Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography–mass spectrometry and ion mobility mass spectrometry

Abstract

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean Cmax of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Metabolic Disposition of Luteolin Is Mediated by the Interplay of UDP-Glucuronosyltransferases and Catechol-O-Methyltransferases in Rats

“…Dio-39-G, the major form of diosmetin that circulates in human plasma, has been reported previously (Silvestro et al, 2013), but little is known of chrysoeriol metabolism. In our study, Chr-49-G and Dio-39-G exhibited higher plasma exposure than Chr-7-G and Dio-7-G (P , 0.05) after oral administration of luteolin in rats.…”

Citrus Flavones: An Update on Sources, Biological Functions, and Health Promoting Properties

“…After oral administration, intestinal microflora enzymes rapidly hydrolyze diosmin into its aglycone, diosmetin, which is then absorbed through the intestinal wall. At systemic circulation level, diosmetin is enzymatically esterified to its relevant metabolite, 3-O-glucuronide, which is further esterified into 3,7-di-O-glucuronide [47,48]. Experimental evidence has shown that diosmin has the ability to inhibit P-glycoprotein [45] and several cytochrome P450 (CYP) enzyme isoforms (CYP1A1, CYP1A2, CYP2B1, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP3A5).…”

The role of rutin and diosmin, two citrus polyhydroxyflavones in disease prevention and treatment

“…In comparison, the quercetin molecule contains five hydroxyl groups without any methoxy group (Figure 1). Pharmacokinetic studies demonstrated that diosmin was rapidly absorbed, followed by hydrolysis through intestinal enzymes to release the aglycone diosmetin after an oral administration of diosmin (Cova et al, 1992;Silvestro et al, 2013). Diosmetin is then rapidly absorbed with a half-life in plasma ranging from 26 to 43 hours in humans (Cova et al, 1992).…”

“…After intestinal absorption and biotransformation, metabolites are eliminated in the urine, mainly as glucuronic acid conjugates. Diosmetin-3-Oglucuronide was identified as the major circulating metabolite of diosmetin in plasma and in urine (Silvestro et al, 2013).…”