Journal of Chromatography B, 856 (2007) 222–228 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 Gong-Jun Yang a,∗, Ping Liu a, Xi-Long Qu a, Min-Juan Xu b, Qi-Shu Qu a, Cheng-Yin Wang a, Xiao-Ya Hu a, Zhi-Yue Wang b,∗ a College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, PR China b College of Animal Science and Technology, Yangzhou University, Yangzhou 225002, PR China Received 31 January 2007; accepted 5 June 2007 Available online 12 June 2007 Abstract The method of high-performance liquid chromatography (HPLC) with UV–vis detection was used and validated for the simultaneous determi- nation 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 × 4.6 mm, 5.0 ␮m) maintained at 35.0 ◦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 ␮g/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50–350.00 ␮g/mL (troxerutin). The detection limits were 0.010–0.050 ␮g/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. © 2007 Elsevier B.V. All rights reserved. Keywords: Flavonoids; Troxerutin; Reversed-phase high-performance liquid chromatography; Rat urine; Chicken plasma 1. Introduction one or more points in the cell cycle, inhibition of DNA synthe- sis, and modulation of signal transduction pathways by altered Flavonoids are a large family of over 4000 ubiquitous sec- expression of key enzymes such as cyclooxygenases and protein ondary plant metabolites, which can be further divided into kinases. Many experimental approaches have been used in these five subclasses including flavonols, flavones, anthocyanins, cat- investigations, including use of cell lines, whole animals and, echins and flavonones [1]. Flavonoids have anti-inflammatory, in a few instances, human cancer patients [4]. For example, in anti-carcinogenic and other beneficial properties and are widely Turkey, the resin which was collected from branches of P. tere- distributed in medicinal plants, fruit juices, teas and health bev- binthus ssp. terebinthus was used as an antiseptic for bronchitis erage resulting in high human consumption [2,3]. Now a large and other respiratory and urinary system diseases because of a number of literatures have been devoted to studies describ- lot of flavonoids in resin [5]. Troxerutin shows a marked affinity ing the potential anticancer activities of flavonoids. In many for the venous wall, it may act to improve capillary function, instances, these effects can be attributed to plausible biochemi- reduce capillary fragility and reduce abnormal leakage. Appli- cal mechanisms including enhanced apoptosis, growth arrest at cations also exist for reducing the occurrence of night cramps and other circulatory problems. Its common usage is mainly in ∗ the treatment of varicose veins and haemorrhoids [6]. Corresponding authors. Tel.: +86 514 7975590x9217/7979045; Up to now, analysis of flavonoids has been accomplished fax: +86 514 7975244. E-mail addresses: [email protected], [email protected] by thin-layer chromatography [7–9], gas chromatography (G.-J. Yang), [email protected] (Z.-Y. Wang). [10,11], capillary electrophoresis [12–17], electrochemical 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.06.002 G.-J. Yang et al. / J. Chromatogr. B 856 (2007) 222–228 223 mensuration [18–20], high-performance liquid chromatography 2. Experimental (HPLC) [1,2,5,6,21–30]. Especially, HPLC was widely used to separate and analyse flavonoids. For example, Wang et al. [26] 2.1. Chemical and reagents reported the determination of flavonols (such as: myricetin, quercetin, kaempferol ) in green and black tea leaves and green Flavonoid standards (see Scheme 1), including rutin, morin, tea infusions by following HPLC method with diode array luteolin, quercetin and kaempferol, which were purchased from detection, and the mobile phase consisted of 30% acetonitrile Sigma–Aldrich. Puerarin and troxerutin (see Scheme 1)was in 0.025 M KH2PO4 buffer solution (v/v), which was adjusted kindly gifted by Yangzhou Institute of Drug Control. Ultrapure to pH 2.5 by 6.0 M HCl. Fang et al. [27] adopted the elu- water at 18.3 M resistance, used for HPLC mobile phase, was tion program to determine quercetin, kaempferol, myricetin, prepared using a Nanopure (New Haven, CT, USA) filtration rhamnetin, isorhamnetin, quercetrin, rutin, morin, galangin, system. HPLC-grade methanol was purchased from Fisher fisetin, apigenin and luteolin in red wine by reversed-phase (Nepean, ON, Canada). All other chemicals were of analytical high-performance liquid chromatography (RP-HPLC) with grade. UV–visible detector. However, to the best of our knowledge, there were few literatures to report the separation of troxerutin 2.2. Chromatography and puerarin or quercetin by RP-HPLC in previous studies. Especially, there was no way to simultaneously separate six The HPLC (Shimadzu, Kyoto, Japan) instrument was flavonoids and troxerutin in rat urine and chicken plasma. equipped with a model series LC-10 ADVP pump, DGU-12 In this paper, the main objective of this study was to develop A degasser, SCL-10 AVP system controller, Rheodyne 7725 a reversed-phase high-performance liquid chromatographic injector with a 20 ␮L loop and a SPD-10AVP UV–Vis detector. method for the simultaneous separation and determination of Separation and determination have been done on a VP-ODS six flavonoids and troxerutin in rat urine and chicken plasma column (5 ␮m particle size, 150 mm × 4.6 mm i.d. Shimadzu, after the oral administration. The proposed HPLC method was Kyoto, Japan). Data acquisition was performed on class-VP accurate, simple and efficient for simultaneous separation and software. The column was thermostatically controlled at 35 ◦C. determination of six flavonoids and troxerutin, and it can be The mobile phase was prepared by mixing water, methanol and applied to determine their content in rat urine and chicken plasma acetic acid in a ratio of 57:43:1 (v/v/v, pH 3.0) at a flow rate samples with satisfactory results. of 0.8 mL/min. The mobile phase was filtered using 0.22 ␮m Scheme 1. Molecular structures of the analytes. 224 G.-J. Yang et al. / J. Chromatogr. B 856 (2007) 222–228 membrane filter (Millipore, Milford, MA) and degassed by calibrator stock solutions, quality control stock solutions, and vacuum prior to use. The detection was set at a wavelength of unknown samples were thawed and vortexed for 30 s. Calibra- 254 nm. An aliquot of 10 ␮L injections were made for each tor and quality control solutions were freshly prepared by adding concentration under the specified chromatographic conditions. 50 ␮L of each stock solution to 450 ␮L of blank chicken plasma, then 1.0 mL of methanol was added. The mixture was vortexed 2.3. Preparation of reference standard solutions for 1.0 min, and centrifugated at 12000 rpm for 10.0 min. Five- hundred microliters of each ultrafiltrate was transferred and Individual standard stock solution of six flaovonids and trox- 10 ␮L of them was injected for analysis. erutin was prepared at a concentration of 1.0 mg/mL in methanol. Standard solutions were diluted to obtain three different con- 2.5. Calibration centrations of six flavonoids (1.0, 5.0, and 20.0 ␮g/mL) and troxerutin (5.0, 25.0, and 100.0 ␮g/mL), and were stored at Calibration curves were produced by injecting prepared rat −20 ◦C. And then the concentrations were determined for 7, urine samples and prepared chicken plasma samples that were 18 and 30 days. Data were compared with results obtained from spiked with six flavonoids concentrations (0.10, 0.50, 1.00, 5.00, freshly prepared standard solution. The change of troxerutin con- 10.00, 30.00, 50.00, 70.00 ␮g/mL) and troxerutin concentrations centration was less than 2.3% after 7 days, 4.0% after 18 days, (0.50, 2.50, 5.00, 25.00, 50.00, 150.00, 250.00, 350.00 ␮g/mL). and the change of six flavonoids concentration was less than 5% An aliquot of 10 ␮L of the resulting solution was injected into for 30 days. HPLC system, and each concentration was analyzed for five times. After determining the peak area, calibration lines of peak 2.4. Sample preparation area versus analyte concentrations were plotted. 2.4.1. Sample preparation – rat urine 2.6. Recovery Six female Wistar rats (weighing 350 ± 10 g, Experimental Animal Research Center of Medicine College, Yangzhou Uni- The relative recovery in rat urine and chicken plasma versity, China) were housed in metabolic cages for collection samples were evaluated at three concentrations 1.00, 5.00 of urine. The rats were provided standard laboratory food and and 20.00 ␮g/mL for six flavonoids and 5.00, 25.00 and water. Before being administered 200 mg/kg of six flaovonids 100.00 ␮g/mL for troxerutin. The rat urine and chicken plasma and troxerutin, the rats were fasted for 24 h but with access to were processed as described under preparation of sample solu- water. Urine samples were collected for a period of 8 h.