Quantitative determination of O-(beta-hydroxyethyl)-rutosides in serum by high-performance liquid chromatography

A procedure for the quantitative determination of O-hydroxyethylated rutosides by high-performance liquid chromatography is described, which can be used for the detection of these modified flavonoids in human serum. Serum samples are processed by the addition of acetone, which removes most of the proteins. After passing the supernatant through a microcolumn of Amberlite XAD-2 and washing with water, the hydroxyethylated rutosides are eluted with methanol. The eluate is concentrated in vacuo. The methanolic solution of the residue is chromatographed on RP-8 columns using UV and fluorescence detectors. The mono- to tetrahydroxyethylated constituents and their corresponding aglycones could be separated with a step gradient, starting with a solvent system of water-methanol-acetic acid (70:30:6) followed by a mixture of water-ethanol-acetic acid (70:30:6). Alternatively, the rutosides can be separated by a linear gradient of water-acetonitrile. An almost linear calibration curve and about 80% recovery are obtained. A detection limit of 1 mg/l is achieved. Pharmacokinetic studies in human volunteers are described.