Isolation and identification of urine metabolites after hydroxytyrosol supplementation in humans

Hydroxytyrosol (HT) is amongst the most potent natural scavengers of olive products found also in a plethora of foodstuffs, nutraceuticals and supplements. Critical factor for HT potency is its metabolisation fate in human system. Usually, the interpretation of the biochemical pathway that determines the bioavailability and activity is mainly based on plasma and urine analysis via LC–MS. However, the lack of appropriate standards, poses the risk of vague or imprecise identification and systematic quantification inaccuracies.

Thus, the goal of the present study is the isolation of HT metabolites from urine and unambiguous identification thereof with NMR. For this purpose, urine samples from women after HT supplementation for a six-month period were used [1].

Urine is a matrix easily obtained in large volumes, providing information about individuals dietary exposure. Urine samples after solid phase extraction-(SPE) treatment were subjected to fractionation using Centrifugal Partition Chromatography-(CPC). CPC lacks solid support, which makes it an excellent choise for biological substrates analysis. After several trials, the best separation was achieved with the biphasic solvent system methyl-t-butyl ether/n-butanol/acetonitrile/water (2:3:1:4, with 8% AA), in ascending mode. For further purification of metabolites semi-preparative RP-HPLC was employed in certain fractions. For the identification both LC-HRMS and NMR were employed. So far, several HT metabolites together with endogenous ones have been identified, in high amount and purity. The unabiguous identification of metabolites and their correlation offer a better insight in HT bioavailability and mechanism of action.

Publication History

Article published online:
13 December 2021

• References

• 1 Bouatra S.. et al. The Human Urine Metabolome. PLoS One. 2013;8(9).