Quantification by UHPLC of total individual polyphenols in fruit juices

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Food Chemistry 138 (2013) 938–949 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Quantification by UHPLC of total individual polyphenols in fruit juices M.C. Díaz-García, J.M. Obón ⇑, M.R. Castellar, J. Collado, M. Alacid Departamento de Ingeniería Química y Ambiental, E.T.S. Ingeniería Agronómica, Universidad Politécnica de Cartagena, Paseo Alfonso XIII, 52, E-30203 Cartagena, Murcia, Spain 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 Fruit juices UHPLC analysis a b s t r a c t The present work proposes a new UHPLC-PDA-fluorescence method able to identify and quantify the main polyphenols present in commercial fruit juices in a 28-min chromatogram. The proposed method improve the IFU method No. 71 used to evaluate anthocyanins profiles of fruit juices. Fruit juices of strawberry, American cranberry, bilberry, sour cherry, black grape, orange, and apple, were analysed identifying 70 of their main polyphenols (23 anthocyanins, 15 flavonols, 6 hydroxybenzoic acids, 14 hydroxycinnamic acids, 4 flavanones, 2 dihydrochalcones, 4 flavan-3-ols and 2 stilbenes). One standard polyphenol of each group was used to calculate individual polyphenol concentration presents in a juice. Total amount of polyphenols in a fruit juice was estimated as total individual polyphenols (TIP). A good 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 juice). This method is useful for authentication analyses and for labelling total polyphenols contents of commercial fruit juices. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Polyphenols are secondary metabolites present in all vegetal tissues, as well as in flowers and fruits. Several thousand of plant polyphenols are known, including a wide variety of molecules that contain at least one aromatic ring with one or more hydroxyl groups in addition to other constituents. They are important antioxidants of human diet (El Gharras, 2009). Recent findings put forward the role of polyphenols in preventing 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 (Link, Balaguer, & Goel, 2010; Sutherland, Rahman, & Appleton, 2006; Yang, Sang, Lambert, & Mao-Jung, 2008). 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: mcdiazgarcia@hotmail.com (M.C. Díaz-García), josemaria. obon@upct.es (J.M. Obón), rosario.castellar@upct.es (M.R. Castellar), jacintacollado @gmail.com (J. Collado), mercedes.alacid@upct.es (M. Alacid). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.061 Focusing on fruit juices, consumers are highly interested in health claims of polyphenols. There is an increasing market in antioxidant fruit juices and formulation of juice mixtures with high polyphenol concentrations. The main commercial antioxidant juices are based on the use of red fruits like bilberry, cranberry, strawberry, cherry, raspberry, mixed with other traditional juices like orange, apple or pineapple. Labelling of commercial fruit juices detailing polyphenol composition would be of great interest for consumers and these data could be also used for epidemiological studies. Juice industry is interested in an official analytical methodology 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 IFU method number 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, which is needed for good anthocyanins resolution (Obón, DíazGarcía, & 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-methods). Thus, it is crucial to have easy and powerful analytical methodology 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 Author's personal copy M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 the difficulty to obtain pure profiles with no peak overlapping in HPLC chromatograms. The use of different detection systems, besides spectrophotometry, as fluorometry and mass spectrometry are important to analyse and to corroborate the identification of every compound. If some unidentified compounds are not taken into account for HPLC quantification, an underestimation of total polyphenol content is obtained. To simplify peak identification and quantification analysis some authors hydrolyze juice polyphenols before HPLC analysis. For example Mattila, Hellstrom, McDougall, 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, procyanidins, flavanones, flavonols, flavan-3-O-ols, flavones and phenolic 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 proanthocyanidins (Naczk & Shahidi, 2006). These assays suffer from nonspecificity, for instance the non-phenolic compounds as ascorbic acid reacts with the Folin-Ciocalteu reagent. Although, these methods 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 PhenolExplorer or USDA Database to compile both, total and individual polyphenol contents in foods measured by different analysis methods. This information is easily accessible and very useful to standardize 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 reliable 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 formic 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. 939 hydroxybenzoic acids, p-Coumaric acid (assay HPLC P98.0%) for hydroxycinnamic acids, (+)-catechin hydrate (assay HPLC P99%) for monomeric flavan-3-ols, resveratrol (assay GC P99%) for stilbenes, being all supplied by Sigma–Aldrich (Madrid, Spain). Anthocyanins standard was pelargonidin 3-O-glucoside (assay HPLC P95%) (callistephin chloride), flavanones standard was hesperidin (assay HPLC P98.5%), flavonols standard was quercetin 3-O-glucoside (assay HPLC P90%), and dihydrochalcones standard was phloridzin (assay HPLC P95%), all were purchased from Extrasynthèse (Genay, France). Ascorbic acid (assay P99.7%) was purchased from Panreac Química S.A. (Barcelona, Spain). Other polyphenols used for identification purposes were flavonols aglycones (kaempferol, rhamnetin, isorhamnetin and myricetin) 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-Orutinoside and quercetin 3-O-rhamnoside were purchased from Sigma-Aldrich (Madrid, Spain). Hydroxybenzoic acids (3,4-dihydroxybenzoic acid, vanillic acid, 4-hydroxybenzoic acid), hydroxycinnamic 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 Panreac 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 from Millipore (Bedford, MA, USA). All other chemicals employed were of analytical grade. 2.2. Fruit samples Seven commercial fruit juices of well known polyphenol composition have been selected for this study. Strawberry puree (Fragaria 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 (Prunus 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 diluted. 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 Teknokroma nylon filters of 0.45 lm (Barcelona, Spain) and used directly for juice analysis. 2. Materials and methods 2.3. UHPLC-PDA-fluorescence analysis methods 2.1. Chemicals Fruit juice analyses were performed in a UHPLC Agilent Technologies modular liquid chromatographic system serie 1200 (Santa Clara, CA, USA) equipped with a binary pump (G1312B), a photodiode array detector (PDA) with multiple wavelength (G1315C), a Phenolic compounds classified as standard compounds used for quantification were: gallic acid (assay HPLC P99%) for Author's personal copy 940 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 fluorescence detector (G1321A), a thermostatized autosampler (G1316A) and a thermostatized automatic injector (G1329B). The two detectors were connected consecutively in the before mentioned sequence (PDA-Fluo). For detection of compounds, the chromatograms were recorded at 260, 280, 320, 360 and 520 nm using the photodiode detector, and 290 nm excitation and 350 nm emission 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 until 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 resolution 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 mobile 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% mobile 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 isocratic 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 order to obtain the relative retention time of each component versus callistephin. Callistephin chloride was prepared ten times concentrated (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 comparison 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 polyphenol group to refer the calculations were: pelargonidin 3-O-glucoside for anthocyanins because it is the same standard that method IFU No. 71 uses; flavonols standard was quercetin 3-O-glucoside 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 selected for dihydrochalcones standard for their presence in apples; and, gallic acid was selected as hydroxybenzoic acids standard because it is the same compound that is used in Folin-Ciocalteu 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 200 mg/L concentration and it was injected three times with the 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 quercetin 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 correspondent standard polyphenol. Finally, the amount of all polyphenols 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 system (Waters, Milford, MA, USA) serie ZQ 4000 linked simultaneously to PDA 2996 photodiode array detector equipped with a quaternary pump, and a thermostatized autosampler injector controlled 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 volume 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 instead of juice. Absorbance was measured at 760 nm. Total polyphenol 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 polyphenol 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 explained 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 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 Author's personal copy M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 941 Fig. 1. Chromatograms of bilberry juice and standard polyphenols analysed by IFU method number 71 (1a and 1b) and by UHPLC-PDA-Fluo method (1c and 1d). 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 anthocyanins 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 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 new method 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 anthocyanins; 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 flavanones, and apple is the main source of dihydrochalcones. Hydroxybenzoic 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, bilberry and black grape, are included as supplementary data. In every figure different chromatograms are shown, according to the detection 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 hydroxybenzoic acids. Monomeric flavan-3-ols and stilbenes were detected by fluorescence with an excitation wavelength of 290 nm and an emission 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 flavan-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 explained 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, EscribanoBailón, 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-rutinoside, (3) quercetin 3-O-glucuronide, (4) quercetin 3-O-glucoside 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) Author's personal copy 942 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 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 Cyanidin 3-O-galactoside Cyanidin 3-O-glucoside Cyanidin 3-O-glucosil-rutinoside Cyanidin 3-O-rutinoside Cyanidin 3-O-sophoroside Delphinidin 3-O-arabinoside Dephinidin 3-O-galactoside Delphinidin 3-O-glucoside Malvidin 3-O-arabinoside Malvidin 3-O-galactoside Malvidin 3-O-glucoside Malvidin 3-O-(6’’-acetyl-glucoside) Malvidin 3-O-(6’’-p-coumaroyl-glucoside) Pelargonidin 3-O-glucoside* Pelargonidin 3-O-rutinoside Peonidin 3-O-arabinoside Peonidin 3-O-galactoside Peonidin 3-O-glucoside Peonidin 3-O-rutinoside Petunidin 3-O-arabinoside Petunidin 3-O-galactoside Petunidin 3-O-glucoside Cy-3ara Cy-3gal Cy-3glu Cy-3glu-rut Cy-3rut Cy-3sho Dp-3ara Dp-3gal Dp-3glu Mv-3ara Mv-3gal Mv-3glu Mv-3ac-glu Mv-3cou-glu Pg-3glu Pg-3rut Pn-3ara Pn-3gal Pn-3glu Pn-3rut Pt-3ara Pt-3gal Pt-3glu 419.25 449.23 449.23 757.41 595.36 647.19 435.21 465.26 465.26 453.33 493.24 493.31 535.33 638.97 433.02 579.17 433.10 463.26 463.26 609.25 449.23 479.28 479.22 419.25 449.23 449.23 757.41 595.36 647.19 435.21 465.26 465.26 453.33 493.24 493.31 535.33 638.97 433.02 579.17 433.10 463.26 463.26 463.24 449.23 479.28 479.22 287.22 287.13 287.13 611.38–287.27 287.10 287.10 303.16 303.16 303.16 331.15 331.22 331.22 331.13 331.03 271.10 270.1 300.90 301.23 301.16 301.2 317.19 317.12 317.19 Flavonols Isorhamnetin 3-O-glucoside Isorhamnetin 3-O-rutinoside Isorhamnetin 7-O-rutinoside Kaempferol 3-O-glucoside* Kaempferol 3-O-rutinoside Myricetin 3-O-arabinoside Myricetin 3-O-glucoside Myricetin 3-O-rutinoside Quercetin 3-O-arabinoside Quercetin 3-O-galactoside Quercetin 3-O-glucoside* Quercetin 3-O-glucosil-rutinoside Quercetin 3-O-glucuronide Quercetin 3-O-rhamnoside Quercetin 3-O-rutinoside* Iso-3glu Iso-3rut Iso-7rut K-glu K-rut My-ara My-glu My-rut Q-ara Q-gal Q-glu Q-glu-rut Q-glucu Q-rha Q-rut 479.02 625.00 625.00 449.23 595.25 451.01 481.00 627.40 435.11 465.26 465.32 773.30 479.22 449.43 465.32 317.18 317.19 317.19 287.13 287.12 319.18 319.18 319.25 303.00 303.16 303.09 303.23 303.16 303.16 303.16 3,4-Dihydroxybenzoic acid* Ellagic acid* Gallic acid* 4-Hydroxybenzoic acid* Syringic acid* Vanillic acid* 3,4dHB Elag Gal 4-HB Syr Van 154.94 303.21 170.88 138.12 199.17 168.90 141.96 303.21 129.9 118.96 139.8 122.7-154.8 Caffeic acid* Caffeoyl glucoside Caffeoyl quinic acid Caftaric acid* Chlorogenic acid* Cinnamic acid* Ferulic acid* Ferulic 4-glucoside acid Neochlorogenic acid p-Coumaric acid* p-Coumaroyl glucose p-Coumaroyl quinic acid p-Coumaroyl tartaric acid Sinapic acid* Caf Caf-glu Caf-qui Caftar Clor Cin Fer Fer4glu Neoclor p-Cou p-Cou-glu p-Cou-qui p-Cou-tar Sin 180.91 342.12 355.51 312.05 354.93 148.86 194.76 356.85 354.86 165.00 327.05 338.87 297.23 224.84 180.91 180.83 180.89 163.03 162.93 148.86 177.01 194.76 163.11 146.85 165.03 146.88 147.05 224.84 Flavanones Didymin* Hesperidin* Naringin* Narirutin* Dyd Hesp Naring Nariru 596.38 611.24 581.33 581.18 596.38 611.24 272.84 273.06 196.12 390.86 435.31 435.06 Dihydrochalcones Phloritzin (Phloretin 20 -O-glucoside)* Phloretin 20 -O-xyloside-glucoside Phlor Phlo-xyl 437.04 568.09 275.17 275.14 199.52–158.06 Flavan-3-ols (+) Catechin* ( ) Epicatechin* ( ) Epigallocatechin ( ) Epigallocatechin 3-gallate* Cat Epi Epigal Epigal-gal 290.93 290.89 307.22 459.02 138.89 138.85 307.22 459.02 165.19–146.98–123.25 165.19–146.98–123.25 197.23 288.95 Stilbenes Resveratrol* Resveratrol glucoside Res Res-glu 228.91 228.82 228.91 228.82 Hydroxybenzoic acids Hydroxycinnamic acids * Pure compounds. 479.15 479.15 196.77 481.22 130.77-79.94 294.08 206.91–141.92 Author's personal copy M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 943 and dihydrochalcones not found. Profiles correspond to p-Coumaroyl 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) 4hydroxybenzoic acid, (3) 3,4-dihydroxybenzoic acid, (4) syringic acid and, (5) ellagic acid. This identification was satisfactory according 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, PhenolExplorer 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-rutinoside, (3) kaempferol 3-O-rutinoside and (4) myricetin 3-O-glucoside. 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 dihydrochalcones not present (profiles correspond to hydroxycinnamic acids: neochlorogenic acid, and chlorogenic acid; and to the anthocyanins: cyanidin 3-O-glucosyl-rutinoside, and cyanidin 3-O-rutinoside); (e) Hydroxybenzoic acids: (1) gallic acid, (2) 3,4dihydroxybenzoic 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 bibliography (Tsanova-Savova et al., 2005; Chaovanalikit & Wrolstad, 2004; USDA Database); (g) Stilbenes: not found. Fig. 2. Chromatograms of strawberry juice analysed by UHPLC-PDA-Fluo method. 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 3.2.3. American cranberry (Vaccinium macrocarpon Ait.) (a) Anthocyanins: (1) cyanidin 3-O-galactoside, (2) cyanidin 3O-arabinoside, (3) peonidin 3-O-galactoside, (4) peonidin 3-O-glucoside and, (5) peonidin 3-O-arabinoside. These results were in agreement with bibliography (Rodríguez-Medina, Segura-Carretero, & Fernández-Gutiérrez, 2009); Naczk & Shahidi, 2006; USDA Database); (b) Flavonols: (1) myricetin 3-O-arabinoside, (2) quercetin 3-O-galactoside, (3) kaempferol 3-O-glucoside, (4) isorhamnetin 3-O-rutinoside and, (5) quercetin 3-O-rhamnoside. This identification 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) determined synapic acid at low concentrations; (d) Flavanones and dihydrochalcones not found. Peak profiles correspond to p-Coumaroyl glucose (hydroxycinnamic acid), vanillic acid (hydroxybenzoic acid) and several anthocyanins; (e) Hydroxybenzoic acids: (1) 3,4dihydroxibenzoic acid, (2) vanillic acid, and (3) ellagic acid. Our identification agrees with bibliography: Rodríguez-Medina et al. (2009), and Phenol-Explorer Database. Wang and Zuo (2011) determined presence of benzoic acid; (f) Monomeric flavan-3-ols: (1) ( )-epigallocatechin, and (2) ( )-epicatechin. This identification Author's personal copy 944 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 3.2.4. Bilberry (Vaccinium myrtillus L. wild) (a) Anthocyanins: (1) dephinidin 3-O-galactoside, (2) delphinidin 3-O-glucoside, (3) cyanidin 3-O-galactoside, (4) delphinidin 3-O-arabinoside, (5) cyanidin 3-O-glucoside, (6) petunidin 3-Ogalactoside, (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) malvidin 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; PhenolExplorer; USDA Database); (b) Flavonols: (1) myricetin 3-O-arabinoside, (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 Database); (c) Hydroxycinnamic acids: (1) p-Coumaroyl glucoside, (2) caffeoyl glucose, (3) chlorogenic acid, (4) caffeic acid, (5) ferulic acid, and (6) synapic acid. Our identification agrees with bibliography (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 anthocyanins; (e) Hydroxybenzoic acids: (1) gallic acid, (2) 3,4-dihydroxybenzoic acid, (3) vanillic acid and, (4) syringic acid. Peak assignments are in agreement with data from bibliography (Rodríguez-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-Oglucoside. Fig. 3. Chromatograms of sour cherry juice analysed by UHPLC-PDA-Fluo method. is suitable with the authors Wang and Zuo (2011), RodríguezMedina 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. 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-Oglucoside, (5) malvidin 3-O-glucoside, (6) malvidin 3-O-(6’’-acetylglucoside), and (7) malvidin 3-O-(600 -p-Coumaroyl-glucoside). Results 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-arabinoside, (2) quercetin 3-O-rutinoside, (3) quercetin 3-O-glucuronide, 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 identification 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 correspond to caftaric acid and p-Coumaroyltartaric acid (hydroxycinnamic 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 (Rodríguez-Medina et al., 2009; Gruz et al., 2008; Russell et al., 2009); (f) Monomeric flavan-3-ols: (1) ( )-epigallocatechin, (2) (+)-catechin, (3) ( )-epigallocatechin 3-gallate, and (4) ( )-epicatechin. This identification was satisfactory according with studied references (Rodríguez-Medina et al., 2009; Tsanova-Savova et al., 2005; Phenol-Explorer; USDA Database); (g) Stilbenes: (5) resveratrol 3-O-glucoside and (6) resveratrol. This identification is suitable with the authors (Piotrowska, Kucinska, & Murias, 2012; Guerrero, Puertas, Fernández, Palma, & Cantos-Villar, 2010). 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) Author's personal copy M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 945 Hydroxycinnamic acids: (1) chlorogenic acid, (2) p-Coumaroyl glucose, (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) ( )-epigallocatechin, (2) (+)-catechin, and (3) ( )-epicatechin; (g) Stilbenes not found. Fig. 4. Chromatograms of orange juice analysed by UHPLC-PDA-Fluo method. Fig. 5. Chromatograms of apple juice analysed by UHPLC-PDA-Fluo method. Compound Group of polyphenols Time (minutes) Relative time (minutes) 946 Table 2 Quantification of polyphenols content as TIP (total individual polyphenols) of commercial fruit juices. Polyphenol content calculated as TIP (mg/100 mL fruit juice) Strawberry Sour cherry American cranberry Bilberry Black grape Orange Apple 4,195 6,452 21,913 39,096 16,643 1,991 9,126 4,086 0,633 5,987 0,391 3,620 0,678 0,241 0,648 3,965 4,012 1,494 1,145 16,123 3,442 4,177 19,852 6,556 1,443 3,416 4,507 0,948 77,928 (a) Ascorbic acid Gallic acid 4-Hydroxybenzoic acid ( )-Epigallocatechin 3,4-Dihydroxybenzoic acid Caffeoyl-quinic acid Neochlorogenic acid Caftaric acid Caffeoyl-glucose acid (+)-Catechin p-Coumaroyl glucose acid p-Coumaroyl quinic acid Caffeic acid Vanillic acid p-Coumaroyl tartaric acid Chlorogenic acid Ferulic acid 4-glucoside ( )-Epigallocatechin gallate Dephinidin 3-O-galactoside Delphinidin 3-O-glucoside Cyanidin 3-O-sophoroside Syringic acid Cyanidin 3-O-glucosyl-rutinoside Delphinidin 3-O-arabinoside ( )-Epicatechin p-Coumaric acid Cyanidin 3-O-glucoside Cyanidin 3-O-galactoside Petunidin 3-O-galactoside Quercetin 3-O-glucosil-rutinoside Ferulic acid Myricetin 3-O-arabinoside Petunidin 3-O-glucoside Pelargonidin 3-O-glucoside Vitamins Hidroxybenzoic acid Hidroxybenzoic acid Monomeric flavan-3-ol Hidroxybenzoic acid Hidroxycinnamic acid Hidroxycinnamic acid Hidroxycinnamic acid Hidroxycinnamic acid Monomeric flavan-3-ol Hidroxycinnamic acid Hidroxycinnamic acid Hidroxycinnamic acid Hidroxybenzoic acid Hidroxycinnamic acid Hidroxycinnamic acid Hidroxycinnamic acid Monomeric flavan-3-ol Anthocyanin Anthocyanin Anthocyanin Hidroxybenzoic acid Anthocyanin Anthocyanin Monomeric flavan-3-ol Hidroxycinnamic acid Anthocyanin Anthocyanin Anthocyanin Flavonol Hidroxycinnamic acid Flavonol Anthocyanin Anthocyanin 1,043 2,200 2,907 3,500 3,846 4,120 4,637 5,342 5,512 5,596 5,751 5,790 6,896 7,310 7,508 7,781 8,012 8,040 8,070 8,845 9,885 9,946 9,960 10,138 10,408 10,694 10,847 10,882 10,979 11,011 11,563 11,722 12,106 12,900 0,081 0,171 0,225 0,271 0,298 0,319 0,359 0,414 0,427 0,434 0,446 0,449 0,535 0,567 0,582 0,603 0,621 0,623 0,626 0,686 0,766 0,771 0,772 0,786 0,807 0,829 0,841 0,844 0,851 0,854 0,896 0,909 0,938 1,000 2,018 6,353 7,212 0,616 3,204 (b) Pelargonidin 3-O-glucoside Peonidin 3-O-galactoside Ellagic acid Myricetin 3-O-rutinoside Cyanidin 3-O-rutinoside Sinapic acid Pelargonidin 3-O-rutinoside Cyanidin 3-O-arabinoside Quercetin 3-O-rutinoside Peonidin 3-O-glucoside Malvidin 3-O-galactoside Petunidin 3-O-arabinoside Quercetin 3-O-arabinoside Quercetin 3-O-glucoside Malvidin 3-O-glucoside Quercetin-3-O-galactoside Quercetin 3-O-glucuronide Anthocyanin Anthocyanin Hidroxybenzoic acid Flavonol Anthocyanin Hidroxycinnamic acid Anthocyanin Anthocyanin Flavonol Anthocyanin Anthocyanin Anthocyanin Flavonol Flavonol Anthocyanin Flavonol Flavonol 12,900 13,009 13,175 13,234 13,243 13,248 13,270 13,923 13,947 14,032 14,172 14,193 14,489 14,841 15,095 15,102 15,500 1,000 1,008 1,021 1,026 1,027 1,027 1,029 1,079 1,081 1,088 1,099 1,100 1,123 1,150 1,170 1,171 1,202 15,131 6,494 7,523 101,175 1,194 4,814 19,496 20,941 2,151 6,562 64,238 7,535 0,512 7,757 5,858 9,973 1,714 3,441 35,863 0,489 3,318 97,717 0,652 34,261 57,410 3,424 0,363 2,705 2,611 2,873 3,792 1,964 73,670 10,874 4,459 0,266 4,764 1,746 1,591 10,908 2,138 1,670 30,658 1,692 48,649 22,084 12,619 10,718 5,740 8,439 1,009 18,662 13,411 0,779 3,757 0,526 0,489 1,141 1,616 3,033 15,131 5,334 1,029 28,051 0,489 1,680 0,441 1,823 41,050 20,403 19,361 20,272 9,939 0,925 3,818 0,154 2,372 1,364 15,857 0,438 43,389 4,529 3,803 12,412 2,234 2,423 5,181 Author's personal copy 6,086 33,176 2,093 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 79,305 40,371 12,809 7,568 Author's personal copy 114,14 1,597 120,481 607,324 1,097 0,641 32,638 1,349 0,856 6,679 227,958 144,933 296,046 0,489 0,513 2,032 0,912 1,796 1,227 2,920 Peonidin 3-O-arabinoside Malvidin 3-O-arabinoside Resveratrol 3-O-glucose Narirutin Peonidin 3-O-rutinoside Isorhamnetin 3-O-rutinoside Kaempferol 3-O-glucoside Naringin Isorhamnetin 7-O-rutinoside Phloretin 20 O xilosil-rutinosido Isorhamnetin 3-O-glucoside Quercetin 3-O-rhamnoside Kaempferol 3-O-rutinoside Myricetin 3-O-glucoside Cinnamic acid Resveratrol Hesperidin Malvidin 3-O-(600 -acetyl-glucoside) Phloritzin Didymin Malvidin 3-O-(600 -p-coumaroyl-glucoside) Anthocyanin Anthocyanin Stilbene Flavanone Anthocyanin Flavonol Flavonol Flavanone Flavonol Dyhidrochalcone Flavonol Flavonol Flavonol Flavonol Hidroxycinnamic acid Stilbene Flavanone Anthocyanin Dyhidrochalcone Flavanone Anthocyanin 16,026 16,484 16,521 16,851 17,145 17,775 17,950 18,065 18,132 19,031 19,246 19,520 19,853 20,379 20,500 20,600 21,803 21,817 24,059 24,970 25,259 1,242 1,278 1,281 1,306 1,329 1,378 1,391 1,400 1,406 1,475 1,492 1,513 1,539 1,580 1,589 1,597 1,690 1,691 1,865 1,936 1,958 TIP value 0,451 1,036 9,372 0,722 10,093 6,858 0,202 5,761 0,397 0,329 2,301 5,904 0,902 2,017 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 947 3.2.7. Apple (Malus pumila P. Mill.) (Fig. 5) (a) Anthocyanins not found; (b) Flavonols not found. Peak correspond to the hydroxycinnamic acid chlorogenic acid; (c) Hydroxycinnamic acids: (1) chlorogenic acid, (2) p-Coumaroyl glucose, and (3) caffeic acid. This identification is suitable with the authors (Fu et al., 2011); Karaman, Tütem, Basßkan, & Apak, 2010; Rodríguez-Medina et al., 2009); (d) Dihydrochalcones: (1) phloridzin 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) ( )-epigallocatechin 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 polyphenol group is approximately from minute 2–13 for hydroxybenzoic 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 anthocyanins; from 11 to 20 min for flavonols; from 16 to 21 min for stilbenes; 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 reported in Section 2. Results were summarized in Table 2a and 2b, where polyphenols were ordered by retention time. Table 2a includes 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 quantified, and included in Table 2a. Highlight the main anthocyanins of each fruit juice shown in Table 2a and b, interesting for authentication purposes. In strawberry 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 anthocyanins are delphinidin 3-O-glucoside (57.41 mg/100 mL), cyanidin 3O-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 from orange (0.3 mg/100 mL). In apple no flavonols 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 and American cranberry have the highest value in hydroxybenzoic acids with 38.2 and 38.7 mg/100 mL, respectively. Black grape and strawberry have a similar value: 26.1 and 25.9 mg/100 mL respectively. Vanillic acid is the main hydroxybenzoic acid in American cranberry with a content of 33.17 mg/100 mL. A high value in hydroxycinnamic acids content was present in bilberry (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 Author's personal copy 948 M.C. Díaz-García et al. / Food Chemistry 138 (2013) 938–949 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 presence 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-3ols 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) was found in bilberry (39.096 mg/100 mL). The ranking of top TIP contents of studied fruit juices was bilberry > 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) > strawberry (140.612 mg GAE/100 mL) > apple (114.212 mg GAE/ 100 mL) > orange (84.059 mg GAE/100 mL). Fig. 6 shows the relationship 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 calculated 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 values 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 values 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 polyphenols (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 origin of fruit samples makes a comparison difficult. 4. Conclusions The proposed UHPLC-PDA-Fluorescence method allows to verify 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 calculated 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 versus 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. Polyphenols data could complete the labelling information of commercial antioxidant fruit juices which offer beneficial effects on consumers’ health. 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