J. Sep. Sci. 2007, 30, 2493 – 2500 J. M. Herrero-Martnez et al. 2493 Jos Manuel Herrero-Martnez Original Paper Fadoua Z. Oumada Mart Ross Elisabeth Bosch Determination of flavonoid aglycones in several Clara Rfols food samples by mixed micellar electrokinetic Departament de Qumica chromatography Analtica, Universitat de Barcelona, Barcelona, Spain The application of mixed micellar electrokinetic chromatography to the separation of ten flavonoid aglycones (catechin, epicatechin, naringenin, morin, fisetin, querce- tin, kaempferol, galangin, apigenin, and chrysin) belonging to four different classes (flavanols, flavanones, flavonols, and flavones), and expected to be prominent in commonly consumed foods, has been developed. A micellar system composed of 25 mM SDS and 25 mM sodium cholate buffered at pH 7.0 provided a simultaneous separation of all compounds in less than 20 min. The procedure could be easily adapted to the determination of some flavonoids from each of these classes in real complex samples (propolis, Ginkgo biloba, etc.). The LODs of these compounds were in the range of 1.2–4 lg/mL, and the peak area and migration time repeatabilities were below 6.0 and 3.1%, respectively. Keywords: Flavonoids / Food analysis / MEKC / Mixed micelles / Received: March 26, 2007; revised: April 18, 2007; accepted: April 22, 2007 DOI 10.1002/jssc.200700124 1 Introduction ual flavonoid glycosides in foods would be desirable, however, most of the reference compounds are not com- Flavonoids are polyphenolic natural products found pre- mercially available. Additionally, differences in flavo- dominantly in plants, comprising an important part of noid glycosides composition from one plant to another our daily diet [1, 2]. These compounds have structures are present, despite these compounds derive only from a based on 2-phenylbenzopyrone (Fig. 1), and differ in their few aglycones. In order to facilitate their study, the con- pattern of hydroxylation, methylation, and glycosila- version of glycosides into aglycones by acid hydrolysis tion, in the degree of unsaturation and in the type and has been described as a practical method for the quanti- position of sugar links [2]. Flavonoids occur in free state tative determination of flavonoids [8–10]. or as aglycones and glycosides. They have been mainly Analysis of flavonoids has been accomplished usually studied due to their antioxidant and free radical scaveng- by HPLC [11, 12]. Nevertheless, most of the chromato- ing activity [3, 4]. Their increasing interest lies in their graphic methods reported in the literature for a simulta- broad pharmacological activities (e.g., antimicrobial, neous measure of the different classes of prominent food spasmolytic, antiallergic, anti-inflammatory, antiviral, flavonoids as their aglycones [13–17] require long anal- anticarcinogenic) [5–7]. Given their wide range of biolog- ysis times, even some works [9, 10, 14] have required two ical functions, there is a growing concern in isolating different chromatographic procedures to separate these and separating the flavonoids from natural sources. The compounds. number of flavonoids present in medicinal plants and Among the modern separation techniques, CE has food products is large and their analysis becomes com- won increasing acclaim for its extremely high efficiency, plex owing to their existence mostly in O-glycosidic small sample volume, high speed, and good resolution. forms. Although a quantitative determination of individ- CE has been applied to the separation of flavonoids since 1991 [18–40]. Nevertheless, many works [25, 31–37] have Correspondence: Dr. Jos Manuel Herrero-Martnez, Departa- focused on the potential of this technique in the meas- ment de Qumica Analtica, Universitat de Barcelona, Av. Diago- urement of the common flavonoids (single or a few sub- nal 647, Barcelona 08028, Spain classes) present in a particular food, and they have paid a E-mail: [email protected], [email protected] little attention to the potential in the characterization of Fax: +34-934021233 the different classes of flavonoids in several matrix sam- Abbreviations: BHT, butylated hydroxytoluene; SC, sodium ples. The analysis of flavonoid glycosides has been cholate addressed by using CZE [19, 21–23, 26] or MEKC [18, 20, i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 2494 J. M. Herrero-Martnez et al. J. Sep. Sci. 2007, 30, 2493 – 2500 Figure 1. Molecular structures of studied fla- vonoid aglycones. 21, 24, 27, 40]. However, analysis of flavonoid aglycones In this work, we developed an analytical method capa- by these techniques has not been studied to the same ble of separating several classes of flavonoid aglycones extent, and only few works on CZE [38] and MEKC [20, (flavanols, flavanones, flavonols, and flavones) com- 28–30, 40] analyses on the flavonoid aglycones have monly present in food samples by mixed MEKC. Within been published. Some of these MEKC procedures have each class, several flavonoids were chosen according to employed organic solvents [28–30, 40] in addition to the their biological interest and abundance in commonly surfactant (usually SDS), as a common additive in order consumed foods. The optimized method was successfully to enhance the separation and selectivity of different applied to the analysis of complex extracts of natural classes of flavonoids in MEKC. Compounds such as flavo- and food products containing several flavonoid agly- noids strongly interact with micelles and consequently cones from each of these classes. selectivity may be varied by modifying the micellar phase. Mixing surfactants with different structural and polar properties to generate mixed micelles has the 2 Materials and methods potential to provide variable micellar phases. Thus, by 2.1 Instrumentation tailoring the micellar environment, solute–micelle interactions can be manipulated to generate the desired MEKC experiments were carried out with a Beckman selectivity [41–44]. Recently, we have reported the suc- instrument P/ACE 5500 (Palo Alto, CA, USA) equipped cessful application of mixed micellar systems to the sep- with a diode array spectrophotometric detector. The sep- aration of procyanidins in food samples [45, 46]. arations were performed at 258C on an uncoated fused- i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2007, 30, 2493 – 2500 Electrodriven Separation 2495 silica capillary (47.0 cm650 lmid6375 lm od) ether layer was separated and evaporated to dryness obtained from Polymicro Technologies (Phoenix, USA). using a rotavapor. The dry residue was dissolved in (0.5 mL) methanol and injected into the CE system. The extraction protocol followed for orange (pulp and 2.2 Reagents and solutions peel) was based on the work of Careri et al. [48]. About All chemicals were of analytical reagent grade unless oth- 3.5 g of orange material was extracted with 4 mL of erwise noted. Sodium dihydrogenphosphate and hydro- methanol, 1 mL of 12 M hydrochloric acid, and 12 mg of genphosphate, hydrochloric acid, sodium hydroxide, BHT, as an antioxidant, in order to obtain a mixture con- diethyl ether, and butylated hydroxytoluene (BHT) were sisting of 1.5 M HCl in a methanol–water solution from Merck (Darmstadt, Germany). SDS from Merck and (50:50, v/v) containing 1500 mg/L BHT. This mixture was sodium cholate (SC) from Fluka (Buchs, Switzerland) mixed and refluxed at 908C for 60 min. After cooling, were used as surfactants. HPLC-grade methanol and ACN methanol was added to a final volume of 10 mL. The were from Merck. All solutions were prepared with mean recovery of flavanones was in the 98 l 2% range, deionized water (Milli-Q deionizer, Millipore, Bedford, while quercetin yielded a recovery higher than 85%. MA, USA). (+)-Catechin hydrate, (–)-epicatechin, querce- All sample solutions were kept at –208C and protected tin dihydrate, kaempferol, and apigenin were obtained from light until their use. from Fluka. Morin, fisetin, chrysin, galangin, and narin- genin were purchased from Sigma (St Louis, MO, USA). 2.4 MEKC procedure Stock solutions (1 mg/mL) of catechin, epicatechin, morin, fisetin, quercetin, kaempferol, and galangin were Before first use, a new capillary was conditioned at 258Cas prepared in methanol. Apigenin and chrysin stock solu- follows: 10 min with 1 M NaOH, 10 min with 0.1 M NaOH, tions were prepared in a methanol–ACN mixture (70:30, 10 min with water, and finally 30 min with the running v/v). All stock solutions were stored at –208C until their buffer. Between runs, the capillary was rinsed with 0.1 M use and protected from daylight. Prior to injection, work- NaOH (1 min), water (1 min), and running buffer (3 min). ing solutions were daily prepared by dilution of stock sol- At the end of each working session, the capillary was utions with methanol. rinsed with deionized water for 10 min. Standards and samples were injected hydrodynamically at 0.5 psi (1 psi = 6894.76 Pa) for 5 s. Before injection, all solutions 2.3 Sample preparation were filtered through a 0.45 lm pore size nylon filter Wine and orange samples were purchased at a local (Whatman, Maidstone, Kent, UK). Detection was carried supermarket, while propolis and Ginkgo biloba were out at 214, 270, 295, and 350 nm. Unless otherwise indi- obtained from a local pharmacy. Different extraction cated, the applied voltage was 20 kV of positive polarity. procedures were carried out according to the matrix The electrophoretic mobility le and the resolution fac- sample. Propolis samples were treated according to the tor Rs were calculated using the following expressions, protocol reported by Cao et al. [37]. About 2 g of raw prop- respectively: olis was extracted with 15 mL of methanol for 10 min in LdLt 1 1 an ultrasonic bath. After centrifugation at 3000 rpm6 l ¼ À e V t t 15 min, the resultant supernatant solution was put in a m eof volumetric flask of 50 mL. The extraction procedure was 2ðtm2 À tm1 Þ Rs ¼ repeated three times.