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.

[HPLC determination of troxerutin in plasma and urine following oral administration in man]. / HPLC-Bestimmung von Troxerutin im Plasma und Harn nach oraler Gabe am Menschen.

A high-performance liquid chromatographic method (HPLC) is described for the analysis of 3',4',7-tri-O-(beta-hydroxy-ethyl)-rutoside (troxerutin) in human plasma and urine. After separation of interfering substances on XAD-2 trihydroxyethylrutoside is converted to tetrahydroxyethylrutoside by 2-chlorethanol in alkaline medium. After HPLC-separation tetrahydroxyethylrutoside is quantified by fluorescence detection. The pharmacokinetics of troxerutin were measured in plasma after oral administration to man. The relative bioavailability of the drug from Venelbin was 97.8 +/- 37.1% compared to an aqueous standard solution.

The metabolism and excretion of 7-mono-0-(beta-hydroxyethyl) rutoside in the dog.

Following i.v. administration of mono-HR to the beagle, plasma levels of both mono-HR and its glucuronide conjugates fell rapidly, neither being detectable 8 h after injection. Following oral administration of 14C-mono-HR, mono-HR-glucuronide was detected in plasma, confirming the absorption of mono-HR, and low levels of 14C were detectable up to 72 h after dosage. Following either oral or i.v. administration of mono-HR, the major route of excretion was fecal elimination of the compound as its aglycone form. Urinary excretion was slight being less than 15% following i.v. dosage and 4% following oral administration. Metabolism of mono-HR was confined to glucuronidation and hydrolytic cleavage of the glycoside side chain. Ring fission products of mono-HR were not detected.

Spectrophotofluorometric analysis of 3',4',7-tris[O-(beta-hydroxyethyl)]rutoside in urine.

A sensitive procedure was developed for 3',4',7-tris[O(beta-hydroxyethyl)]rutoside (I) in urine. The method is based on the fluoresence behavior of the I-aluminum complex in absolute methanol. This complex has activation and emission wavelengths of 420 and 480 nm, respectively. Optimum conditions for the reaction were investigated. The fluorescence was linear (r = 0.998) in the range of 0.1-4.0 microgram of I/ml. At concentrations below 0.1 microgram/ml, a shift in the emission wavelength was observed. Replicate studies (n = 9) of spiked urine samples, each containing 0.4 microgram of I/ml, showed good precision with a relative standard deviation of 0.009. Overall percent recovery (+/- SEM) from five urine samples was 99.5 +/- 1.34%. Following a single 500-mg po dose of I to individuals, only traces of I were found in the urine. However, beta-glucuronidase treatment of urine resulted in a total cumulative urine excretion of 26.53 mg of I after 78.6 hr.

Determination of individual hydroxyethyl rutosides in various animal body fluids by thin-layer chromatography and scanning densitometry.

A method is described for the accurate determination in plasma of the beta-hydroxyethylrutosides by measurement of the fluorescence of their borocitrate complexes by scanning densitometry following separation by thin-layer chromatography. A modification is also described for estimation of individual hydroxyethylrutosides and their glucuronide conjugates in samples of bile and urine.

The disposition and metabolism of 3',4',7-tri-O-(beta-hydroxyethyl)rutoside and 7-mono-O-(beta-hydroxyethyl)rutoside in the mouse.

1. Following intravenous administration of 3',4',7-tri-O-(beta-hydroxy[14C2]ethyl)rutoside or 7-mono-O-(beta-hydroxy[14C2]ethyl)rutoside to male mice, 68% of the dose of each is excreted in faeces as the corresponding hydroxyethyl-quercetin within 72 h of dosage. Mean urinary excretions of mono- and tri-hydroxyethylrutosides in 72 h were 27 and 21% respectively. Unchanged rutosides and their glucuronides were detected in urine. 2. In biliary-cannulated animals, the mean biliary excretion of both tri- and mono-hydroxyethylrutosides was 71%, in 24 h of dosage. In both cases most 14C was excreted in 3 h, as unchanged rutosides and glucuronide conjugates. 3. Fall of blood 14C concn, was rapid for both compounds. Neither compound was detected in brain but there was short-term accumulation in liver and kidney, and 2--3 h after dosage, most 14C for both compounds was associated with the gastro-intestinal contents. 4. Animals killed 72 h after dosage of either compound contained less than 7% of dose, mostly in the colon and caecal contents. 5. Foetuses removed 3 h after dosage of either compound to the dams did not contain 14C; foetuses removed 5 min after dosage contained low levels of 14C, substantially below the maternal blood level and equiv. to less than 0-1% of dose in each case. No 14C was detected in amniotic fluid.