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High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring

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Abstract

Trimethylamine N-oxide (TMAO) has attracted interest as circulating levels have reported prognostic value in patients with cardiovascular conditions, such as heart failure. With continual advances in accurate mass measurements, robust methods that can employ the capabilities of time of flight mass spectrometers would offer additional utility in the analysis of complex clinical samples. A Waters Acquity UPLC was coupled to a Waters Synapt G2-S high-resolution mass spectrometer. TMAO was measured in plasma by stable-isotope dilution-hydrophilic interaction liquid chromatography-time of flight mass spectrometry with multiple reaction monitoring (LC-ToF-MRM). Two transitions were monitored: m/z 76.1 to 58.066/59.073 and m/z 85.1 to 66.116/68.130. The method was assessed for linearity, lower limits of detection and quantitation, and reproducibility. A selected cohort of patients with systolic heart failure (SHF; n = 43) and healthy controls (n = 42) were measured to verify the assay is suitable for the analysis of clinical samples. Quantitative analysis of TMAO using LC-ToF-MRM enabled linearity to be established between 0.1 and 75 μmol/L, with a lower limit of detection of 0.05 μmol/L. Relative standard deviations reported an inter-day variation of ≤20.8 % and an intra-day variation of ≤11.4 % with an intra-study quality control variation of 2.7 %. Run times were 2.5 min. Clinical application of the method reported that TMAO in SHF was elevated compared to that of healthy controls (p < 0.0005). LC-ToF-MRM offers a highly selective method for accurate mass measurement of TMAO with rapid and reproducible results. Applicability of the method was shown in a selected cohort of patient samples.

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Acknowledgments

This work was supported by the John and Lucille van Geest Foundation and the National Institute for Health Research Leicester Cardiovascular Biomedical Research Unit.

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    Authors and Affiliations

    1. Department of Cardiovascular Sciences and NIHR Leicester Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, LE3 9QP, UK

      Liam M. Heaney, Donald J. L. Jones, Richard J. Mbasu, Leong L. Ng & Toru Suzuki

    2. Department of Cancer Studies, University of Leicester, RKCSB, Leicester, LE2 7LX, UK

      Donald J. L. Jones & Richard J. Mbasu

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    1. Liam M. Heaney
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    2. Donald J. L. Jones
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    Correspondence to Donald J. L. Jones or Toru Suzuki.

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    Liam M. Heaney and Donald J. L. Jones contributed equally to this work.

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    Table S1

    Standard addition experiments to assess recovery of trimethylamine N-oxide (TMAO) spiked into a pre-treated and untreated plasma matrix (PDF 7 kb)

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    Heaney, L.M., Jones, D.J.L., Mbasu, R.J. et al. High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring. Anal Bioanal Chem 408, 797–804 (2016). https://doi.org/10.1007/s00216-015-9164-6

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