Published: May 18, 2011 r2011 American Chemical Society 6353 dx.doi.org/10.1021/jf200625z | J. Agric. Food Chem. 2011, 59, 6353–6359 ARTICLE pubs.acs.org/JAFC A Fast Method Coupling Ultrahigh Performance Liquid Chromatography with Diode Array Detection for Flavonoid Quantification in Citrus Fruit Extracts Alexander Medina-Rem on, †,‡ Sara Tulipani, †,§ Maria Rotch  es-Ribalta, †,§ Maria De Lourdes Mata-Bilbao, † Cristina Andres-Lacueva, †,§ and Rosa M. Lamuela-Raventos* ,†,‡ † Nutrition and Food Science Department, XaRTA, INSA, Pharmacy School, University of Barcelona, Barcelona, Spain ‡ CIBER 06/003 Physiopathology of Obesity and Nutrition, (CIBEROBN), and RETICS RD06/0045/0003, Instituto de Salud Carlos III, Spain § Ingenio-CONSOLIDER program, FUN-C-FOOD, CSD2007-063, Barcelona, Spain ABSTRACT: Flavonoids are a widely distributed group of polyphenolic compounds present in an extensive range of edible plants, notably Citrus species. This article reports a rapid, optimized, and validated method for the separation and quantification of flavonoids in three Citrus fruit extracts by ultrahigh performance liquid chromatography (UHPLC) using a photodiode array detector. This new procedure allowed the simultaneous separation and quantification of 11 selected flavonoids in 5.5 min, 8.2 times faster than that by HPLC analysis. The solvent consumption for each individual analysis was also reduced almost 6.2-fold. The most abundant component in the analyzed samples was naringin (299.06544.36 mg 100 g 1 ), followed by rutin (116.60256.33 mg 100 g 1 ) and quercetin (7.78251.49 mg 100 g 1 ). Isoquercitrin was found in a lower proportion (60.0581.88 mg 100 g 1 ). The method was completely validated, providing a sensitive analysis for flavonoid detection and showing satisfactory data for all the parameters tested. This methodology is cheaper, more environmentally friendly, and easier to perform than others previously described. KEYWORDS: Citrus, flavonoids, Citrus fruit extracts, UHPLC, LC/MS/MS ’ INTRODUCTION Flavonoids are a widely distributed group of polyphenolic compounds that have been reported to act as antioxidants in various biological systems. 1 They are present in a wide range of edible plants, fruits, vegetables, teas, wines, and fruit juices, especially those from Citrus species. 2,3 Flavonoids in plants usually occur in a glycosylated form, mainly with glucose or rhamnose, but they can also be linked with galactose, arabinose, xylose, glucuronic acid, or other sugars. 1,4 Because the solid parts of Citrus fruit have a very high flavanone content, the whole fruit may contain up to 5 times as much as a glass of orange juice. 5 The amount of flavonoids present in Citrus fruit extracts varies according to the genetic background, the processing methods and maturity since immature fruits have higher amounts of polyphenols than mature fruits. 6 The concentrations also depend on the age of the plant, as the highest levels are detected in tissues showing pronounced cell divisions. 2,3,7 Because of the beneficial health effects of flavonoids present in fruits and vegetables, 8 their simultaneous identification and quantification is very important for many areas of science. Liquid chromatographic (LC) methodologies represent, to date, the most widely used approach to phenolic analysis. 9 High performance liquid chromatography (HPLC) of Citrus fruit juices shows peaks for flavanone glycosides that vary from one fruit to another. Numerous methods for the detection and quantification of flavonoid compounds in Citrus fruit have already been developed. 1013 According to Moln ar-Perl et al. 14 and Kocevar et al., 15 the number of flavonoids detected simulta- neously by most HPLC methods are separated in 45 or 50 min. However, Chen et al. 16 have developed a rapid ultrahigh performance liquid chromatography (UHPLC) method for the simultaneous determination of flavonoids in different species of Epimedium using a 12 min gradient elution. Sp  acil et al. 17 compared a conventional HPLC system and a UHPLC system, equipped with columns containing similar stationary phases, in the analysis of wine and tea samples. While both analytical methods gave good results, the UHPLC system appeared to be superior. UHPLC methods not only are faster, more sensitive, and more efficient but also are more reliable and ecological. 17 This article reports a rapid, optimized, and validated method for the separation and quantification of flavonoids in Citrus fruit extracts by UHPLC using a photodiode array (PDA) detector with a very rapid sample preparation in order to decrease the time and cost of sample analysis; its many advantages include high resolution, speed, an exceptionally small sample volume, and short running time for the separation. 18 This method was applied in three Citrus fruits extracts to characterize the flavonoids with an efficient separation. ’ MATERIALS AND METHODS Standards and Reagents. All samples and standards were handled with no exposure to light. Naringin (naringenin-7-O-rhamnoglucoside), Received: February 14, 2011 Accepted: May 18, 2011 Revised: May 16, 2011