44E-7 |
Development of a rapid and sensitive method for extraction and quantification of diterpenes by RP-HPLC |
E. CHEN, B. Stankovic, and W. Zhou. Wisconsin Center for Space Automation & Robotics, Univ. of Wisconsin, Madison, 1415 Engineering Dr., Madison, WI 53706 Ginkgolides are diterpenes with pharmacological properties that are accumulated in the leaves and roots of the ginkgo tree (Ginkgo biloba L.). Their pharmacological properties are related to the prevention of cardiovascular disease and stroke, and enhancement of memory. Analysis of the ginkgolides (and of the closely related sesquiterpenoid bilobalide) is difficult due to their poor UV absorbance, difficulties in obtaining efficient extraction from the tissue matrix, low sensitivity and poor reproducibility of quantitation. In the absence of a widely accepted procedure for reproducible detection of these compounds, the comparison of different batches of leaves and standardization of extracts are difficult to achieve. The objectives of the present work were to establish a simple and effective extraction method for ginkgolides based on pH-dependent diterpene ionization, and to develop a highly sensitive method for analysis of extracted diterpenes using RP-HPLC. Extraction of diterpenes was performed in three steps, using H2O, Na2HPO4 buffer (pH 8.0) and ethyl acetate. The extract was dried, the residue was dissolved in methanol, filtered, and a 10 mL sample was analyzed by HPLC. Liquid chromatography (Hitachi D-7000) coupled with a refractive index detector was employed for analysis. A Spherisorb C18 column was used for separation, carried out isocratically at 35oC with water-methanol (70.5:29.5, v/v) Five diterpenes and bilobalide were quantitatively extracted using this method, from both ginkgo leaves and from commercially available ginkgo extracts. The recovery rate was 98-101% with RSD 2.2-3.8%. The diterpenes and bilobalide were successfully resolved using RP-HPLC. The detection limit of diterpenes was 0.05-0.1 mg, with a linear range of 0.1-12 mg. This detection limit represents a 10-fold improvement over previously reported methods, suggesting that rapid extraction of ginkgolides based on pH-dependent diterpene ionization, in conjunction with RP-HPLC, is a viable technique for routine quantification of diterpenes in biological samples.
Session 44E, Nutraceuticals & Functional Foods
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