Quantitative analyses of metabolites using uniformly 13C-labelled components as internal standards
Quantification and quality control of metabolite concentrations such as kaempferol, chlorogenic acid, quinic acid, quercetin etc. in plant cells or human tissues is often laborious, inaccurate, and inefficient. Concentrations of (unstable) metabolites are often strongly affected by (unintended) differences in sample preparation and analysis, resulting from different laboratory protocols and available expertise. Moreover, obtaining reliable results is often hampered by interacting effects of the metabolites with the food matrix (Havlik et al, 2020).
Stable Isotope Solution: Isotope Dilution Mass Spectrometry (IDMS)
Many of these difficulties can be tackled by applying a stable isotope dilution technique (Erk et al, 2009). Uniformly stable isotope labelled analogues of the metabolites are used as internal standards (IS). Degradation of metabolites during the analyses and differences in handling of the samples during processing are accounted for by applying a uniformly 13C-labelled internal standard. In tiny amounts (e.g. 10 ng aliquots per sample), they provide a means for rapid, sensitive, and selective metabolite identification and quantification in different kinds of experiments (Erk et al, 2009; Weidel et al, 2014).
Results from publications with IsoLife’s products
During the last decade, several novel stable isotope methods for the quantification of metabolites such as quinic acid, chlorogenic acid, quercetin, and kaempferol using 13C-labelled internal standards have been established (Erk et al, 2009; Moradi-Afrapoli et al, 2014; Nakabayashi et al, 2015; Weidel et al, 2014).
13C chlorogenic acid and 13C quinic acid have been used to develop rapid and quantitative methods to determine these compounds in, for instance, apple and potato products of different varieties (Hagl et al, 2011; Weidel et al, 2014). In vitro analyses of 13C chlorogenic acid and its metabolites were done by Scherbl et al. (2017) after consumption of coffee, either or not accompanied by breakfast. Consumption of breakfast appeared to induce retarded, but continuous release of chlorogenic acid in the gastrointestinal tract.
Also for 13C flavonoids like 13C kaempferol and 13C quercetin HPLC/MS, methods have been developed in which these compounds are used as internal standards (Fernández-del-Río et al, 2020; Havlik et al, 2020; Moradi-Afrapoli et al, 2016; Zabela et al, 2016). For kaempferol, a major flavonoid in human diet, it became clear that it is rapidly metabolised, has a poor oral bioavailability, and shows an extreme short half-life time of 3-4 minutes in plasma (Zabela et al, 2016).
Figure 1. Typical MRM chromatograms of kaempferol quantifier (A), kaempferol qualifier (B), 13C15-labeled kaempferol (C) in rat plasma (Zabela et al, 2016).
Food matrix and/or co-ingestion of other food sources seem to be highly important for the functioning of the gut microbiota and the colonic absorption and bioavailability of organic acids and flavonoids (Havlik et al, 2020; Pinta et al, 2018; Scherbl et al, 2017; Weidel et al, 2014; Zabela et al, 2016). Elucidating biotransformations by the gut microflora, determining bioavailability, and establishing biokinetics are highly relevant for understanding effects of nutrition on human metabolism (Pinta et al, 2018).
Erk T, H Bergmann, E Richling. 2009.
A novel method for the quantification of quinic acid in food using stable isotope dilution analysis.
Journal of AOAC International 92: 730-733.
Fernández-del-Río L, E Soubeyrand, GJ Basset, CF Clarke. 2020.
Metabolism of the flavonol kaempferol in kidney cells liberates the B-ring to enter coenzyme Q biosynthesis.
Molecules 25: 2955.
Hagl S, H Deusser, B Soyalan, C Janzowski, F Will, H Dietrich, F Albert, S Rohner, E Richling. 2011.
Colonic availability of polyphenols and D-(-)-quinic acid after apple smoothie consumption.
Molecular Nutrition & Food Research 55: 368-377.
Havlik J, V Marinello, A Gardyne, M Hou, W Mullen, DJ Morrison, T Preston, E Combet, CA Edwards. 2020.
Dietary fibres differentially impact on the production of phenolic acids from rutin in an in vitro fermentation model of the human gut microbiota.
Nutrients 12: 1577.
Moradi-Afrapoli F, M Oufir, FR Walter, MA Deli, M Smiesko, V Zabela, V Butterwecke, M Hamburger. 2016.
Validation of UHPLC–MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier and intestinal drug permeability studies.
Journal of Pharmaceutical and Biomedical Analysis 128: 264-274.
Nakabayashi R, H Tsugawa, M Kitajima, H Takayama, K Saito. 2015.
Boosting sensitivity in liquid chromatography–Fourier transform ion cyclotron resonance–Tandem mass spectrometry for product ion analysis of monoterpene indole alkaloids.
Frontiers in Plant Science 6: 1127.
Scherbl D, M. Renouf, C. Marmet, L. Poquet, I. Cristiani, S. Dahbane, S. Emady-Azar, J. Sauser, J. Galan, F. Dionisi, E. Richling. 2017.
Breakfast consumption induces retarded release of chlorogenic acid metabolites in humans.
European Food Research and Technology 243: 791-806.
Weidel E, M Schantz, E Richling. 2014.
A rapid method for quantifying chlorogenic acid levels in potato samples.
Journal of AOAC International 97: 902-907.
Zabela V, C Sampath, M Oufir, F Moradi-Afrapoli, V Butterweck, M Hamburger. 2016.
Pharmacokinetics of dietary kaempferol and its metabolite 4-hydroxyphenylacetic acid in rats.
Fitoterapia 115: 189-197.