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Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography–triple quadrupole mass spectrometry

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Abstract

Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)–based liquid chromatography–multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

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Acknowledgments

This work was partially supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (R01 DK123499).

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  1. Guan-yuan Chen

    Present address: Graduate Institute of Forensic Medicine, National Taiwan University, Taipei, Taiwan

Authors and Affiliations

  1. Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, 28081, USA

    Guan-yuan Chen & Qibin Zhang

  2. Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, 27412, USA

    Qibin Zhang

Authors
  1. Guan-yuan Chen
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Correspondence to Qibin Zhang.

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Deidentified, commercial human plasma samples were used in this work. Research conducted with unidentified samples is not human subjects research and is not regulated by the Federal Policy for the Protection of Human Subjects (45 CFR Part 46).

The authors declare that they have no conflicts of interest.

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Chen, Gy., Zhang, Q. Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography–triple quadrupole mass spectrometry. Anal Bioanal Chem 412, 2841–2849 (2020). https://doi.org/10.1007/s00216-020-02514-x

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  • DOI: https://doi.org/10.1007/s00216-020-02514-x

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