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Simultaneous determination of the flavonoid aglycones diosmetin and hesperetin in human plasma and urine by a validated GC/MS method: in vivo metabolic reduction of diosmetin to hesperetin.

Diosmetin and hesperetin are the aglycones of the flavonoid glycosides diosmin and hesperidin which occur naturally in citrus fruit. A GC/MS method for the simultaneous determination of diosmetin and hesperetin in human plasma and urine has been developed and validated. The method was linear in the 2-300 ng/mL concentration range for both diosmetin and hesperetin in plasma and urine (r > 0.999). The precision of the method was better than 6.01 and 7.16% for diosmetin and hesperetin, respectively, and the accuracy was 96.76-100.40% and 95.00-105.50% for diosmetin and hesperetin, respectively. The lower limit of quantitation was found to be 2 ng/mL for both analytes in plasma and urine. Recovery of diosmetin, hesperetin and internal standard naringenin was greater than 82.5%. The method has been applied for the determination of diosmetin and hesperetin in plasma and urine samples obtained from a healthy male subject following a single oral 1000 mg dose of the flavonoid glycoside diosmin. The presence of hesperetin in plasma and urine samples indicates the metabolic reduction of diosmetin to its flavanone analogue hesperetin through reduction of the 2,3 double bond of the C-ring by the enzymes of bacteria of the intestinal microflora.