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  Journal of Chromatography A, 1011 (2003) 163– Fast separation and determination of tyrosol, hydroxytyrosol andother phenolic compounds in extra-virgin olive oil by capillaryzone electrophoresis with ultraviolet-diode array detection a, a b b *Matteo Bonoli , Marina Montanucci , Tullia Gallina Toschi , Giovanni Lercker a ` Dipartimento di Scienze degli Alimenti ,  Universita di Bologna ,  Via Ravennate 1020,  Cesena 47023,  Italy b ` Dipartimento di Scienze degli Alimenti ,  Universita di Bologna ,  Viale Fanin 40,  Bologna 40127,  Italy Received 6 March 2003; received in revised form 10 June 2003; accepted 16 June 2003 Abstract Olive oil is the main source of fat in the Mediterranean diet, and its consumption has been related to a low incidence of coronary heart disease and certain cancers. Recent findings demonstrate that olive oil phenolics are powerful in vitro and invivo antioxidants and display other biological activities that could partially account for the observed healthful effects of theMediterranean diet. A detailed method optimization plan was carried out to separate the most popular phenols in olive oil forfour separation parameters: buffer concentration, buffer pH, applied voltage and temperature. Consequently, an analyticalmethod capable of separating 21 different phenols and polyphenols by capillary zone electrophoresis was developed; theseparation was performed within 10 min, using a 40 cm 3 50  m m capillary, with a 45 m  M   sodium tetraborate buffer (pH9.60), at 27 kVand 30 8 C. The optimized method was applied to methanolic extracts of several Italian extra-virgin olive oilsobtained by different technologies in order to characterize and to compare their antioxidant profile. Positive correlations of phenolic compounds found by capillary zone electrophoresis (CZE) and two colorimetric indexes (total polyphenols and o -diphenols) were found and discussed. ©  2003 Elsevier B.V. All rights reserved. Keywords :   Olive oil; Food analysis; Phenols; Polyphenols; Tyrosols 1. Introduction  est in olive oil may be partly related to its uniquetaste; however, particular interest is due to itsVirgin olive oil is a fundamental ingredient of the nutritional properties. In fact, the saturated-to-unsatu-Mediterranean diet and, over the past few years, its rated fatty acid ratio and the presence of naturaldiffusion and consumption has spread remarkably antioxidants could prevent certain human diseases. Inoutside the Mediterranean basin. The growing inter- most cases, health and dietary benefits induced byconsumption of virgin olive oil are due to thesynergistic activity among the several minor con-stituents of virgin olive oil, such as vitamins ( a - and g -tocopherols and  b -carotene), phytosterols, pig- * Corresponding author. Tel.:  1 39-547-636-121; fax:  1 39-547- ments, terpenic acids, flavonoids (such as luteolin 382-348.  E  - mail address : (M. Bonoli).  and quercetin), squalene, and phenolic compounds 0021-9673/03/$ – see front matter  ©  2003 Elsevier B.V. All rights reserved.doi:10.1016/S0021-9673(03)01100-2  164  M  .  Bonoli et al .  /   J  .  Chromatogr  .  A 1011 (2003) 163–172 (phenols and polyphenols) [1–5]. In particular, phen-  2. Experimental olic compounds are strong antioxidants and are alsoresponsible for the astringency and bitterness of olive 2.1.  Reagents and chemicals oils.Hydroxytyrosol and tyrosol are the main phenolic HPCE-grade water, HPCE-grade 0.1  M   NaOH,compounds in extra-virgin olive oil. According to HPCE-grade 1  M   NaOH, HPCE-grade 0.1  M   HCl,several authors, the antioxidant activity of virgin sodium tetraborate, and HPLC-grade water wereolive oil is directly related to their concentration from Fluka (Buchs, Switzerland). All HPLC-grade[5–7]. To extract the polar fraction, namely the organic solvents were from Merck (Darmstadt, Ger-phenolic and polyphenolic compounds, a simple and many).fast methanol– n -hexane-based liquid–liquid extrac-tion is usually followed [8]. Several extraction 2.2.  Standards and samples methods of the polar fraction from virgin olive oilhave been compared in a previous study [9]. The following commercial products were used:Several methods have been developed to analyze protocatechuic acid, 3,4-dihydroxyphenylacetic acid,phenolic species in olive oil. TLC, NMR and, tyrosol, 2,3-dihydroxyphenylethanol, 4-hydroxy-especially, HPLC have been used for this purpose; phenylacetic acid, 4-hydroxybenzoic acid, caffeicthe latter technique is able to provide the phenolic acid, vanillic acid, dihydrocaffeic acid, siringic acid,profile of virgin olive oil [8,10–16]. These analytical  p -coumaric acid, ferulic acid,  o -coumaric acid, gen-techniques often need general complex sample prep- tisic acid and cinnamic acid were from Fluka. Gallicaration protocols, and HPLC analysis is time-con- acid, luteolin, taxifolin and quercetin were fromsuming. On the other hand, capillary electrophoresis Sigma (St. Louis, MO, USA); oleuropein glycoside`has proven to be a fast, valid and reliable tool for was from Extrasynthese (Genay, France).food analysis, especially for analysis of phenolic One mg/ml standard stock solutions were pre-compounds [17–20]. pared in HPLC-grade methanol. Appropriate dilu-The main advantages of the use of capillary tions (from 1 mg/ml to 0.001 mg/ml) for eight-pointelectrophoresis even in its basic mode, namely calibration curves were made. Two solutions atcapillary zone electrophoresis (CZE), for phenol different concentration (100  m g/ml and 5  m g/ml) of analysis are its high separation power, which leads to each standard were prepared to perform the re-fast and well-resolved separations of similar com- peatability study.pounds, and the possibility to use a low and specific The samples were defined as not filtered Protecteddetection wavelength that leads to high sensitivity. Denomination of Origin (PDO) ‘‘Aprutino Pescar-Despite these characteristics, CZE has been rarely ese’’ extra-virgin olive oils. The sampling wasused for the separation of phenols and polyphenols in performed at two different harvesting periods (Oc-olive oils. The first goal of this work was, therefore, tober 2001 and November 2001), in order to de-to develop the fastest and the simplest CZE method termine the effect of ripening time on the amount of that allowed to separate simultaneously hydroxy- phenolic compounds. The analyzed samples weretyrosol and tyrosol, as well as the main and the most from 95%  c v .  Dritta  and 5%  c v .  Leccino  olive fruits.common antioxidant compounds of virgin olive oil. Samples of virgin olive oil obtained by severalThe second aim of this work was to compare the processing systems were chosen, in order to investi-amount of phenolic compounds in several Italian gate the relationship between phenolic amounts andvirgin olive oil samples differing by their technolo- type of processing. The processing systems were:gy, in order to characterize and to compare their two traditional pressure systems (Pressure1Oct andantioxidant contents. Statistical correlations among Pressure2Oct for October, and Pressure1Nov andphenolic contents found by CZE and two colorimet- Pressure2Nov for November), two continuous cen-ric indexes (total polyphenols and  o -diphenols) are trifugation systems (Centrifugation1Oct andalso reported and discussed. Centrifugation2Oct for October, and   M  .  Bonoli et al .  /   J  .  Chromatogr  .  A 1011 (2003) 163  –  172  165 Centrifugation1Nov and Centrifugation2Nov for 2.5.  Spectrophotometric determination of total November) and the last one was an ‘‘ECO’’ continu-  phenols ous centrifugation system (EcoOct for October, andEcoNov for November), characterized by recycling The total phenol content of extracts was deter-of mill waste water during malaxation. mined by the Folin-Ciocalteu spectrophotometricmethod at 750 nm [22], using a gallic acid cali-bration curve. The spectrophotometric analysis was 2.3.  Synthesis of hydroxytyrosol  repeated three times for each extract ( n 5 3).Hydroxytyrosol was prepared by chemical reduc- 2.6.  Spectrophotometric determination of o -tion of 3,4-dihydroxyphenylacetic acid, according to  diphenols Baraldi et al. [21]. Briefly, to an ice-cooled andstirred slurry of LiAlH (5.12 g) in dry THF (200 According to Mateos et al. [10], 0.5 ml of  4 ml), 3,4-dihydroxyphenylacetic acid (7.6 g) was phenolic extract obtained from olive oil by liquid–added portion wise during half an hour. After the liquid extraction was dissolved in 5 ml of CH OH– 3 addition was completed, the suspension was heated water (1:1, v/v); a mixture of 4 ml of the solutionunder reflux for 6 h, cooled in an ice bath, and the and 1 ml of a 5% solution of sodium molybdatehydride excess eliminated by careful addition of dihydrate in CH CH OH–water (1:1, v/v) was 3 2 water (100 ml) and 10% HCl (100 ml). The organic shaken vigorously. After 15 min, the absorbance atlayer was separated and the aqueous acid phase was 370 nm was measured using gallic acid for theextracted with ethyl acetate (4 3 100 ml). The com- calibration curve using a glass cuvette. The spectro-bined organic extracts were dried with magnesium photometric analysis was repeated three times forsulfate and concentrated in vacuum. The oily residue each extract ( n 5 3).was chromatographed on a silica gel column (1 cmdiameter 3 20 cm height), eluting with ethyl acetate– 2.7.  Instrumentation light petroleum (b.p. 40–70 8 C) (1:1, v/v) to give3,4-dihydroxyphenylethanol (hydroxytyrosol), as a A Beckman capillary electrophoresis instrumentcolorless oil (4.6 g, 66% yield). P/ACE 5500 (Beckman Instruments, Fullerton, CA,USA), equipped with a diode array detector, wasused. Data acquisition and processing were accom- 2.4.  Liquid  – liquid extraction of phenolic from real  plished using a PC equipped with Beckman P/ACE samples  Station software. The capillary cartridge containinguncoated fused-silica tubing (50  m m I.D. 3 375  m mThe extraction was performed according to the O.D.) was supplied from Beckman. Total capillaryprocedure described by Pirisi et al. [8]. Briefly, 2 g length was 47 cm, whereas effective length wasof oil were weighed in a centrifuge tube and added 40 cm. UV detection was performed at 200 nm. Peak with 1 ml of   n -hexane and 2.0 ml of CH OH–water identification was performed by spiking the samples 3 (60:40, v/v). The mixture was stirred for 2 min in a with standard compounds and by spectral analysis.vortex apparatus, and the tube was centrifuged at3000 rev./min (30 cm diameter) for 5 min. The 2.8.  CZE conditions methanol layer was separated and the extractionrepeated twice. The extracts were combined and New capillaries were conditioned by flushing 1  M  evaporated to dryness under reduced pressure and sodium hydroxide solution (5 min), 0.1  M   sodiumlow temperature ( , 35 8 C). Samples were dissolved hydroxide (5 min), HPCE-grade water (5 min) andin 1 ml of CH OH–water (1:1, v/v) and filtered running buffer (5 min). The capillary not in use was 3 through a 0.45  m m nylon filter for capillary electro- stored in water to prevent buffer crystallization.phoresis analysis. The optimized running buffer was 45 m  M   sodium  166  M  .  Bonoli et al .  /   J  .  Chromatogr  .  A 1011 (2003) 163  –  172 tetraborate (pH 9.6), prepared by dissolving an as described previously [21]. Taxifolin was added toappropriate amount of solid salt in HPLC-grade the mixture only at the end of the optimization plan.water. The buffer was sonicated for 10 min and, These compounds were chosen because they havethen, filtered through a 0.2  m m cellulose acetate been found to be the main phenolic compounds insyringe filter (Orange Scientific, Waterloo, Belgium). virgin olive oil.Samples were injected hydrodynamically at the The optimization of the CZE method was evalu-anodic end in low pressure mode (0.5 p.s.i.) for 3 s ated in terms of resolution of five critical pairs(1 p.s.i. 5 6894.76 Pa). (tyrosol/2,3-dihydroxyphenylethanol, syringic acid/ Electrophoretic separations were carried out at ferulic acid,  p -coumaric acid/quercetin, quercetin/ positive power supply of 27 kV for 10 min, main- vanillic acid, 3,4-dihydroxyphenylacetic acid/gallictaining the capillary temperature at 30 8 C; this re- acid) and total analysis time. Coelution of othersulted in a current of   | 110  m A. Before each phenolic compounds was also verified by spikinginjection, the capillary was rinsed in high pressure technique.mode (20 p.s.i.) with 0.1  M   HCl (2 min), HPCE-grade water (2 min) and re-equilibrated with runningbuffer (2 min). After each electrophoretic cycle, the 3.2.  Effect of buffer concentration ,  pH  ,  voltage capillary was rinsed with HPCE-grade water (2 min).  and temperature on phenol migration time and  All washing steps were performed at 30 8 C. The  peak resolution running buffer was changed after three runs.All samples were injected in capillary electro- First of all, in order to define the operative modephoresis seven times ( n 5 7). to adopt, the types of electrolytes and, eventually, thesurfactants to mix were chosen. In fact, in theliterature, the most efficient operative mode toseparate phenolic compounds has been found to be 3. Results and discussion borate-based CZE, but borate–phosphate-basedmicellar electrokinetic chromatography (MEKC) 3.1.  Optimization of CZE method   methods with sodium dodecylsulfate (SDS) as micel-lar agent have been also used [23–30]. Therefore, inA detailed optimization plan was carried out order to simplify operative conditions, a borate-basedbecause specific references about separation and CZE method was developed.determination of phenols and polyphenols in virgin The applied voltage and the temperature were setolive oil by capillary electrophoresis have not been at 20 kV and 30 8 C, respectively, and the followingpublished yet [23–30]. concentrations of tetraborate were used: 20, 30, 40,Four separation parameters were investigated dur- 45, 50, 75, 100, 150 and 200 m  M  . When theing the optimization study in order to verify the tetraborate concentration was increased, it led tobehavior of the analyzed phenolic compounds: buffer longer analysis times and improved peak resolutionconcentration, buffer pH, applied voltage and tem- due to its specific complexing effect on the poly-perature during the electrophoretic run. hydroxylated species (phenols and polyphenols)A phenolic compound mixture was prepared: [29,30]. In fact, tetraborate complexes vicinal hy-tyrosol, 2,3-dihydroxyphenylethanol, oleuropein gly- droxyl groups on the polyphenol ring resulting in acoside, hydroxytyrosol, dihydrocaffeic acid, cin- new charged species, which will be electrophorizednamic acid, 4-hydroxyphenylacetic acid, gentisic by their differences in the charge-to-mass ratio.acid, syringic acid, ferulic acid, luteolin, taxifolin, However, over a concentration of 75 m  M  , several o -coumaric acid,  p -coumaric acid, quercetin, vanillic compounds coeluted and analysis time was con-acid, 4-hydroxybenzoic acid, caffeic acid, 3,4- siderably increased; in addition, over 150 m  M  ,dihydroxyphenylacetic acid, gallic acid and current problems occurred. It was found that 45 m  M  protocatechuic acid. Hydroxytyrosol was synthesized tetraborate concentration represented the best com-
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