Simultaneous determination of diosmin and diosmetin in human plasma by ion trap liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study.

Diosmetin (3',5,7-trihydroxy-4'-methoxyflavone) is the aglycone of the flavonoid glycoside diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-ramnoglucoside). Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the simultaneous determination of diosmin and diosmetin in human plasma was developed and validated. Plasma samples were incubated with beta-glucuronidase/sulphatase. The analytes were isolated by liquid-liquid extraction with tert-butyl methyl ether at pH 2, and separated on a C(18) reversed-phase column using a mixture of methanol/1% formic acid (58:42, v/v) at a flow rate of 0.5ml/min. APCI in the positive ion mode and multiple reaction monitoring (MRM) method was employed. The selected transitions for diosmin, diosmetin and the internal standard (7-ethoxycoumarin) at m/z were: 609.0-->463.0, 301.2-->286.1 and 191, respectively. A good linearity was found in the range of 0.25-500ng/ml (R(2)>0.992) for both compounds. The intra-batch assay precision (CV) for diosmin and diosmetin ranged from 1.5% to 11.2% and from 2.8% to 12.5%, respectively, and the inter-batch precision were from 5.2% to 11.5% and 8.5% to 9.8%, respectively. The accuracy was well within the acceptable range the accuracies (from -2.7% to 4.2% and -1.6% to 3.5% for diosmin and diosmetin, respectively). The mean recoveries of diosmin, diosmetin and the internal standard were 87.5%, 89.2% and 67.2%. Stability studies showed that diosmin and diosmetin were stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of diosmin in healthy volunteers following a single oral administration (Daflon).