S.L.H. is supported by United States National Institutes of Health (NIH) and Office of Dietary Supplement Grant R01 HL103866, and NIH Grant P20 HL113452. S.L.H. is also partially supported by a gift from the Leonard Krieger Fund. Z.W. is partially supported by an American Heart Association Scientist Development Grant (12SDG12050473). Mass spectrometry instrumentation used is housed in a facility partially supported by a Center of Innovation Award from AB SCIEX.

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Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

Abstract

Section snippets

Reagents

Research subjects

Optimization of parameters for mass spectrometric assay of TMAO and d9-TMAO

Discussion

Competing financial interests

Acknowledgments

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr. A

Biochim. Biophys. Acta

Cell Metab.

Optimization of parameters for mass spectrometric assay of TMAO and d₉-TMAO