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

• Published in: Analytical and bioanalytical chemistry Vol. 405; no. 25; pp. 8295 - 8310
• Main Authors: Silvestro, Luigi, Tarcomnicu, Isabela, Dulea, Constanta, Attili, Nageswara Rao B. N., Ciuca, Valentin, Peru, Dan, Rizea Savu, Simona
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2013; Springer; Springer Nature B.V
• Subjects: Amino acids; Analgesics; Analysis; Analytical Chemistry; Biochemistry; Bioflavonoids; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, Liquid - methods; Enzymes; Extraction; Flavones; Flavonoids; Flavonoids - blood; Flavonoids - metabolism; Flavonoids - urine; Food Science; Glucuronides - blood; Glucuronides - metabolism; Glucuronides - urine; Glycosides; Hemorrhoids; Human; Humans; Ionic mobility; Laboratory Medicine; Limit of Detection; Linearity; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Metabolites; Methods; Monitoring/Environmental Analysis; Pharmacokinetics; Plasma; Research Paper; Scientific imaging; Urine; United States > US; Peru; Germany; Diosmetin; Liquid chromatography; Diosmetin glucuronides; Pharmacokinetics; Differential ion-mobility mass spectrometry; Mass spectrometry
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-013-7237-y

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
C
max
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.