Quantification by UHPLC of total individual polyphenols in fruit juices

a do idad s a in c No. ber in av as ls i juice). This method is useful for authentication analyses and for labelling total polyphenols contents of bolites its. Se wide v with ents. T 2009). (Link, Balaguer, & Goel, 2010; Sutherland, Rahman, & Appleton, 2006; Yang, Sang, Lambert, & Mao-Jung, 2008). which is needed for good anthocyanins resolution (Obón, Díaz- García, & Castellar, 2011). The International Fruit Juice Association (IFU) does not have a polyphenol method to analyse profiles and quantify polyphenols (http://www.ifu-fruitjuice.com/ifu-meth- ods). Thus, it is crucial to have easy and powerful analytical meth- odology to measure polyphenol content of commercial fruit juices. HPLC is the preferred method for separation and quantification of individual polyphenols in fruits, using detection systems based on spectrophotometry, fluorometry, and/or mass spectrometry. The amount and diversity of polyphenols in vegetal tissues show Abbreviations: PDA-Fluo, photodiode array detector-fluorimetric detector; TIP, total individual polyphenols; TP, total polyphenols; UHPLC, ultra high-performance liquid chromatography; GAE, gallic acid equivalents; IFU, International Federation of Fruit Juice Producers; USDA, United States Department of Agriculture. ⇑ Corresponding author. Tel.: +34 968 32 55 64; fax: +34 968 32 55 55. E-mail addresses: [email protected] (M.C. Díaz-García), josemaria. [email protected] (J.M. Obón), [email protected] (M.R. Castellar), jacintacollado Food Chemistry 138 (2013) 938–949 Contents lists available at Food Che journal homepage: www.else @gmail.com (J. Collado), [email protected] (M. Alacid). Recent findings put forward the role of polyphenols in prevent- ing diseases such as chronic inflammatory diseases, some kind of cancers, cardiovascular diseases, antimicrobial and anti-cariogenic effects, or neurodegenerative diseases (Dai, Borenstein, Wu, Jackson, & Larson, 2006; De Pascual-Teresa & Sánchez-Ballesta, 2008; Pan, Lai, & Ho, 2010; Scalbert, Manach, Morand, Remesy, & Jimenez, 2005). Effects of polyphenols on health require full knowledge on their chemistry, occurrence in foods, metabolism and bioavailability, mechanisms of biological activity, or surrogate markers of health consumers and these data could be also used for epidemiological studies. Juice industry is interested in an official analytical method- ology to establish polyphenol profiles and its quantification in fruit juices. According to the CODEX-STAN 247-2005 for Fruit Juices and Nectars (http://www.codexalimentarius.net), the IFUmethod num- ber 71 (1998) type I determine anthocyanins profile for red fruit juice authentication. Anthocyanins are the polyphenol compounds responsible of the red colour of these juices. This HPLC method use very acid mobile phases of formic acid with a pH value close to 2.3, 1. Introduction Polyphenols are secondary meta tissues, as well as in flowers and fru polyphenols are known, including a contain at least one aromatic ring groups in addition to other constitu oxidants of human diet (El Gharras, 0308-8146/$ - see front matter � 2012 Elsevier Ltd. A http://dx.doi.org/10.1016/j.foodchem.2012.11.061 commercial fruit juices. � 2012 Elsevier Ltd. All rights reserved. present in all vegetal veral thousand of plant ariety of molecules that one or more hydroxyl hey are important anti- Focusing on fruit juices, consumers are highly interested in health claims of polyphenols. There is an increasing market in anti- oxidant fruit juices and formulation of juicemixtureswith high pol- yphenol concentrations. The main commercial antioxidant juices are based on the use of red fruits like bilberry, cranberry, straw- berry, cherry, raspberry, mixed with other traditional juices like or- ange, apple or pineapple. Labelling of commercial fruit juices detailing polyphenol composition would be of great interest for Fruit juices UHPLC analysis correlation (r2 = 0.966) was observed between calculated TIP, and total polyphenols (TP) determined by the well-known colorimetric Folin-Ciocalteu method. In this work, the higher TIP value corresponded to bilberry juice (607.324 mg/100 mL fruit juice) and the lower to orange juice (32.638 mg/100 mL fruit Quantification by UHPLC of total individu M.C. Díaz-García, J.M. Obón ⇑, M.R. Castellar, J. Colla Departamento de Ingeniería Química y Ambiental, E.T.S. Ingeniería Agronómica, Univers a r t i c l e i n f o Article history: Received 18 August 2012 Received in revised form 13 November 2012 Accepted 16 November 2012 Available online 29 November 2012 Keywords: Polyphenol analysis Anthocyanins a b s t r a c t The present work propose main polyphenols present improve the IFU method strawberry, American cran identifying 70 of their ma hydroxycinnamic acids, 4 fl polyphenol of each group w Total amount of polypheno ll rights reserved. l polyphenols in fruit juices , M. Alacid Politécnica de Cartagena, Paseo Alfonso XIII, 52, E-30203 Cartagena, Murcia, Spain new UHPLC-PDA-fluorescence method able to identify and quantify the ommercial fruit juices in a 28-min chromatogram. The proposed method 71 used to evaluate anthocyanins profiles of fruit juices. Fruit juices of ry, bilberry, sour cherry, black grape, orange, and apple, were analysed polyphenols (23 anthocyanins, 15 flavonols, 6 hydroxybenzoic acids, 14 anones, 2 dihydrochalcones, 4 flavan-3-ols and 2 stilbenes). One standard used to calculate individual polyphenol concentration presents in a juice. n a fruit juice was estimated as total individual polyphenols (TIP). A good SciVerse ScienceDirect mistry vier .com/locate / foodchem are important to analyse and to corroborate the identification of every compound. If some unidentified compounds are not taken Che into account for HPLC quantification, an underestimation of total polyphenol content is obtained. To simplify peak identification and quantification analysis some authors hydrolyze juice polyphe- nols before HPLC analysis. For example Mattila, Hellstrom, McDou- gall, Dobson, and Pihlava (2011) made an alkaline hydrolysis in their studies to determinate polyphenol content in European blackcurrant juices. Sometimes it is needed a pre-concentration and a purification of polyphenols from its complex matrix before instrumental analysis by HPLC (Wang, Gong, Chen, Han, & Li, 2012). In addition, different specific methods exist for analysis of juice profiles of the different polyphenol types, like anthocyanins, procy- anidins, flavanones, flavonols, flavan-3-O-ols, flavones and pheno- lic acids (Ignat, Volf, & Popa, 2011). Special emphasis is focused on a general fast HPLC method for all polyphenol analysis (De Villiers, Kalili, Malan, & Roodman, 2010; Rodríguez-Medina, Segura-Carretero, & Fernández-Gutiérrez, 2009; Valls, Millán, Martí, Borrás, & Arola, 2009) valid to quantify a total phenolic index (Tsao & Yang, 2003), but in general no quantification of the resulting compounds in juices has been done. Quantification of total phenolic in juices is usually done by colorimetric methods. These simple assays are used to determine different structural groups present in phenolic compounds. The Folin-Ciocalteu assay is widely used to measure total phenolics (Singleton, Orthofer, & Lamuela-Raventós, 1999), while the vanillin and proanthocyanidin assays are used to estimate total proantho- cyanidins (Naczk & Shahidi, 2006). These assays suffer from non- specificity, for instance the non-phenolic compounds as ascorbic acid reacts with the Folin-Ciocalteu reagent. Although, these meth- ods provide very useful qualitative and quantitative information, their main disadvantage is that they only give an estimation of the total phenolic content and do not give quantitative measurement of individual polyphenol content. An important effort has been done by databases such as Phenol- Explorer or USDA Database to compile both, total and individual polyphenol contents in foods measured by different analysis meth- ods. This information is easily accessible and very useful to stan- dardize polyphenol profiles obtained for the same food by different authors (Scalbert et al., 2011; USDA Database). The aim of this research was to present a unique, fast and reli- able UHPLC method valid to identify all kind of polyphenols and other interesting compounds like ascorbic acid (vitamin C) present in fruit juices. The principal objective was to improve HPLC IFU method number 71 (1998) with the simultaneous use of PDA and fluorescence detection. Trifluoracetic acid was used instead of for- mic acid in mobile phases. Besides, HPLC–MS was used to check the identification of doubtful compounds. The method proposed was applied to quantify the total individual polyphenols (TIP) in seven pure fruit juices. The selection of analysed fruit juices was done to cover most of compounds of polyphenol groups. This method allows obtaining TIP value valid for labelling fruit juices and a ‘‘fingerprint’’ of the tested fruit juice. This is one way to fulfill quality criteria and to check any adulteration of fruit juices. 2. Materials and methods 2.1. Chemicals the difficulty to obtain pure profiles with no peak overlapping in HPLC chromatograms. The use of different detection systems, be- sides spectrophotometry, as fluorometry and mass spectrometry M.C. Díaz-García et al. / Food Phenolic compounds classified as standard compounds used for quantification were: gallic acid (assay HPLC P99%) for hydroxybenzoic acids, p-Coumaric acid (assay HPLC P98.0%) for hydroxycinnamic acids, (+)-catechin hydrate (assay HPLC P99%) for monomeric flavan-3-ols, resveratrol (assay GCP99%) for stilb- enes, being all supplied by Sigma–Aldrich (Madrid, Spain). Antho- cyanins standard was pelargonidin 3-O-glucoside (assay HPLC P95%) (callistephin chloride), flavanones standard was hesperidin (assay HPLCP98.5%), flavonols standard was quercetin 3-O-gluco- side (assay HPLCP90%), and dihydrochalcones standard was phlo- ridzin (assay HPLCP95%), all were purchased from Extrasynthèse (Genay, France). Ascorbic acid (assayP99.7%) was purchased from Panreac Química S.A. (Barcelona, Spain). Other polyphenols used for identification purposes were flavo- nols aglycones (kaempferol, rhamnetin, isorhamnetin and myrice- tin) purchased from Sigma–Aldrich (Madrid, Spain), flavonols glycosides (kaempferol 3-O-glucoside, kaempferol 3-O-rutinoside, cynarin, quercetin 3-O-galactoside, kaempferol 7-O-glucoside, isorhamnetin 3-O-glucoside and isorhamnetin-3-O-rutinoside, purchased from Extrasynthèse (Genay, France). Quercetin 3-O- rutinoside and quercetin 3-O-rhamnoside were purchased from Sigma-Aldrich (Madrid, Spain). Hydroxybenzoic acids (3,4-dihy- droxybenzoic acid, vanillic acid, 4-hydroxybenzoic acid), hydroxy- cinnamic acids (chlorogenic acid, caffeic acid, ferulic acid, syringic acid, cinnamic acid, ellagic acid), and monomeric flavan-3-ols ((�)- epigallocatechin, (�)-epicatechin, (�)-epigallocatechin 3-gallate) were purchased from Sigma–Aldrich (Madrid, Spain). Flavanones (narirutin, naringin, didymin) were purchased from Extrasynthèse (Genay, France). Acetonitrile HPLC grade (assay 99.9%) was purchased from Pan- reac Química S.A. (Barcelona, Spain); trifluoroacetic acid for HPLC (assay 99%) and formic acid for HPLC (assay 98%) were purchased from Sigma–Aldrich (Madrid, Spain); Folin-Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Water was purified in a Milli-Q water purification system fromMillipore (Bedford, MA, USA). All other chemicals employed were of analytical grade. 2.2. Fruit samples Seven commercial fruit juices of well known polyphenol com- position have been selected for this study. Strawberry puree (Fra- garia x ananassa) with 6.8�Brix, orange juice (Citrus sinensis L. Osbeck) with 10.8�Brix, apple juice (Malus pumila P. Mill.) with 11.1�Brix, and black grape juice concentrate (Vitis vinifera L.) with 65.6�Brix, were kindly supplied by J. García Carrión S.A. (Jumilla, Spain). Bilberry juice concentrate (Vaccinium myrtillus L. wild) with 64.5�Brix, was kindly supplied by Grünewald Fruchtsaft (Stainz, Austria), and American cranberry juice concentrate (Vaccinium macrocarpon Ait.) with 62.2�Brix was supplied by Oceans Spray (Lakeville, Massachusetts, USA). Sour cherry juice concentrate (Pru- nus cerasus L.) with 60.2�Brix, was kindly supplied by Mondi Food (Rijkevorsel, Belgium). All fruit juices were frozen at �20 �C until use. Concentrated fruit juice samples (60.2–65.6�Brix) were 5 times diluted in water to obtain a similar �Brix value to non concentrated fruit juices, while purees and fruit juices (6.8–11�Brix) were not di- luted. In all cases, samples were centrifuged at 15,000g at 10 �C during 15 min in a Z383K Hermle centrifuge (Wehingen, Germany) to remove any solid residue. Supernatants were filtered with Tek- nokroma nylon filters of 0.45 lm (Barcelona, Spain) and used di- rectly for juice analysis. 2.3. UHPLC-PDA-fluorescence analysis methods Fruit juice analyses were performed in a UHPLC Agilent Tech- mistry 138 (2013) 938–949 939 nologies modular liquid chromatographic system serie 1200 (Santa Clara, CA, USA) equipped with a binary pump (G1312B), a photodi- ode array detector (PDA) with multiple wavelength (G1315C), a 200 mg/L concentration and it was injected three times with the Che fluorescence detector (G1321A), a thermostatized autosampler (G1316A) and a thermostatized automatic injector (G1329B). The two detectors were connected consecutively in the before men- tioned sequence (PDA-Fluo). For detection of compounds, the chro- matograms were recorded at 260, 280, 320, 360 and 520 nm using the photodiode detector, and 290 nm excitation and 350 nm emis- sion were the conditions used in fluorescence detector. UHPLC was run by Agilent Chem-Station for LC&LC/MS Systems. The modified UHPLC IFU method analyses use a Zorbax SB-C18, 3.5 lm, 15 cm � 4.6 mm i.d. column (Agilent, Santa Clara, CA, USA), a two-phase gradient system of formic acid/water (10/90, v/v) as mobile phase A, and formic acid/acetonitrile/water (10/50/40, v/ v) as mobile phase B. The gradient started with 12% mobile phase B at isocratic elution for 0.7 min, reaching 30% mobile phase B at 17.3 min, 100% mobile phase B at 23.3 min, at isocratic elution un- til 25.3 min. The gradient reached the initial conditions at 28.6 min, being maintained at isocratic elution for 2 min. The total flow rate was 1 mL/min, and 4 lL was the injection volume. The temperature of analysis was 25 �C. In this paper we propose a modified method with a high resolu- tion column Zorbax SB-C18, 1.8 lm, 10 cm � 4.6 mm i.d. column (Agilent, Santa Clara, CA, USA), use of two-phase gradient system of trifluoroacetic acid/water (0.5/99.5, v/v) as mobile phase A, and trifluoroacetic acid/acetonitrile/water (0.5/50/49.5, v/v) as mo- bile phase B. The gradient started with 92% mobile phase A and 8% mobile phase B, reaching 18% mobile phase B at 1.2 min, 32% mo- bile phase B at 14 min, 60% mobile phase B at 28 min, 100% mobile phase B at 34 min, at isocratic elution until 38.8 min. The gradient reached the initial conditions at 39.2 min, being maintained at iso- cratic elution for 0.8 min. The total flow rate was 1 mL/min. The temperature of analysis was 25 �C. Recorder chromatograms time was 28 min, and a typical working pressure was 290 bars. Callistephin chloride was used as internal standard, and thus two different analyses were done for each juice sample. In the first analysis the autosampler injected simultaneously 2 lL of juice sample and 2 lL of a standard callistephin solution (50 mg/L) in or- der to obtain the relative retention time of each component versus callistephin. Callistephin chloride was prepared ten times concen- trated (500 mg/l) in HCl 0,01 N, stored frozen and diluted with water before using. In a second analysis, only 2 lL of juice sample was injected to evaluate the feasible presence of callistephin in the juice. All juice samples and standards were analysed by triplicate, thus retention times and peak area of each compound calculated as the average. The polyphenols were tentatively identified according to their elution order, retention time of standard pure compounds, UV– Vis or fluorescence spectra characteristics, and comparing with the main phenolic composition of each analysed juice obtained after a deep bibliographic revision. A complete UV–Vis spectrum database of all juice components was built up, being used to asses peak identification. The quantification of polyphenol was calculated by the compar- ison between area values obtained for the components of every fruit juice analysed and the peak area of the selected standard for each polyphenol group. Standard selected from each polyphe- nol group to refer the calculations were: pelargonidin 3-O-gluco- side for anthocyanins because it is the same standard that method IFU No. 71 uses; flavonols standard was quercetin 3-O-glu- coside because it is present in the great majority of fruits studied as it indicates in the bibliography; p-Coumaric acid was selected for hydroxycinnamic acids because it is present in several of studied fruits according to the bibliography; hesperidin was the flavanone selected due to its higher presence in oranges; phloridzin was se- 940 M.C. Díaz-García et al. / Food lected for dihydrochalcones standard for their presence in apples; and, gallic acid was selected as hydroxybenzoic acids standard be- cause it is the same compound that is used in Folin-Ciocalteu method optimised to obtain its calibration curve. Standards for calibration curves were freshly prepared using as solvents water for p-coumaric acid, and gallic acid, and pure methanol for querce- tin 3-O-glucoside, hesperidin, phloridzin, (+)-catechin, and resveratrol. The calculations of the total individual polyphenols (TIP) were carried out in the following way: inside each polyphenol group, areas of each identified pick were quantified referring it to the cor- respondent standard polyphenol. Finally, the amount of all poly- phenols was added, and TIP expressed in mg/100 mL of juice. 2.4. HPLC–MS analysis method HPLC–MS analyses were carried out using an Alliance 2695 sys- tem (Waters, Milford, MA, USA) serie ZQ 4000 linked simulta- neously to PDA 2996 photodiode array detector equipped with a quaternary pump, and a thermostatized autosampler injector con- trolled by Empower 2002 software (Waters, Milford, MA, USA) for data acquisition and processing. PDA detection was set at 240–650 nm. The mass range selected was m/z 50–1000, mode ESI(+), capillary voltage was 3.5 kV and, cone voltage was 15. A Zorbax SB-C18, 5 lm, 25 cm � 4.6 mm i.d. column (Agilent, Santa Clara, CA, USA) was used for all samples. The elution gradient conditions were the same as the before mentioned modified method but the times were multiplied 5 times, because the chromatographic column has 5 times higher volume than the column used in UHPLC method. Working pressure was close to 100 bars, flow rate was 1 mL/min, and injection vol- ume was 10 lL. 2.5. Total phenolic quantification (TP) Determination of total phenolic compounds was performed by the Folin-Ciocalteu method according to the method of Skerget et al. (2005). In brief, 0.5 mL of juice sample were mixed with 2.5 mL of Folin-Ciocalteu reagent diluted 10 times with water, after that (within a time interval from 0.5 to 8 min) 2 mL of Na2CO3 (75 g/L) was added. The sample was incubated for 5 min at 50 �C and then cooled. For a blank, 0.5 mL of distilled water was used in- stead of juice. Absorbance was measured at 760 nm. Total polyphe- nol content is expressed as mg GAE (Gallic Acid Equivalents) per 100 mL fruit juice. Gallic acid was selected as polyphenol reference because it represents the main response of all the major polyphe- nol compounds in fruit and vegetables as aglycones and conjugates (quercetin and quercitrin, (+)-catechin and procyanidin mixture, and caffeic and chlorogenic acid) (George, Brat, Alter, & Amiot, 2005). Final TP value was corrected subtracting the ascorbic acid concentration determined by the UHPLC-PDA-Fluo method ex- plained before (wavelength: 243 nm), because Folin-Ciocalteu method quantifies this non-polyphenol compound. 3. Results and discussion 3.1. Optimization of UHPLC-PDA-fluorescence method for polyphenol analysis method. As for monomeric flavan-3-ols, (+)-catechin was selected to be the majority compound in the fruits; and standard stilbene was resveratrol because its presence in the black grapes is higher. Ascorbic acid was also selected as standard for the quantification of this vitamin in the fruits. Each standard was prepared up to mistry 138 (2013) 938–949 This study proposes the use of a unique standard method for the analysis of total individual polyphenol present in fruit juices by UHPLC-PDA-fluorescence. This is an alternative of IFU method IFU Che number 71 (1998) to determine all kind of polyphenols, not only anthocyanins. As formic acid used in the IFU method is involved in the HPLC equipment corrosion, the method propose the use of trifluoroacetic acid to acidify the mobile phases. Critical factors such as column type, eluent gradient or injection volumes were studied and the best results obtained compared to IFU method number 71. Bilberry juice was selected to evaluate the anthocya- nins resolution because of its high number of these compounds. A sample problem with a standard of each important polyphenol group studied was also analysed. The standard polyphenols gallic acid, (+)-catechin, p-coumaric acid, pelargonidin 3-O-glucoside, quercetin 3-O-glucoside, hesperidin, phloridzin and resveratrol were mixed and dissolved in methanol up to a concentration of 0.02 mg/mL. Detection wavelengths selected were 520 nm to determine the anthocyanins, and 280 nm to the other polyphenols mixture. Chromatograms obtained by both methods are shown in Fig. 1. Fig. 1a and b show the results obtained with IFU method, and Fig. 1c and d with the method proposed. In Fig. 1a and c we can identify the main 14 anthocyanins present in bilberry juice with Fig. 1. Chromatograms of bilberry juice and standard polyphenols analysed by M.C. Díaz-García et al. / Food the only difference that peaks number 7 and 8 in chromatogram 1a has inverted their elution order position in chromatogram 1c. If we compare Fig. 1b and d we can observe that the proposed method identifies the different polyphenol standards better than IFU method. Formic acid employed in IFU method causes base line derivation at the end of the chromatogram, however a very stable base line is observed with the use of trifluoroacetic acid. For this reason we propose the newmethod detailed in Material and Methods, because it can analyse all polyphenol components in fruit juice with an only injection and with better resolution. 3.2. Identification and quantification of polyphenols in fruit juices by UHPLC-PDA-Fluo method Seven of the most consumed fruit juices have been selected. All of them have a well known polyphenol composition. Five were red fruit juices (bilberry, American cranberry, strawberry, sour cherry and black grape) and the other two were orange and apple. These fruits were chosen because bilberry is a rich source of anthocya- nins; strawberry, sour cherry and cranberry are rich sources of flavonols and monomeric flavan-3-ols, black grape is well known to be a rich source of stilbenes, orange is a rich source of flavanon- es, and apple is the main source of dihydrochalcones. Hydroxyben- zoic and hydroxycinnamic acids are present in all studied fruits. All fruit juices have been analysed by the proposed optimised method by UHPLC-PDA-Fluo and also by HPLC–MS as reported in Section 2. The results of polyphenol UHPLC-PDA-Fluo analysis of strawberry, sour cherry, orange and apple juices are shown in Figs. 2–4, respectively. Chromatograms of American cranberry, bil- berry and black grape, are included as supplementary data. In every figure different chromatograms are shown, according to the detec- tion wavelength of the different compounds studied. Absorbance wavelength detection was 520 nm for anthocyanins identification, 360 nm for flavonols, 320 nm for hydroxycinnamic acids, 280 nm for flavanones and dihydrochalcones, and 260 nm for hydroxyben- zoic acids. Monomeric flavan-3-ols and stilbenes were detected by fluorescence with an excitationwavelength of 290 nm and an emis- sion wavelength 350 nm. These fluorescence conditions were determinate in previous studies, as a compromise between the best excitation and emission wavelengths for the main monomeric fla- van-3-ols and stilbenes, as far as only one injection per sample was done (Obón et al., 2011). Only the main components have been identified, and peaks with areas lower than 2% of total areas have not been taken into account. Peak identification was done as ex- method number 71 (1a and 1b) and by UHPLC-PDA-Fluo method (1c and 1d). mistry 138 (2013) 938–949 941 plained in Section 2. UV–Vis spectra of different polyphenols, and their UV-maximum wavelength are summarized in Obón et al. (2011), and were very useful for peak assignment. HPLC–MS data analyses were used to corroborate peak assignment. Table 1 shows the fragmentation patterns obtained for each polyphenol analysed. This table is also very useful for polyphenol assignment. Main polyphenols identified in chromatograms for each fruit juice are specified bellow. Data analysis results are also presented for American cranberry, bilberry, and black grape, although the correspondent chromatograms are offered as supplementary data. Cited references thereafter were used to evaluate peak identifications. 3.2.1. Strawberry (Fragaria x ananassa) (Fig. 2) (a) Anthocyanins: (1) cyanidin 3-O-glucoside, (2) pelargonidin 3-O-glucoside and, (3) pelargonidin 3-O-rutinoside. These results were in agreement with bibliography (Lopes da Silva, Escribano- Bailón, Pérez, Rivas-Gonzalo, & Santos-Buelga, 2007; Stintzing, Trichterborn, & Carle, 2006; Phenol-Explorer; USDA Database); (b) Flavonols: (1) Myricetin 3-O-rutinoside, (2) quercetin 3-O-rutino- side, (3) quercetin 3-O-glucuronide, (4) quercetin 3-O-gluco- side and, (5) kaempferol 3-O-glucoside. Our identification agrees with bibliography (Zheng, Wang, Wang, & Zheng, 2007; Gil, Holcroft, & Kader, 1997; Phenol-Explorer; USDA Database); (c) Table 1 MS-data of polyphenols identified from HPLC–MS analysis. Polyphenol group Compound Abbreviation [M�H]+ Base peak MSn (m/z) Anthocyanins Cyanidin 3-O-arabinoside Cy-3ara 419.25 419.25 287.22 Cyanidin 3-O-galactoside Cy-3gal 449.23 449.23 287.13 Cyanidin 3-O-glucoside Cy-3glu 449.23 449.23 287.13 Cyanidin 3-O-glucosil-rutinoside Cy-3glu-rut 757.41 757.41 611.38–287.27 Cyanidin 3-O-rutinoside Cy-3rut 595.36 595.36 287.10 Cyanidin 3-O-sophoroside Cy-3sho 647.19 647.19 287.10 Delphinidin 3-O-arabinoside Dp-3ara 435.21 435.21 303.16 Dephinidin 3-O-galactoside Dp-3gal 465.26 465.26 303.16 Delphinidin 3-O-glucoside Dp-3glu 465.26 465.26 303.16 Malvidin 3-O-arabinoside Mv-3ara 453.33 453.33 331.15 Malvidin 3-O-galactoside Mv-3gal 493.24 493.24 331.22 Malvidin 3-O-glucoside Mv-3glu 493.31 493.31 331.22 Malvidin 3-O-(6’’-acetyl-glucoside) Mv-3ac-glu 535.33 535.33 331.13 Malvidin 3-O-(6’’-p-coumaroyl-glucoside) Mv-3cou-glu 638.97 638.97 331.03 Pelargonidin 3-O-glucoside* Pg-3glu 433.02 433.02 271.10 Pelargonidin 3-O-rutinoside Pg-3rut 579.17 579.17 270.1 Peonidin 3-O-arabinoside Pn-3ara 433.10 433.10 300.90 Peonidin 3-O-galactoside Pn-3gal 463.26 463.26 301.23 Peonidin 3-O-glucoside Pn-3glu 463.26 463.26 301.16 Peonidin 3-O-rutinoside Pn-3rut 609.25 463.24 301.2 Petunidin 3-O-arabinoside Pt-3ara 449.23 449.23 317.19 Petunidin 3-O-galactoside Pt-3gal 479.28 479.28 317.12 Petunidin 3-O-glucoside Pt-3glu 479.22 479.22 317.19 Flavonols Isorhamnetin 3-O-glucoside Iso-3glu 479.02 317.18 Isorhamnetin 3-O-rutinoside Iso-3rut 625.00 317.19 479.15 Isorhamnetin 7-O-rutinoside Iso-7rut 625.00 317.19 479.15 Kaempferol 3-O-glucoside* K-glu 449.23 287.13 196.77 Kaempferol 3-O-rutinoside K-rut 595.25 287.12 Myricetin 3-O-arabinoside My-ara 451.01 319.18 Myricetin 3-O-glucoside My-glu 481.00 319.18 Myricetin 3-O-rutinoside My-rut 627.40 319.25 481.22 Quercetin 3-O-arabinoside Q-ara 435.11 303.00 Quercetin 3-O-galactoside Q-gal 465.26 303.16 Quercetin 3-O-glucoside* Q-glu 465.32 303.09 Quercetin 3-O-glucosil-rutinoside Q-glu-rut 773.30 303.23 Quercetin 3-O-glucuronide Q-glucu 479.22 303.16 Quercetin 3-O-rhamnoside Q-rha 449.43 303.16 Quercetin 3-O-rutinoside* Q-rut 465.32 303.16 Hydroxybenzoic acids 3,4-Dihydroxybenzoic acid* 3,4dHB 154.94 141.96 118.96 Ellagic acid* Elag 303.21 303.21 Gallic acid* Gal 170.88 129.9 4-Hydroxybenzoic acid* 4-HB 138.12 Syringic acid* Syr 199.17 139.8 122.7-154.8 Vanillic acid* Van 168.90 Hydroxycinnamic acids Caffeic acid* Caf 180.91 180.91 Caffeoyl glucoside Caf-glu 342.12 180.83 Caffeoyl quinic acid Caf-qui 355.51 180.89 Caftaric acid* Caftar 312.05 163.03 Chlorogenic acid* Clor 354.93 162.93 Cinnamic acid* Cin 148.86 148.86 130.77-79.94 Ferulic acid* Fer 194.76 177.01 Ferulic 4-glucoside acid Fer4glu 356.85 194.76 Neochlorogenic acid Neoclor 354.86 163.11 294.08 p-Coumaric acid* p-Cou 165.00 146.85 p-Coumaroyl glucose p-Cou-glu 327.05 165.03 p-Coumaroyl quinic acid p-Cou-qui 338.87 146.88 p-Coumaroyl tartaric acid p-Cou-tar 297.23 147.05 Sinapic acid* Sin 224.84 224.84 206.91–141.92 Flavanones Didymin* Dyd 596.38 596.38 196.12 Hesperidin* Hesp 611.24 611.24 390.86 Naringin* Naring 581.33 272.84 435.31 Narirutin* Nariru 581.18 273.06 435.06 Dihydrochalcones Phloritzin (Phloretin 20-O-glucoside)* Phlor 437.04 275.17 199.52–158.06 Phloretin 20-O-xyloside-glucoside Phlo-xyl 568.09 275.14 Flavan-3-ols (+) Catechin* Cat 290.93 138.89 165.19–146.98–123.25 (�) Epicatechin* Epi 290.89 138.85 165.19–146.98–123.25 (�) Epigallocatechin Epigal 307.22 307.22 197.23 (�) Epigallocatechin 3-gallate* Epigal-gal 459.02 459.02 288.95 Stilbenes Resveratrol* Res 228.91 228.91 Resveratrol glucoside Res-glu 228.82 228.82 * Pure compounds. 942 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 Che M.C. Díaz-García et al. / Food Hydroxycinnamic acids: (1) p-Coumaroyl glucoside, (2) caffeic acid, (3) chlorogenic acid, (4) p-Coumaric acid and, (5) cinnamic acid that it was detected in 260 nm chromatogram. Identifications were in agreement with other authors (Zheng et al., 2007, Breitfellner, Solara, & Sontag, 2003; Phenol-Explorer Database); (d) Flavanones Fig. 2. Chromatograms of strawberry juice analysed by UHPLC-PDA-Fluo method. and dihydrochalcones not found. Profiles correspond to p-Couma- royl glucose and caffeic acid (hydroxycinnamic acids), quercetin 3-O-glucuronide (flavan-3-ol) and pelargonidin 3-O-glucoside (anthocyanins); (e) Hydroxybenzoic acids: (1) gallic acid, (2) 4- hydroxybenzoic acid, (3) 3,4-dihydroxybenzoic acid, (4) syringic acid and, (5) ellagic acid. This identificationwas satisfactory accord- ing with studied references (Russell, Labat, Scobbie, Duncan, & Duthie, 2009; Zheng et al., 2007); (f) Monomeric flavan-3-ols: (1) (�)-epigallocatechin, (2) (+)-catechin and (3) (�)-epicatechin. This identification is suitable with the authors (Tsanova-Savova, Ribarova, & Gerova, 2005; Arts, Van de Putte, & Hollman, 2000, Phenol- Explorer and, USDA Database); (g) Stilbenes: assignments was: (4) resveratrol 3-O-glucoside. 3.2.2. Sour cherry (Prunus cerasus L.) (Fig. 3) (a) Anthocyanins: (1) cyaniding 3-O-sophoroside, (2) cyanidin 3-O-glucosyl-rutinoside, (3) cyanidin 3-O-glucoside, (4) cyanidin 3-O-rutinoside and, (5) peonidin 3-O-rutinoside. Peak assignments are in agreement with data from bibliography (Bonerz, Würth, & Dietrich, 2007; Chaovanalikit & Wrolstad, 2004; Goiffon, Mouly, & Gaydou, 1999; Phenol-Explorer; USDA Database); (b) Flavonols: (1) quercetin 3-O-glucosyl-rutinoside, (2) quercetin 3-O-rutino- side, (3) kaempferol 3-O-rutinoside and (4) myricetin 3-O-gluco- side. Results are in agreement with studied references (Fu et al., 2011; Ferretti, Bacchetti, Belleggia, & Neri, 2010; Chaovanalikit & Wrolstad, 2004); (c) Hydroxycinnamic acids (1) caffeoylquinic acid, (2) neochlorogenic acid, (3) p-Coumaroylquinic acid, (4) chlorogenic acid, (5) caffeic acid and, (6) ferulic acid. These results were in agreement with bibliography (Chaovanalikit & Wrolstad, 2004; Phenol-Explorer Database); (d) Flavanones and dihydrochal- cones not present (profiles correspond to hydroxycinnamic acids: neochlorogenic acid, and chlorogenic acid; and to the anthocya- nins: cyanidin 3-O-glucosyl-rutinoside, and cyanidin 3-O-rutino- side); (e) Hydroxybenzoic acids: (1) gallic acid, (2) 3,4- dihydroxybenzoic acid and (3) vanillic acid. This identification was satisfactory according with studied reference (Chaovanalikit & Wrolstad, 2004); (f) Monomeric flavan-3-ols: (1) (+)-catechin, and (2) (�)-epicatechin. Our identification agrees with bibliogra- phy (Tsanova-Savova et al., 2005; Chaovanalikit & Wrolstad, 2004; USDA Database); (g) Stilbenes: not found. 3.2.3. American cranberry (Vaccinium macrocarpon Ait.) (a) Anthocyanins: (1) cyanidin 3-O-galactoside, (2) cyanidin 3- O-arabinoside, (3) peonidin 3-O-galactoside, (4) peonidin 3-O-glu- coside and, (5) peonidin 3-O-arabinoside. These results were in agreement with bibliography (Rodríguez-Medina, Segura-Carrete- ro, & Fernández-Gutiérrez, 2009); Naczk & Shahidi, 2006; USDA Database); (b) Flavonols: (1)myricetin 3-O-arabinoside, (2) querce- tin 3-O-galactoside, (3) kaempferol 3-O-glucoside, (4) isorhamnetin 3-O-rutinoside and, (5) quercetin 3-O-rhamnoside. This identifica- tion was satisfactory according with studied references (Wang & Zuo, 2011, and Phenol-Explorer Database); (c) Hydroxycinnamic acids: (1) caffeoyl glucose, (2) p-Coumaroyl glucose, (3) caffeic acid, (4) chlorogenic acid, (5) feruloyl glucose, (6) ferulic acid, and (7) cinnamic acid. Peak assignments are in agreement with data from bibliography (Wang & Zuo, 2011); Rodríguez-Medina et al., 2009; Phenol-Explorer Database). Besides, Wang and Zuo (2011) deter- mined synapic acid at low concentrations; (d) Flavanones and dihydrochalcones not found. Peak profiles correspond to p-Couma- royl glucose (hydroxycinnamic acid), vanillic acid (hydroxybenzoic acid) and several anthocyanins; (e) Hydroxybenzoic acids: (1) 3,4- dihydroxibenzoic acid, (2) vanillic acid, and (3) ellagic acid. Our identification agrees with bibliography: Rodríguez-Medina et al. mistry 138 (2013) 938–949 943 (2009), and Phenol-Explorer Database. Wang and Zuo (2011) deter- mined presence of benzoic acid; (f) Monomeric flavan-3-ols: (1) (�)-epigallocatechin, and (2) (�)-epicatechin. This identification Che 944 M.C. Díaz-García et al. / Food is suitable with the authors Wang and Zuo (2011), Rodríguez- Medina et al. (2009); USDA Database); (g) Stilbenes no peak assignment although Wang and Zuo (2011) determined presence of resveratrol 3-glucoside, and Huang & Mazza (2011) found resveratrol in this fruit. Fig. 3. Chromatograms of sour cherry juice analysed by UHPLC-PDA-Fluo method. 3.2.4. Bilberry (Vaccinium myrtillus L. wild) (a) Anthocyanins: (1) dephinidin 3-O-galactoside, (2) delphin- idin 3-O-glucoside, (3) cyanidin 3-O-galactoside, (4) delphinidin 3-O-arabinoside, (5) cyanidin 3-O-glucoside, (6) petunidin 3-O- galactoside, (7) petunidin 3-O-glucoside, (8) cyanidin 3-O-arabinoside, (9) peonidin 3-O-galactoside, (10) petunidin 3-O-arabinoside, (11) malvidin 3-O-galactoside, (12) peonidin 3-O-glucoside, (13) mal- vidin 3-O-glucoside, and (14) malvidin 3-O-arabinoside. Results are in agreement with studied references (Rodríguez-Medina et al., 2009; Tian, Giusti, Stoner, & Schwartz, 2005; Phenol- Explorer; USDA Database); (b) Flavonols: (1) myricetin 3-O-arabi- noside, (2) quercetin 3-O-arabinoside, (3) quercetin 3-O-galactoside, (4) quercetin 3-O-glucuronide, and (5) kaempferol 3-O-rutinoside. Peak assignments are in agreement with data from bibliography (Rodríguez-Medina et al., 2009; Phenol-Explorer; USDA Data- base); (c) Hydroxycinnamic acids: (1) p-Coumaroyl glucoside, (2) caffeoyl glucose, (3) chlorogenic acid, (4) caffeic acid, (5) feru- lic acid, and (6) synapic acid. Our identification agrees with bibli- ography (Rodríguez-Medina et al., 2009; Naczk & Shahidi, 2006; Phenol-Explorer Database); (d) Flavanones and dihydrochalcones not present. Peak profiles correspond to caffeoyl glucose and chlorogenic acid (hydroxycinnamic acids) and several anthocya- nins; (e) Hydroxybenzoic acids: (1) gallic acid, (2) 3,4-dihydroxy- benzoic acid, (3) vanillic acid and, (4) syringic acid. Peak assignments are in agreement with data from bibliography (Rod- ríguez-Medina et al., 2009; Cho, Howard, Prior, & Clark, 2004); (f) Monomeric flavan-3-ols: (1) (+)-catechin, and (2) (�)-epicatechin. Results are in agreement with studied references (Tsanova-Savova et al., 2005; USDA Database); (g) Stilbenes: (3) resveratrol 3-O- glucoside. 3.2.5. Black grape (Vitis vinifera L.) (a) Anthocyanins: (1) delphinidin 3-O-glucoside, (2) cyanidin 3-O-glucoside, (3) petunidin 3-O-glucoside, (4) peonidin 3-O- glucoside, (5) malvidin 3-O-glucoside, (6) malvidin 3-O-(6’’-acetyl- glucoside), and (7) malvidin 3-O-(600-p-Coumaroyl-glucoside). Re- sults are in agreement with studied references (Gómez-Alonso, García-Romero, & Hermosín-Gutiérrez, 2007; Wu & Prior, 2005; Phenol-Explorer Database); (b) Flavonols: (1) myricetin 3-O-arabi- noside, (2) quercetin 3-O-rutinoside, (3) quercetin 3-O-glucuro- nide, and (4) isorhamnetin 3-O-glucoside. These results were in agreement with bibliography (Rodríguez-Medina et al., 2009; Gómez-Alonso et al., 2007; USDA Database). (c) Hydroxycinnamic acids: (1) caftaric acid, (2) chlorogenic acid, (3) caffeic acid, (4) p-Coumaroyltartaric acid, and (5) p-Coumaric acid. This identifica- tion is suitable with the authors (Rodríguez-Medina et al., 2009; Gruz, Novak, & Strnad, 2008, and Phenol-Explorer Database); (d) Flavanones and dihydrochalcones: not found. Peak profiles corre- spond to caftaric acid and p-Coumaroyltartaric acid (hydroxycin- namic acid) and the anthocyanin malvidin 3-O-glucoside. (e) Hydroxybenzoic acids: (1) gallic acid, (2) 4-hydroxybenzoic acid, (3) 3,4-dihydroxybenzoic acid, (4) vanillic acid, and (5) syringic acid. Results are in agreement with studied references (Rodrí- guez-Medina et al., 2009; Gruz et al., 2008; Russell et al., 2009); (f) Monomeric flavan-3-ols: (1) (�)-epigallocatechin, (2) (+)-cate- chin, (3) (�)-epigallocatechin 3-gallate, and (4) (�)-epicatechin. This identification was satisfactory according with studied refer- ences (Rodríguez-Medina et al., 2009; Tsanova-Savova et al., 2005; Phenol-Explorer; USDA Database); (g) Stilbenes: (5) resvera- trol 3-O-glucoside and (6) resveratrol. This identification is suitable with the authors (Piotrowska, Kucinska, & Murias, 2012; Guerrero, Puertas, Fernández, Palma, & Cantos-Villar, 2010). mistry 138 (2013) 938–949 3.2.6. Orange (Citrus sinensis L. Osbeck) (Fig. 4) (a) Anthocyanins: the variety of orange studied have not anthocyanins; (b) Flavonols: (1) isorhamnetin 7-rutinoside; (c) Hydroxycinnamic acids: (1) chlorogenic acid, (2) p-Coumaroyl glu- cose, (3) caffeic acid, and (4) feruloyl glucose. Peak assignments are in agreement with data from bibliography (Fu et al., 2011; Klimczak, Malecka, Szlachta, & Gliszczynska-Swiglo, 2007); (d) Flavanones: (1) narirutin, (2) naringin, (3) hesperidin, and (4) dydimin. Our identification agrees with bibliography (Naczk & Shahidi, 2006; USDA Database; Phenol-Explorer); (e) Hydroxybenzoic acids: (1) gallic acid; (f) Monomeric flavan-3-ols: (1) (�)-epigallo- catechin, (2) (+)-catechin, and (3) (�)-epicatechin; (g) Stilbenes not found. M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 945 Fig. 4. Chromatograms of orange juice analysed by UHPLC-PDA-Fluo method. Fig. 5. Chromatograms of apple juice analysed by UHPLC-PDA-Fluo method. Table 2 Quantification of polyphenols content as TIP (total individual polyphenols) of commercial fruit juices. Compound Group of polyphenols Time (minutes) Relative time (minutes) Polyphenol content calculated as TIP (mg/100 mL fruit juice) Strawberry Sour cherry American cranberry Bilberry Black grape Orange Apple (a) Ascorbic acid Vitamins 1,043 0,081 2,018 4,195 21,913 39,096 1,991 0,391 0,678 Gallic acid Hidroxybenzoic acid 2,200 0,171 6,353 6,452 16,643 9,126 3,620 4-Hydroxybenzoic acid Hidroxybenzoic acid 2,907 0,225 7,212 4,086 (�)-Epigallocatechin Monomeric flavan-3-ol 3,500 0,271 0,616 1,194 0,633 0,241 0,648 3,4-Dihydroxybenzoic acid Hidroxybenzoic acid 3,846 0,298 3,204 6,494 4,814 19,496 5,987 Caffeoyl-quinic acid Hidroxycinnamic acid 4,120 0,319 7,523 Neochlorogenic acid Hidroxycinnamic acid 4,637 0,359 101,175 Caftaric acid Hidroxycinnamic acid 5,342 0,414 20,941 Caffeoyl-glucose acid Hidroxycinnamic acid 5,512 0,427 2,151 40,371 (+)-Catechin Monomeric flavan-3-ol 5,596 0,434 6,562 2,093 12,809 3,965 4,012 1,145 p-Coumaroyl glucose acid Hidroxycinnamic acid 5,751 0,446 64,238 79,305 7,568 1,494 16,123 p-Coumaroyl quinic acid Hidroxycinnamic acid 5,790 0,449 0,512 Caffeic acid Hidroxycinnamic acid 6,896 0,535 7,535 7,757 6,086 9,973 3,442 1,443 3,416 Vanillic acid Hidroxybenzoic acid 7,310 0,567 5,858 33,176 1,714 4,177 p-Coumaroyl tartaric acid Hidroxycinnamic acid 7,508 0,582 19,852 Chlorogenic acid Hidroxycinnamic acid 7,781 0,603 3,441 35,863 0,489 97,717 6,556 4,507 77,928 Ferulic acid 4-glucoside Hidroxycinnamic acid 8,012 0,621 3,318 0,948 (�)-Epigallocatechin gallate Monomeric flavan-3-ol 8,040 0,623 0,652 2,611 Dephinidin 3-O-galactoside Anthocyanin 8,070 0,626 34,261 Delphinidin 3-O-glucoside Anthocyanin 8,845 0,686 57,410 3,424 Cyanidin 3-O-sophoroside Anthocyanin 9,885 0,766 2,873 Syringic acid Hidroxybenzoic acid 9,946 0,771 3,792 0,363 2,705 1,964 Cyanidin 3-O-glucosyl-rutinoside Anthocyanin 9,960 0,772 73,670 Delphinidin 3-O-arabinoside Anthocyanin 10,138 0,786 30,658 (�)-Epicatechin Monomeric flavan-3-ol 10,408 0,807 10,874 4,764 1,746 1,692 0,526 2,423 5,181 p-Coumaric acid Hidroxycinnamic acid 10,694 0,829 4,459 0,489 Cyanidin 3-O-glucoside Anthocyanin 10,847 0,841 0,266 1,591 48,649 1,141 Cyanidin 3-O-galactoside Anthocyanin 10,882 0,844 10,908 22,084 Petunidin 3-O-galactoside Anthocyanin 10,979 0,851 12,619 Quercetin 3-O-glucosil-rutinoside Flavonol 11,011 0,854 2,138 Ferulic acid Hidroxycinnamic acid 11,563 0,896 1,670 10,718 8,439 Myricetin 3-O-arabinoside Flavonol 11,722 0,909 5,740 1,009 1,616 Petunidin 3-O-glucoside Anthocyanin 12,106 0,938 18,662 3,033 Pelargonidin 3-O-glucoside Anthocyanin 12,900 1,000 15,131 (b) Pelargonidin 3-O-glucoside Anthocyanin 12,900 1,000 15,131 Peonidin 3-O-galactoside Anthocyanin 13,009 1,008 13,411 3,757 Ellagic acid Hidroxybenzoic acid 13,175 1,021 5,334 0,779 Myricetin 3-O-rutinoside Flavonol 13,234 1,026 1,029 Cyanidin 3-O-rutinoside Anthocyanin 13,243 1,027 28,051 Sinapic acid Hidroxycinnamic acid 13,248 1,027 0,489 Pelargonidin 3-O-rutinoside Anthocyanin 13,270 1,029 1,680 Cyanidin 3-O-arabinoside Anthocyanin 13,923 1,079 1,823 41,050 Quercetin 3-O-rutinoside Flavonol 13,947 1,081 0,441 3,818 0,154 Peonidin 3-O-glucoside Anthocyanin 14,032 1,088 20,403 19,361 2,372 Malvidin 3-O-galactoside Anthocyanin 14,172 1,099 20,272 Petunidin 3-O-arabinoside Anthocyanin 14,193 1,100 9,939 Quercetin 3-O-arabinoside Flavonol 14,489 1,123 0,925 Quercetin 3-O-glucoside Flavonol 14,841 1,150 1,364 Malvidin 3-O-glucoside Anthocyanin 15,095 1,170 43,389 12,412 Quercetin-3-O-galactoside Flavonol 15,102 1,171 15,857 4,529 Quercetin 3-O-glucuronide Flavonol 15,500 1,202 0,438 3,803 2,234 946 M .C.D íaz-G arcía et al./Food Chem istry 138 (2013) 938– 949 Pe on id in 3- O -a ra bi n os id e A n th oc ya n in 16 ,0 26 1, 24 2 9, 37 2 M al vi di n 3- O -a ra bi n os id e A n th oc ya n in 16 ,4 84 1, 27 8 10 ,0 93 R es ve ra tr ol 3- O -g lu co se St il be n e 16 ,5 21 1, 28 1 6, 85 8 2, 30 1 2, 01 7 N ar ir u ti n Fl av an on e 16 ,8 51 1, 30 6 5, 90 4 Pe on id in 3- O -r u ti n os id e A n th oc ya n in 17 ,1 45 1, 32 9 1, 03 6 Is or h am n et in 3- O -r u ti n os id e Fl av on ol 17 ,7 75 1, 37 8 1, 22 7 K ae m pf er ol 3- O -g lu co si de Fl av on ol 17 ,9 50 1, 39 1 0, 45 1 2, 92 0 N ar in gi n Fl av an on e 18 ,0 65 1, 40 0 0, 39 7 Is or h am n et in 7- O -r u ti n os id e Fl av on ol 18 ,1 32 1, 40 6 0, 32 9 Ph lo re ti n 20 O xi lo si l- ru ti n os id o D yh id ro ch al co n e 19 ,0 31 1, 47 5 0, 90 2 Is or h am n et in 3- O -g lu co si de Fl av on ol 19 ,2 46 1, 49 2 0, 20 2 Q u er ce ti n 3- O -r h am n os id e Fl av on ol 19 ,5 20 1, 51 3 2, 03 2 K ae m pf er ol 3- O -r u ti n os id e Fl av on ol 19 ,8 53 1, 53 9 0, 91 2 0, 72 2 M yr ic et in 3- O -g lu co si de Fl av on ol 20 ,3 79 1, 58 0 1, 79 6 C in n am ic ac id H id ro xy ci n n am ic ac id 20 ,5 00 1, 58 9 0, 51 3 0, 48 9 R es ve ra tr ol St il be n e 20 ,6 00 1, 59 7 5, 76 1 0, 85 6 H es pe ri di n Fl av an on e 21 ,8 03 1, 69 0 6, 67 9 M al vi di n 3- O -( 60 0 - ac et yl -g lu co si de ) A n th oc ya n in 21 ,8 17 1, 69 1 1, 09 7 Ph lo ri tz in D yh id ro ch al co n e 24 ,0 59 1, 86 5 1, 34 9 D id ym in Fl av an on e 24 ,9 70 1, 93 6 0, 64 1 M al vi di n 3- O -( 60 0 - p- co u m ar oy l- gl u co si de ) A n th oc ya n in 25 ,2 59 1, 95 8 1, 59 7 TI P va lu e 14 4, 93 3 29 6, 04 6 22 7, 95 8 60 7, 32 4 12 0, 48 1 32 ,6 38 11 4, 14 M.C. Díaz-García et al. / Food Che 3.2.7. Apple (Malus pumila P. Mill.) (Fig. 5) (a) Anthocyanins not found; (b) Flavonols not found. Peak cor- respond to the hydroxycinnamic acid chlorogenic acid; (c) Hydroxycinnamic acids: (1) chlorogenic acid, (2) p-Coumaroyl glu- cose, and (3) caffeic acid. This identification is suitable with the authors (Fu et al., 2011); Karaman, Tütem, Bas�kan, & Apak, 2010; Rodríguez-Medina et al., 2009); (d) Dihydrochalcones: (1) phlorid- zin 20-O-xyloside, and (2) phloridzin. Peak assignments are in agreement with data from bibliography (Rodríguez-Medina et al., 2009); (e) Hydroxybenzoic acids: (1) syringic acid; (f) Monomeric flavan-3-ols: (1) (�)-epigallocatechin, (2) (+)-catechin, (3) (�)-epi- gallocatechin 3-gallate, and (4) (�)-epicatechin. This identification was satisfactory according with studied references (Karaman et al., 2010; Rodríguez-Medina et al., 2009; Tsanova-Savova et al., 2005; USDA Database); (g) Stilbenes: (5) resveratrol 3-O-glucoside, and (6) resveratrol. UHPLC-PDA-Fluo separation of every kind of polyphenol in all fruit juice analysis, showed an efficient separation with no peaks overlapping of main compounds. The elution order of every poly- phenol group is approximately from minute 2–13 for hydroxyben- zoic acids; from 3 to 10 min for monomeric flavan-3-ols; from 5 to 19 min for hydroxycinnamic acids; from 8 to 25 min for anthocya- nins; from 11 to 20 min for flavonols; from 16 to 21 min for stilb- enes; from 16 to 25 min for flavanones; and from 22 to 25 min for dihydrochalcones. In total, seventy (70) different polyphenols were identified in the seven fruit analysed. All these polyphenols were quantified and its concentrations in the fruit juices were calculated as re- ported in Section 2. Results were summarized in Table 2a and 2b, where polyphenols were ordered by retention time. Table 2a in- cludes polyphenols of lower retention times than internal standard (tr = 12.9 min, pelargonidin 3-glucoside), and Table 2b those of higher retention times. Apart from polyphenols cited, ascorbic acid was detected at 243 nm. It eluted at 1.043 min. It was also quanti- fied, and included in Table 2a. Highlight the main anthocyanins of each fruit juice shown in Table 2a and b, interesting for authentication purposes. In straw- berry is pelargonidin 3-O-glucoside (15.13 mg/100 mL), in sour cherry is cyanidin 3-O-glucosyl-rutinoside (73.67 mg/100 mL), in black grape is malvidin 3-O-glucoside (12.41 mg/100 mL), in American cranberry is peonidin 3-O-glucoside (20.40 mg/ 100 mL). Regarding to bilberry anthocyanins, the mains anthocya- nins are delphinidin 3-O-glucoside (57.41 mg/100 mL), cyanidin 3- O-glucoside (48.65 mg/100 mL), and malvidin 3-O-glucoside (43.39 mg/100 mL). Total individual polyphenols (TIP) was calculated as explained in Section 2. Bilberry has the highest TIP quantity of anthocyanins (372.2 mg/100 mL) followed by sour cherry (107.2 mg/100 mL), American cranberry (55.9 mg/100 mL), black grape (25.1 mg/ 100 mL), and strawberry (17.1 mg/100 mL). Orange and apple do not have anthocyanins as they are not red fruits. About flavonols, American cranberry (27.7 mg/100 mL), bilberry (10.9 mg/100 mL), and sour cherry (8.6 mg/100 mL), have higher quantity than black grape (4.2 mg/100 mL), or strawberry (3.7 mg/100 mL). The lowest value is fromorange (0.3 mg/100 mL). In apple noflavonols has been found. Regarding to flavonols content, quercetin 3-O-galactoside with 15.85 mg/100 mL in American cranberry is the highest value determined.Bilberry andAmericancranberryhave thehighest value in hydroxybenzoic acids with 38.2 and 38.7 mg/100 mL, respec- tively. Black grape and strawberry have a similar value: 26.1 and 25.9 mg/100 mL respectively. Vanillic acid is the main hydroxyben- zoic acid in American cranberrywith a content of 33.17 mg/100 mL. A high value in hydroxycinnamic acids content was present in bil- mistry 138 (2013) 938–949 947 berry (164.5 mg/100 mL), sour cherry (154.5 mg/100 mL), and American cranberry (102.5 mg/100 mL). Apple and strawberry have lower similar values (97.4 and 80.2 mg/100 mL respectively). Black grape has a quantity of 51.6 mg/100 mL value and the lowest value was orange with 8.4 mg/100 mL. Highlight the important presence of chlorogenic acid in bilberry (97.71 mg/100 mL) and apple (77.93 mg/100 mL). Neochlorogenic acid has the major content in sour cherry (101.17 mg/100 mL). Orange is the only fruit with pres- ence of flavanones with a quantity of 13.6 mg/100 mL. Quantity in hesperidin and narirutin are 6.67 and 5.90 mg/100 mL, respectively. Apple is the only fruit where dihydrochalcones could be identify with a value of 2.2 mg/100 mL. The highest quantities in flavan-3- ols were present in strawberry (18.1 mg/100 mL), bilberry (14.5 mg/100 mL), and apple (9.6 mg/100 mL). Sour cherry, orange and black grape have a similar values: 6.8; 6.7 and 5.7 mg/100 mL, respectively. The lowest value is in American cranberry (2.9 mg/ 100 mL). Stilbenes are in black grape (8.1 mg/100 mL), bilberry (6.8 mg/100 mL), and apple (2.8 mg/100 mL). The rest of fruits have no stilbenes. The highest ascorbic acid concentration (vitamin C) 948 M.C. Díaz-García et al. / Food Che was found in bilberry (39.096 mg/100 mL). The ranking of top TIP contents of studied fruit juices was bil- berry > sour cherry > American cranberry > strawberry > black grape > apple > orange. 3.3. Correlation between total individual polyphenols (TIP) measured by UHPLC-PDA-Fluo versus total polyphenols (TP) measured by colorimetric Folin-Ciocalteu method Total Polyphenols (TP) were estimated using the Folin-Ciocalteu colorimetric method and subtracting the value of ascorbic acid amount, as explained in Section 2. Ranking of top TP content of juices was bilberry (384.504 mg GAE/100 mL) > sour cherry (244.725 mg GAE/100 mL) > American cranberry (192.537 mg GAE/100 mL) > black grape (167.479 mg GAE/100 mL) > straw- berry (140.612 mg GAE/100 mL) > apple (114.212 mg GAE/ 100 mL) > orange (84.059 mg GAE/100 mL). Fig. 6 shows the rela- tionship between values of TP (colorimetric method) and TIP (UHPLC-PDA-Fluo) obtained from fruit juice analysis. There is a good correlation between both analysis methods, and R2 value cal- culated was 0.9661. Slope is lower than unit which means that as an average TIP values are higher than TP values. These TIP values indicate that the selection of standards for quantification of each polyphenol group was good. The red fruits juices strawberry, sour cherry, American cranberry and bilberry juices had higher TIP val- ues than TP. Black grape and orange juices had higher TP than TIP values. In Apple juices both TIP and TP offered a similar value. Phenol-Explorer Database offers the following ranking of TP val- ues for whole fruit: Canada blueberry (656 mg/100gFW) > sour cherry (352 mg/100gFW) > American cranberry (315mg/100 gFW) > strawberry (289 mg/100gFW) > orange (278.59 mg/ Fig. 6. Correlation between total polyphenols content of fruit juices measured as total polyphenols (TP) by a colorimetric method, and as total individual polyphe- nols (TIP) by UHPLC-UV-Fluo method. 100gFW) > black grape (184.97 mg/100gFW) > apple (130.92 mg/ 100 mL). Although a general trend is followed difference in the ori- gin of fruit samples makes a comparison difficult. 4. Conclusions The proposed UHPLC-PDA-Fluorescence method allows to ver- ify the authenticity of fruit juices and to quantify its polyphenol composition. This method shows a better polyphenol identification regarding to the IFU method No. 71 with a chromatogram time of only 28 min. Total Individual Polyphenol (TIP) is a parameter cal- culated as the sum of the individual polyphenol content presents in each fruit juice. Quantification of the different polyphenol groups is also feasible. Use of UHPLC methods are encouraged ver- sus colorimetric methods for quantification of total polyphenols. In this research we had identified and quantified in a single analysis 70 polyphenols presents in seven fruit juices. This simple method can be extended for the polyphenols analysis of a high diversity of fruits, being of special interest for juice industry. Poly- phenols data could complete the labelling information of commer- cial antioxidant fruit juices which offer beneficial effects on consumers’ health. Acknowledgements The present work has been supported by projects and a grant from ‘‘Fundación Séneca’’ (18476/BPS/11 and 08702-PI-08) and ‘‘Ministerio de Ciencia y Tecnología’’ (AGL2007-60455). The authors thank J. García Carrión, S.A. (Jumilla, Spain) for supplying the fruit juices. References Arts, I. C., Van de Putte, B., & Hollman, P. (2000). Catechin content of foods commonly consumed in the Netherlands. Journal of Agricultural and Food Chemistry, 48, 1746–1751. Bonerz, D., Würth, K., & Dietrich, H. (2007). Analytical characterization and the impact of ageing on anthocyanin composition and degradation in juices from five sour cherry cultivars. European Food Research Technology, 224, 355–364. Breitfellner, F., Solara, S., & Sontag, G. (2003). Radiation induced chemical changes of phenolic compounds in strawberries. Radiation Physics and Chemistry, 67, 497–499. Chaovanalikit, A., & Wrolstad, R. E. (2004). Anthocyanin and polyphenolic composition of fresh and processed cherries. Journal of Food Science, 69, 73–83. Cho, M. J., Howard, L. R., Prior, R. L., & Clark, J. R. (2004). Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by high-performance liquid chromatography/mass spectrometry. Journal of the Science of Food and Agriculture, 84, 1771–1782. Dai, Q., Borenstein, A. R., Wu, Y., Jackson, J. C., & Larson, E. B. (2006). Fruit and vegetable juices and Alzheimer’s disease: The Kame Project. The American Journal of Medicine, 119(9), 751–759. De Pascual-Teresa, S., & Sánchez-Ballesta, M. T. (2008). Anthocyanins: From plant to health. A review. Phytochemistry, 7, 281–299. De Villiers, A., Kalili, K. M., Malan, M., & Roodman, J. (2010). Improving HPLC separation of polyphenols. LC GC EUROPE, 23(9), 466. El Gharras, H. (2009). Polyphenols: Food sources, properties and applications – A review. International Journal of Food Science and Technology, 44(12), 2512–2518. Ferretti, G., Bacchetti, T., Belleggia, A., & Neri, D. (2010). Cherry antioxidants: From farm to table. Review. Molecules, 15, 6993–7005. Fu, L., Xu, B., Xu, X., Gan, R., Zhang, Y., Xia, E., et al. (2011). Antioxidant capacities and total phenolic contents of 62 fruits. Food Chemistry, 129, 345–350. George, S., Brat, P., Alter, P., & Amiot, M. J. (2005). Rapid determination of polyphenols and vitamin C in plant derived products. Journal of Agricultural and Food Chemistry, 53, 1370–1373. Gil, M. I., Holcroft, D. M., & Kader, A. A. (1997). Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments. Journal of Agricultural and Food Chemistry, 45, 1662–1667. Goiffon, J. P., Mouly, P. P., & Gaydou, E. M. (1999). Anthocyanic pigment determination in red fruit juices, concentrated juices and syrups using liquid chromatography. Analytica Chimica Acta, 382, 39–50. Gómez-Alonso, S., García-Romero, E., & Hermosín-Gutiérrez, I. (2007). HPLC analysis of diverse grape and wine phenolics using direct injection and multidetection by DAD and fluorescence. Journal of Food Composition and mistry 138 (2013) 938–949 Analysis, 20, 618–626. Gruz, J., Novak, O., & Strnad, M. (2008). Rapid analysis of phenolic acids in beverages by UPLC–MS/MS. Food Chemistry, 111, 789–794. Guerrero, R. F., Puertas, B., Fernández, M. I., Palma, M., & Cantos-Villar, E. (2010). Induction of stilbenes in grapes by UV-C: Comparison of different subspecies of Vitis. Innovative Food Science and Emerging Technologies, 11, 231–238. (Accessed 01.08.12). (Accessed 01.08.12). Huang, X., & Mazza, G. (2011). Simultaneous analysis of serotonin, melatonin, piceid and resveratrol in fruits using liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1218, 3890–3899. Ignat, I., Volf, I., & Popa, V. I. (2011). A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chemistry, 126, 1821–1835. Karaman, S�., Tütem, E., Bas�kan, K. S., & Apak, R. (2010). Comparison of total antioxidant capacity and phenolic composition of some apple juices with combined HPLC–CUPRAC assay. Food Chemistry, 120, 1201–1209. Klimczak, I., Malecka, M., Szlachta, M., & Gliszczynska-Swiglo, A. (2007). Effect of storage on the content of polyphenols, vitamin C and the antioxidant activity of orange juices. Journal of Food Composition and Analysis, 20, 313–322. Link, A., Balaguer, F., & Goel, A. (2010). Cancer chemoprevention by dietary polyphenols: Promising role for epigenetics. Biochemical Pharmacology, 80(12 SI), 1771–1792. Lopes da Silva, F., Escribano-Bailón, M. T., Pérez, J. J., Rivas-Gonzalo, J. C., & Santos- Buelga, C. (2007). Anthocyanin pigments in strawberry. LWT – Food Science and Technology, 40, 374–382. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41(5), 1523–1542. Mattila, P. H., Hellstrom, J., McDougall, G., Dobson, G., & Pihlava, J. M. (2011). Polyphenol and vitamin C contents in European commercial blackcurrant juice products. Food Chemistry, 127, 1216–1223. Obón, J. M., Díaz-García, M. C., & Castellar, M. R. (2011). Red fruit juice quality and authenticity control by HPLC. Journal of Food Composition and Analysis, 6, 760–771. Pan, M. H., Lai, C. S., & Ho, C. T. (2010). Anti-inflammatory activity of natural dietary flavonoids. Food and Function, 1, 15–31. Scalbert, A., Andres-Lacueva, C., Arita, M., Kroon, P., Manach, C., Urpi-Sarda, M., et al. (2011). Databases on food phytochemicals and their health-promoting effects. Journal of Agricultural and Food Chemistry, 59(9), 4331–4348. Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin- Ciocalteu reagent. Methods in Enzymology, 299, 152–178. Skerget, M., Kotnik, P., Hadolin, M., Hras, A. R., Simonic, M., & Knez, Z. (2005). Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chemistry, 89, 191–198. Stintzing, F. C., Trichterborn, J., & Carle, R. (2006). Characterisation of anthocyanin– betalain mixtures for food colouring by chromatic and HPLC-DAD–MS analyses. Food Chemistry, 94, 296–309. Sutherland, B., Rahman, R., & Appleton, I. (2006). Mechanisms of action of green tea catechins, with a focus on ischemia-induced neurodegeneration. Review. Journal of Nutritional Biochemistry, 17, 291–306. Tian, Q., Giusti, M. M., Stoner, G. D., & Schwartz, S. J. (2005). Screening for anthocyanins using HPLC coupled to electrospray ionization tandem mass spectrometry with precursorion analysis, production analysis, common- neutral-loss analysis, and selected reaction monitoring. Journal of Chromatography, A, 1091, 72–82. Tsanova-Savova, S., Ribarova, F., & Gerova, M. (2005). (+)-Cathechin and (�)- epicatechin in Bulgarian fruits. Journal of Food Composition and Analysis, 18, 691–698. Tsao, R., & Yang, R. (2003). Optimization of a new mobile phase to know the complex and real polyphenolic composition: Towards a total phenolic index using high-performance liquid chromatography. Journal of Chromatography, A, 1018, 29–40. USDA Database for the Flavonoid Content of Selected Foods. Release 2.1. Accessed in 2012. Valls, J., Millán, S., Martí, M. P., Borrás, E., & Arola, L. (2009). Advanced separation methods of food anthocyanins, isoflavones and monomeric flavan-3-ols. Review. Journal of Chromatography, A, 1216, 7143–7172. Wang, C., & Zuo, Y. (2011). Ultrasound-assisted hydrolysis and gas chromatography- M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 949 Phenol-Explorer Database. Accessed in 2011. Piotrowska, H., Kucinska, M., & Murias, M. (2012). Biological activity of piceatannol: Leaving the shadow of resveratrol. Mutation Research, 750, 60–82. Rodríguez-Medina, I. C., Segura-Carretero, A., & Fernández-Gutiérrez, A. (2009). Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices. Journal of Chromatography, A, 1216, 4736–4744. Russell, W., Labat, A., Scobbie, L., Duncan, G., & Duthie, G. (2009). Phenolic acid content of fruits commonly consumed and locally produced in Scotland. Food Chemistry, 115, 100–104. Scalbert, A., Manach, C., Morand, C., Remesy, C., & Jimenez, L. (2005). Dietary polyphenols and the prevention of diseases. Critical Reviews in Food Science and Nutrition, 45, 287–306. mass spectrometric determination of phenolic compounds in cranberry products. Food Chemistry, 128, 562–568. Wang, L., Gong, L. H., Chen, C. J., Han, H. B., & Li, H. H. (2012). Column- chromatographic extraction and separation of polyphenols, caffeine and theanine from green tea. Food Chemistry, 131, 1539–1545. Wu, X., & Prior, R. (2005). Systematic identification and characterization of anthocyanins by HPLC–ESI-MS/MS in common fruits in the United States: Fruits and berries. Journal of Agricultural and Food Chemistry, 53, 2589– 2599. Yang, C. S., Sang, S., Lambert, J. D., & Mao-Jung, L. (2008). Bioavailability issues in studying the health effects of plant polyphenolic compounds. Molecular Nutrition & Food Research, 52, S139–S151. Zheng, Y., Wang, S. Y., Wang, C. Y., & Zheng, W. (2007). Changes in strawberry phenolics, anthocyanins, and antioxidant capacity in response to high oxygen treatments. LWT – Food Science and Technology, 40, 49–57. Quantification by UHPLC of total individual polyphenols in fruit juices 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Fruit samples 2.3 UHPLC-PDA-fluorescence analysis methods 2.4 HPLC–MS analysis method 2.5 Total phenolic quantification (TP) 3 Results and discussion 3.1 Optimization of UHPLC-PDA-fluorescence method for polyphenol analysis 3.2 Identification and quantification of polyphenols in fruit juices by UHPLC-PDA-Fluo method 3.2.1 Strawberry (Fragaria x ananassa) (Fig. 2) 3.2.2 Sour cherry (Prunus cerasus L.) (Fig. 3) 3.2.3 American cranberry (Vaccinium macrocarpon Ait.) 3.2.4 Bilberry (Vaccinium myrtillus L. wild) 3.2.5 Black grape (Vitis vinifera L.) 3.2.6 Orange (Citrus sinensis L. Osbeck) (Fig. 4) 3.2.7 Apple (Malus pumila P. Mill.) (Fig. 5) 3.3 Correlation between total individual polyphenols (TIP) measured by UHPLC-PDA-Fluo versus total polyphenols (TP) measured by colorimetric Folin-Ciocalteu method 4 Conclusions Acknowledgements References