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A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR

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Abstract

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol.

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Acknowledgements

The CORSAIRE platform from Biogenouest is acknowledged. The authors are grateful to Dr. Jonathan Farjon for his help in implementing NUS in the adiabatic HSQC sequence.

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Funding

L.H. received financial support from the Research Council of Saint-Joseph University of Beirut and CEISAM. S.R. received financial support from the French Research Ministry. The authors received funding from the Région Pays de la Loire (Grant RFI-Food 4.2-Project AIMM).

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Authors and Affiliations

  1. CNRS, CEISAM UMR 6230, Faculté des Sciences, Université de Nantes, BP 92208, 2 rue de la Houssinière, 44000, Nantes, France

    Lenny Haddad, Sophie Renou, Gérald S. Remaud & Serge Akoka

  2. Research Unit: Technologies et Valorisation Agroalimentaire (TVA), Laboratory of Metrology and Isotopic Fractionation, Faculty of Science, Saint Joseph University of Beirut, Mar Mikhael, P.O. Box 17-5208, Beirut, 1104 2020, Lebanon

    Lenny Haddad, Toufic Rizk & Joseph Bejjani

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  1. Lenny Haddad
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  2. Sophie Renou
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  3. Gérald S. Remaud
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Contributions

Lenny Haddad: investigation, data curation, formal analysis, writing—original draft; Sophie Renou: investigation, data curation, formal analysis, writing—original draft; Gérald S. Remaud: conceptualization, resources, supervision, writing—review and editing, funding acquisition; Toufic Rizk: project administration, funding acquisition; Joseph Bejjani: conceptualization, supervision, resources, writing—review and editing, funding acquisition; Serge Akoka: conceptualization, methodology, supervision, writing—review and editing

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Correspondence to Serge Akoka.

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Haddad, L., Renou, S., Remaud, G.S. et al. A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR. Anal Bioanal Chem 413, 1521–1532 (2021). https://doi.org/10.1007/s00216-020-03135-0

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  • DOI: https://doi.org/10.1007/s00216-020-03135-0

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