Simultaneous determination of benzophenones and gentisein in Hypericum annulatum Moris by high-performance liquid chromatography

The content of the benzophenones, hypericophenonoside, neoannulatophenonoside, annulatophenonoside, annulatophenone, acetylannulatophenonoside and the xanthone derivative gentisein have been determined in aerial parts, leaves, flowers and stems of Hypericum annulatum Moris. Extraction of samples with methanol by magnetic stirring at room temperature allowed a good recovery of analytes (from 90.70% for gentisein to 103.81% for annulatophenonoside) and the precision of the entire procedure was < 6.05%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 290 nm. The mobile phase comprised methanol and 20 mm potassium dihydrogen phosphate (adjusted to a pH of 3.19 with o-phosphoric acid), and gradient elution mode was applied. The detection limits were 0.03, 0.02 and 0.001 microg/mL for hypericophenonoside, acetylannulatophenonoside and gentisein, respectively. The total amounts of the phenolic compounds assayed ranged from 10.92 mg/g in stems to 82.86 mg/g in leaves. Hypericophenonoside was the dominant benzophenone present in the majority of the plant samples, being present in amounts between 7.54 +/- 0.25 mg/g in stems and 64.22 +/- 2.44 mg/g in leaves. Hypericophenonoside accounted for up to 77.50% of the components found in the leaves, whereas annulatophenonoside (6.29 +/- 0.15 mg/g) and acetylannulatophenonoside (8.95 +/- 0.09 mg/g) were detected in much lower quantities. In contrast to leaves, flowers showed a tendency towards higher contents of gentisein (9.35 +/- 0.07 mg/g) and neoannulatophenonoside (4.72 +/- 0.04 mg/g) than the other parts assayed.