ISSN 1061-9348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 10, pp. 906–911. © Pleiades Publishing, Ltd., 2013. 906 1 Green tea (Camellia sinensis) originates from Chi- na and has anticancer, antiinflammatory and antioxi- dative activity. Green tea has attracted significant at- tention for its health benefits in a variety of disorders, ranging from cancer to weight loss [1]. Tea is the most consumed drink in the world after water [2]. A plant extract of tea contains quercetin as a bio- active compound [3, 4]. The estimated amounts of quercetin are in the range of 2–2.5 g/kg of the plant material. Otherwise, a quercetin (3,3',4',5,7-pen- tahydroxyflavone) belongs to a group of polyphe- nols and is the most abundant dietary flavonoid [5  8]. The structural elements of quercetin are three rings and five hydroxyl groups, which are the building blocks for other flavonoids. Several chromatographic techniques have been employed to separate, characterize, and quantify indi- vidual polyphenols in teas [9]. The different methods for monitoring the quercetin concentration in the green tea extract were described in the literature. Wang et al. [10] optimized the conditions for hydrolyzing and determining flavonols in tea leaves and tea infu- sions in an isocratic elution HPLC system. The two high resolution, gradient elution reverse-phased HPLC systems for the separation of over 30 flavonols, flavan-3-ols, and related compounds in green and black teas and their identification by diode array de- tection and electrospray mass spectrometry (MS) us- 1 The article is published in the original. ing an ion trap instrument with a MS n facility was de- scribed [11]. Analysis of the constituents of extracts of tea (Camellia sinensis L.) and Rooibos (Aspalathus lin- earis) leaves using thin-layer chromatography (TLC) was conducted by Ligor et al. [12]. Recently, Horie and Kohata [13] suggested capillary electrophoresis as a rapid technique for the estimation of those compo- nents of green tea that contribute to tea quality. The primary advantage of capillary electrophoresis is the relatively short analysis time (about 10 min compared to 30 min for HPLC). Direct analysis of infusions of green tea with electrospray ionization mass spectrom- etry operated in the negative ionization mode met with limited success in terms of the identification of prom- inent polyphenols and other minor components [14]. In the literature, there are the different methods for simultaneous monitoring of polyphenols in tea, but with a long time of analysis and complex procedure. Thus, the object was to develop and validate a simple RP-HPLC method for rapid, precise, accurate and sensitive determination of quercetin in the ethanol extracts from green tea. EXPERIMENTAL Samples and reagents. Standard quercetin sample was purchased from Merck Chemicals Ltd. (United Kingdom). Methanol was HPLC grade, while ethanol (96%) was analytical grade. Those were obtained from Sigma-Aldrich (United States). Development and Validation of a New RP-HPLC Method for Determination of Quercetin in Green Tea 1 Ivan M. Savic, Vesna D. Nikolic, Ivana M. Savic, Ljubisa B. Nikolic, and Mihajlo Z. Stankovic Faculty of Technology, University of Nis Bulevar oslobodjenja 124, Leskovac, 16000 Serbia Received February 14, 2012; in final form December 13, 2012 Abstract—Green tea (Camellia sinensis) contains quercetin as a bioactive compound. Quercetin has anti- inflammatory and anticancer effects. The aim of this paper was to develop and validate an RP-HPLC method for determination of quercetin in green tea simpler and faster than other available methods. RP- HPLC analysis was performed by isocratic elution with a flow rate of 1.0 mL/min. Pure methanol was used as a mobile phase, while the quantification was effected at 370 nm. The separation was performed at 35°C using a C 18 column (4.6 × 250 mm, 5 μm). The results showed that the peak area response is linear within the concentration range of 10–70 μg/mL (r = 0.9986). The values of LOD and LOQ were 1.2 and 4 μg/mL, respectively. For the intra-day and intra-instrument reproducibility, RSD were in the range of 0.05–0.84% and 0.89–1.55%, respectively. The results of accuracy for the different concentrations of quercetin (40, 50 and 60 μg/mL) were 101.3, 98.4, and 98.2%. The developed and validated method was successfully applied to the determination of quercetin in green tea extract. Keywords: development, validation, RP-HPLC, quercetin, green tea DOI: 10.1134/S1061934813100080 ARTICLES