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Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography–mass spectrometry and hydrophilic interaction liquid chromatography–mass spectrometry

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Abstract

Ultrahigh-performance supercritical fluid chromatography–mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

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Acknowledgments

We would like to acknowledge the help of the group of Bohuslav Melichar (University Hospital Olomouc, Czech Republic) with the sample collection and other people involved in the sample processing from University of Pardubice (Vladimíra Nováková Mužáková, Jana Kňavová, and Iva Fousová).

Funding

This work was supported by the grant project no. 18-12204S funded by the Czech Science Foundation.

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Authors and Affiliations

  1. Faculty of Chemical Technology, Department of Analytical Chemistry, University of Pardubice, Studentská 573, 532 10, Pardubice, Czech Republic

    Denise Wolrab, Michaela Chocholoušková, Robert Jirásko, Ondřej Peterka & Michal Holčapek

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  1. Denise Wolrab
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  2. Michaela Chocholoušková
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  3. Robert Jirásko
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  5. Michal Holčapek
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Correspondence to Michal Holčapek.

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The study was approved by the ethical committee at University Hospital Olomouc, Czech Republic, and all healthy volunteers signed informed consent.

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The authors declare that they have no conflict of interest.

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Published in the topical collection Current Progress in Lipidomics with guest editors Michal Holčapek, Gerhard Liebisch, and Kim Ekroos.

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Wolrab, D., Chocholoušková, M., Jirásko, R. et al. Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography–mass spectrometry and hydrophilic interaction liquid chromatography–mass spectrometry. Anal Bioanal Chem 412, 2375–2388 (2020). https://doi.org/10.1007/s00216-020-02473-3

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