The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection.

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.

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.

[Chromatographic studies of four water-soluble rutosides]. / Chromatographische Untersuchungen an vier wasserlöslichen Rutosiden

The water-soluble rutosides O-(beta-hydroxyethyl)-rutosides (HR), trihydroxyethylrutoside (tri-HR) and tetrahydroxyethylrutoside (tetra-HR), with and without NaCl, were separated by means of both thin-layer chromatographic and paper-chromatographic methods; separation of mono-HR was carried out using only the product with NaCl. The results of these studies show: 1. According to the procedure employed, the chromatograms of the tested rutosides showed several spots up to a maximum of 12 in each product. 2. Under certain methodical conditions the chromatograms of tri-HR and tetra-HR with and without NaCl differed; in the chromatograms of the tetra products either 12 or 9 components were detected. 7 spots were found in the chromatogram of tri-I, and 6 spots were observed in that of tri-II. The tetra-HR with NaCl was found to contain a substance which reacted with the diazonium salt fast blue salt B adopting a red coloring. This substance might be responsible for the biological effects, such as the influence exerted on yeast cell respiration. 3. At present, difficulties are still encountered in separating the substance. A comparison of its chromatograms with those of rutin, quercetin, and the bivalent phenols hydroquinone, catechol, and resorcin suggests the substance to be a mono-isomer or a di-isomer of rutin with a resorcin-like structure in the A ring. 4. Preliminary studies on yeast cell respiration using tetra-HR seem to confirm the biological effectiveness of the substance.