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. 2023 Feb 15;71(6):3033-3039.
doi: 10.1021/acs.jafc.2c07145. Epub 2023 Jan 31.

Separation of Isomeric Forms of Urolithin Glucuronides Using Supercritical Fluid Chromatography

Ana M Ares  1 , Laura Toribio  1 , Rocío García-Villalba  2 , Jose M Villalgordo  3 , Yusuf Althobaiti  4 , Francisco A Tomás-Barberán  2   4 , José Bernal  1
Affiliations

Separation of Isomeric Forms of Urolithin Glucuronides Using Supercritical Fluid Chromatography

Ana M Ares et al. J Agric Food Chem. .

Abstract

Urolithins are gut microbiota metabolites produced in humans after consuming foods containing ellagitannins and ellagic acid. Three urolithin metabotypes have been reported for different individuals depending on the final urolithins produced. After absorption, they are conjugated with glucuronic acid (phase II metabolism), and these are the main circulating metabolites in plasma and reach different tissues. Different regioisomeric isomers of urolithin glucuronides have been described. Still, their identification and quantification in humans have not been properly reported due to resolution limitations in their analysis by reversed-phase high-performance liquid chromatography. In the present study, we report a novel method for separating these isomers using supercritical fluid chromatography. With this method, urolithin A 3- and 8-glucuronide, isourolithin A 3- and 9- glucuronide, and urolithin B 3-glucuronide (8-hydroxy urolithin 3-glucuronide; 3-hydroxy urolithin 8-glucuronide; 3-hydroxyurolithin 9-glucuronide; 9-hydroxyurolithin 3-glucuronide; and urolithin 3-glucuronide) were separated in less than 15 min. The proposed method was applied to successfully analyze these metabolites in urine samples from different volunteers belonging to different metabotypes.

Keywords: chromatographic separation; glucuronides; gut microbiota metabolites; supercritical fluid chromatography; urolithins.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of the urolithin glucuronides.
Figure 2
Figure 2
Representative SFC-UV chromatograms obtained from a mixture of urolithin glucuronide standards (100 mg/L). Chromatographic conditions are detailed in subsection SFC-UV conditions. 1: urolithin A 3-glucuronide; 2: urolithin A 8-glucuronide; 3: isourolithin A 9- glucuronide; 4: urolithin B 3-glucuronide; and 5: isourolithin A 3-glucuronide.
Figure 3
Figure 3
Representative SFC-UV chromatograms obtained from volunteers belonging to different urolithin metabotypes: (A) sample 8, metabotype A; (B) sample 7, metabotype B; and (C) sample 10, metabotype 0. Chromatographic conditions are detailed in subsection SFC-UV conditions. 1: urolithin A 3-glucuronide; 2: urolithin A 8-glucuronide; 3: isourolithin A 9- glucuronide; 4: urolithin B 3-glucuronide; and 5: isourolithin A 3-glucuronide.
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