A validated HPLC determination of the flavone aglycone diosmetin in human plasma

Abstract

Diosmetin, 3′,5,7-trihydroxy-4′-methoxyflavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal microflora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid–liquid extraction and separated on a C₈ reversed-phase column with methanol–water–acetic acid (55:43:2, v/v/v) as the mobile phase at 43°C. Peaks were monitored at 344 nm. The method was linear in the 10–300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%. Copyright © 2004 John Wiley & Sons, Ltd.