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Method Article
Protocol for the analysis of phenolic compounds using nano-liquid chromatography-mass spectrometry and Caco-2 assays: from the evaluation of the uptake to the enterocyte metabolism
María del Mar Contreras
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avenida Fuentenueva s/n, 18071 Granada, Spain
Corresponding Author
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When using the Caco-2 model, the analysis of phenolic compounds may be a challenge for those presenting low uptake rates, as well as the determination of cellular accumulation or metabolism is not an easier task. In this sense, the combination of nano-liquid chromatography (nano-LC) with high resolution mass spectrometry (MS) is a potential analytical alternative. Here we provide a nano-LC-MS protocol for analysing phenolic compounds in samples from Caco-2 assays that could be applied routinely. On the one hand, the samples from transport experiments are simply acidified. On the other hand, trace metabolites at cellular levels can be detected after a simple step of preconcentration. As an example, the absorption and enterocyte metabolism of N-feruloyltyramine is determined. Our outcomes show that the mass accuracy using lock-mass calibration is satisfactory in order to measure accurate m/z values and obtain the molecular formula of N-feruloyltyramine metabolites. For quantitative purposes, the method is adequate in terms of linearity, repeatability, and sensitivity. Consequently, the application of this global methodology may provide clues to understand the physiological distribution and mechanism of action of phenolic compounds.
Keywords
Bioavailability, Caco-2 cells, mass spectrometry, miniaturized technique, nano-liquid chromatography, phenolic compounds, uptake
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Table 1 Table 1 _N_-feruloyltyramine calibration curve: number of points (n), calibration curve, concentration range, coefficient of determination and limits of detection (LOD) and quatification (LOQ).
- supplement0.pdf
Table 2 Table 2 Accuracy, intra- and inter-day repeatability of _N_-feruloyltyramine.
- supplement0.pdf
Table 3 Table 3 Quantification of _N_-feruloyltyramine in the receiver chambers of the Caco-2 assay in the apical (Ap) to basolateral (Bl) and Bl to Ap transport directions.
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Associated Publications
Nano-liquid chromatography coupled to time-of-flight mass spectrometry for phenolic profiling: A case study in cranberry syrups
by María del Mar Contreras, David Arráez-Román, Alberto Fernández-Gutiérrez, +1
Talanta (04 November, 2015)
Further exploring the absorption and enterocyte metabolism of quercetin forms in the Caco-2 model using nano-LC-TOF-MS
by María del Mar Contreras, Isabel Borrás-Linares, María Herranz-López, +2
ELECTROPHORESIS (30 November, 2015)
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This is a preprint. It has not completed peer review.
Method Article
Protocol for the analysis of phenolic compounds using nano-liquid chromatography-mass spectrometry and Caco-2 assays: from the evaluation of the uptake to the enterocyte metabolism
María del Mar Contreras
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avenida Fuentenueva s/n, 18071 Granada, Spain
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Nov, 2015
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
When using the Caco-2 model, the analysis of phenolic compounds may be a challenge for those presenting low uptake rates, as well as the determination of cellular accumulation or metabolism is not an easier task. In this sense, the combination of nano-liquid chromatography (nano-LC) with high resolution mass spectrometry (MS) is a potential analytical alternative. Here we provide a nano-LC-MS protocol for analysing phenolic compounds in samples from Caco-2 assays that could be applied routinely. On the one hand, the samples from transport experiments are simply acidified. On the other hand, trace metabolites at cellular levels can be detected after a simple step of preconcentration. As an example, the absorption and enterocyte metabolism of N-feruloyltyramine is determined. Our outcomes show that the mass accuracy using lock-mass calibration is satisfactory in order to measure accurate m/z values and obtain the molecular formula of N-feruloyltyramine metabolites. For quantitative purposes, the method is adequate in terms of linearity, repeatability, and sensitivity. Consequently, the application of this global methodology may provide clues to understand the physiological distribution and mechanism of action of phenolic compounds.
Figure 1
Figure 2
Figure 3
Figure 4
Associated Publications
Nano-liquid chromatography coupled to time-of-flight mass spectrometry for phenolic profiling: A case study in cranberry syrups
by María del Mar Contreras, David Arráez-Román, Alberto Fernández-Gutiérrez, +1
Talanta (04 November, 2015)
Further exploring the absorption and enterocyte metabolism of quercetin forms in the Caco-2 model using nano-LC-TOF-MS
by María del Mar Contreras, Isabel Borrás-Linares, María Herranz-López, +2
ELECTROPHORESIS (30 November, 2015)