Food Chemistry 138 (2013) 1062–1071

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Food Chemistry

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Identification of phenolic constituents in red chicory salads (Cichorium intybus)by high-performance liquid chromatography with diode array detection andelectrospray ionisation tandem mass spectrometry

Chiara Carazzone, Dora Mascherpa, Gabriella Gazzani, Adele Papetti ⇑Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy

a r t i c l e i n f o

Article history:Received 30 July 2012Received in revised form 8 November 2012Accepted 16 November 2012Available online 23 November 2012

Keywords:HPLC-PDA–ESI/MSn

FlavonoidsPhenolic acidsCichorium intybus var. silvestre

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.11.060

⇑ Corresponding author. Tel.: +39 0382 987863; faxE-mail addresses: chiara.carazzone@unipv.it (C. C

unipv.it (D. Mascherpa), gabriella.gazzani@unipv.itunipv.it (A. Papetti).

a b s t r a c t

Phenolic acids and flavonoids extracted from several types of Cichorium intybus var. silvestre salads(‘‘Chioggia’’, ‘‘Treviso’’, ‘‘Treviso tardivo’’, and ‘‘Verona’’) were characterised by high-performance liquidchromatography–electrospray ionisation/mass spectrometry. Among the 64 compounds detected, severalhydroxycinnamic acid derivatives including 8 mono- and dicaffeoylquinic acids, 3 tartaric acid deriva-tives, 31 flavonol and 2 flavone glycosides, as well as 10 anthocyanins were characterised based on UVspectra and MSn fragmentation patterns. Furthermore, several isomers of caffeic acid derivatives weredistinguished for the first time by their specific mass spectral data. This is the first study reporting theglycosylation type and position of mono- and diglycosylated flavonoids in red salads.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

High intakes of dietary fruit and vegetables are associated witha reduced incidence of chronic diseases (Margett & Buttriss, 2003).Their health-promoting factors, i.e. phytochemicals, have a widerange of biological action, including antioxidant, anticancer, anti-inflammatory, and a-glucosidase inhibition activities. The mostinteresting of such compounds are phenolic acids, including chlor-ogenic acids, and flavonoids such as anthocyanins, flavonols, flava-none, and flavan-3-ols. Every day humans ingest large amounts ofplant polyphenols (up to 1 g/day), which generally occur as glyco-sides, because glycosylation makes them less reactive and morewater-soluble, hence easier to store in the cell vacuole. The flavo-noids responsible for blue, red or purple leaf colours widespreadin the plant kingdom are anthocyanins. Their structures are basedon the C15 skeleton of anthocyanidins (consisting of a chromanering bearing a second aromatic ring B in position 2), which areglycosylated and/or acylated at specific hydroxylated positions(Delgado-Vargas & Paredes-Lopez, 2003). Cyanidin, delphinidin,malvidin, pelargonidin, peonidin, and petunidin are the six mostcommon anthocyanin aglycones; glucose, rhamnose, galactose, xy-lose, and arabinose are the most prevalent sugars, often acylatedwith aromatic or aliphatic acids. Anthocyanins (of which US resi-

ll rights reserved.

: +39 0382 422975.arazzone), dora.mascherpa@(G. Gazzani), adele.papetti@

dents take up to 180–215 mg/day) have been found to afford arte-rial protection, inhibit platelet aggregation, and protect endothelialtissue, thus reducing the risk of coronary heart disease (Tsuda,Horio, & Osawa, 2003; Wang & Mazza, 2002; Youdim, McDonald,Kalt, & Joseph, 2002).

Red berries, red cabbage, red onion, and eggplant are amongthe best explored plant food matrices containing anthocyanins(Dobson et al., 2012), whereas the polyphenol content of red chic-ory salads still needs to be fully characterised. Chicory (Cichoriumintybus L.) is a diploid plant species of the Asteraceae family; theCichorieae tribe includes approximately 100 genera and many hun-dred species of which some genera are used as salad greens, e.g.Cicerbita, Cichorium, Lactuca, Scorzonera, Taraxacum, and Tragopo-gon. ‘‘Radicchio’’ chicories are especially popular in northeasternItaly, where due to their resistance to low temperatures they aremostly consumed as raw salad in winter, when few fresh vegeta-bles are available. Red radicchio salads, denominated ‘‘Chioggia’’,‘‘Treviso’’, ‘‘Treviso tardivo’’, and ‘‘Verona’’ radicchio, are allC. intybus var. silvestre. They have a distinctive, agreeable tasteand are popular on food markets including those of central Europeand the United States. Radicchio ‘‘Treviso tardivo’’, a late wintersalad, has earned Protected Geographical Indication (PGI) andProtected Designation of Origin (PDO) status.

A complete characterization of the polyphenols found in thesevegetables is not yet available. The aim of this work was a qualita-tive investigation of the phenolic acid and flavonoid content of thefour cultivar of C. intybus var. silvestre. High-performance liquidchromatography (HPLC) using a photodiode-array detector (PDA)

C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071 1063

coupled with electrospray ionisation/mass spectrometry (ESI/MSn)is the method of choice for such studies. MSn is the most effectivetool to elucidate polyphenol structures, since it provides informa-tion on the molecular mass (MM) and fragmentation pattern ofanalytes. Compared to the structural characterization of flavonoidglycosides achieved with MS methods, HPLC–ESI/MSn providesinformation on the aglycone part, the types of carbohydrates pres-ent, the stereochemistry of terminal monosaccharide units, the se-quence of the glycan part, and interglycosidic linkages (Ferreres,Llorach, & Gil-Izquierdo, 2004). One of the most useful MSn ap-proaches applied to the identification of unknown compounds isdata-dependent acquisition, which employs user-specified criteriato select the ion of interest for subsequent fragmentation. The re-sults obtained set the basis for further investigation and profiling,as well as can be a useful tool in evaluating polyphenol dietaryintake.

2. Materials and methods

2.1. Materials and chemicals

HPLC-grade and HPLC–MS grade water and methanol were pur-chased from Sigma–Aldrich (Saint Louis, MO), as were standard5-O-caffeoylquinic acid (chlorogenic acid, 5-CQA), malic acid,caffeic acid, keampferol, kaempferol-3-O-glucoside, and querce-tin-3,7-di-O-glucoside. Quinic acid was purchased from AcrosOrganics (Geel, Belgium), cyanidin-3-O-glucoside and malvidin-3-O-glucoside from Extrasynthese (Genay, France). HPLC-gradewater, used for sample preparation, was obtained with a Milli-Qwater purification system (Millipore, Billerica, MA). Filtrationmembranes (0.45-lm cellulose acetate/cellulose nitrate mixedesters) were purchased from Millipore.

2.2. Polyphenolic extract preparation from vegetable samples

Five clumps each of the 4 types of C. intybus var. silvestre(‘‘Chioggia’’, ‘‘Treviso’’, ‘‘Treviso tardivo’’, and ‘‘Verona’’) were pur-chased at a local market in the autumn (September–December).Fresh leaves (20 g) were washed, cut into small pieces, suspendedin 12.5 mL of MeOH–HCOOH (99:1, v/v) and shaken for 1 h in anice bath in the dark. The mixture was then centrifuged for 5 minat 8750g; the insoluble residue was re-extracted 3 times with afresh aliquot of the same mixture. The extracts obtained werepooled, filtered through a 0.45-lm membrane and then directly in-jected into the HPLC-PDA–ESI/MSn.

2.3. Liquid chromatography–tandem mass spectrometry

HPLC-PDA–ESI/MSn analyses were performed using a ThermoFinnigan Surveyor Plus HPLC apparatus equipped with a quater-nary pump, a Surveyor UV–vis photodiode-array detector (PDA),a Surveyor Plus autosampler, and a vacuum degasser connectedto an LCQ Advantage Max ion trap mass spectrometer (all fromThermo Fisher Scientific, Waltham, MA, USA) through an ESIsource.

Separation was achieved on a Gemini� C18 analytical column(150 � 2.0 mm i.d., 5 lm) with a Hypersil Gold C18 guard column(10 � 2.1 mm i.d., 5 lm; both from Phenomenex, Torrance, CA).The mobile phase consisted of A (0.1% formic acid in water) andB (methanol) at a flow rate of 0.3 mL/min (injection volume10 lL). Gradient elution was carried out using the following time-table: from 2% B to 5% B in ten minutes, then to 40% B in 50 min, to60% B in 10 min, and to 100% B in 10 min. An isocratic elution with100% B was then carried out for 10 more minutes. The resulting to-tal run time was 90 min, followed by column reconditioning

(Papetti et al., 2008). The sample tray and column temperatureswere set at 4 �C.

The chromatogram was recorded at several wavelengths, char-acteristic of different classes of polyphenols, such as 280 nm forphenolic acids, 320 nm for hydroxycinnamic acids, 370 nm forflavonols, and 520 nm for anthocyanins. Spectral data were ac-quired in the range of 200–600 nm for all peaks.

The ion trap operated in data-dependent, full scan (100–1000m/z), zoom scan, and MSn mode to obtain fragment ion m/z witha collision energy of 35% and an isolation width of 3 m/z. Data-dependent acquisition, where user-specified criteria are appliedto select the ion of interest for subsequent fragmentation, areamong the most useful approaches employed to identify unknowncompounds by MS. Using this approach single stage MS providesthe putative molecular mass that can be used in combination withUV detection for a first tentative structure assignment; structureelucidation and confirmation can then be obtained by tandemMS analysis via the fragmentation pathway. When greater discrim-ination was required additional targeted MS2 and MS3 experimentswere performed on selected pseudomolecular ions.

The negative and positive parameters of the ion mode ESIsource had previously been optimised by flow injection analysis,using 5-CQA, kaempferol, and cyanidin-3-O-glucoside (5 ppm in0.1% formic acid–methanol solution, 50:50, v/v) to a ionisationvoltage of 3.5 kV, a capillary temperature of 260 �C, a sheath gasflow rate of 50 arbitrary units (AU), and an auxiliary gas flow rateof 20 AU.

The Thermo Fisher Scientific Excalibur 2.0 software was usedfor data acquisition and processing.

Three independent assays were performed to analyse eachmethanolic extract from vegetables by HPLC/PDA–ESI/MSn; no rel-evant variations attributable to the nature of the detected frag-ments or their relative intensities were observed.

3. Results and discussion

Compound attribution to each class based on chromatographicbehaviour, UV–visible (UV–vis) spectra and mass spectra, and com-parisons with the literature are addressed below and summarisedin Table 1.

Chromatographic peaks were preliminarily classified intohydroxycinnamic acid, flavonol, and flavone derivatives, mainlyglycosides, according to PDA UV–vis spectra, when concentrationand resolution allowed to recover them. Phenolic compounds exhi-bit absorbance maxima in the 275–285 nm wavelength region dueto the aromatic ring in their molecular structure. Phenolic acidsand flavonoids have characteristic UV–vis absorbances: hydroxy-benzoic acids are detected at 280 nm, hydroxycinnamic acids at320 nm, flavonols between 350 and 385 nm, and flavones in the277–295 nm range with a shoulder at 300–330 nm (Olsen, Aaby,& Borge, 2009). The chromatographic retention time of each phe-nolic compound compared to that of the external standard, whenavailable, was used to support its identity. When commercial stan-dards were not available, the analytes were identified by combin-ing MSn data with the respective literature data.

Polyphenols in nature generally occur as sugar conjugates, usu-ally O-glycosides. In MS/MS analysis, cleavage of the glycosidiclinkage and concomitant H rearrangement leads to elimination ofthe sugar residue, namely 162 amu (hexose; glucose or galactose),146 amu (deoxyhexose; rhamnose), 132 amu (pentose; xylose orarabinose), and 176 amu (glucuronic acid). The MS2 and MS3 prod-uct ion spectra of flavonol 3,7-di-O-glycosides using negative ionESI-MS demonstrate that they can be differentiated from isomericmono-O-diglycosides and the glycosylation positions determined(Ablajan et al., 2006).

Table 1UV–vis, MS and MSn data of polyphenolic compounds in Cichorium intybus var. silvestre.

No UV kmax (nm) m/z MSn m/z Identification

1⁄,a 210 133 MS2[133]: 115 (100), 89 (3) Malic acid2⁄ 260 179 MS2[179]: 135 (100) Caffeic acid3⁄,a 220, 272 191 MS2[191]: 173 (50), 127 (30), 111 (100) Quinic acid4a 250, 323

303sh353 MS2[353]: 191 (100), 179 (50), 135 (5) 3-Caffeoylquinic acid

5⁄ 246, 324310sh

353 MS2[353]: 191 (100), 179 (3) 5-Caffeoylquinic acid

6a 252, 324303sh

353 MS2[353]: 191 (15), 179 (60), 173 (100) 4-Caffeoylquinic acid

7a,b,c 246, 324310sh

353 MS2[353]: 191 (100), 179 (2) cis-5-Caffeoylquinic acid

8a,d 232, 277, 321 311 MS2[311]: 179 (41), 149 (100), 135 (3) cis-Caftaric9d 241, 327

310sh311623

MS2[311]: 179 (80), 149 (100), 135 (5)MS2[623]: 491 (100), 311 (94)

trans-Caftaric acid

10a,c,d 245, 320 335 MS2[335]: 179 (10), 161 (100), 135 (45) 5-Caffeoylshikimic acid11d 241, 321

300sh337 MS2[337]: 191 (100) 5-p-Coumaroylquinic acid

12a 256, 352 727+ MS2[727]: 479 (100), 303 (28)MS3[479]: 303(100)

Quercetin-3-O-glucuronide-7-O-(600-O-malonyl)-glucoside

13d 264, 366 697+ MS2[697]: 535 (65), 449 (100), 287 (30) Kaempferol-3-O-glucosyl-7-O-(600-O-malonyl)-glucoside14 519 MS2[519]: 259 (100), 215 (25) Dimer of unknown acid15a 234, 324 363 MS2[363]: 207 (30), 155 (100), 137 (46) Dimethoxycinnamoyl shikimic acid16b,c,d 265, 366 463+ MS2[463]: 287 (100) Unknown kaempferol derivative17b,c 263, 340 611+ MS2[611]: 449 (20), 431 (40), 287 (100) Kaempferol-3-O-sophoroside18a,b,c 249, 268, 345 519 MS2[519]: 315 (100)

MS3[315]: 300 (100)Isorhamnetin-7-O-(600-O-acetyl)-glucoside

19 242, 323301sh

367 MS2[367]: 191 (100), 173 (10) 5-O-feruloyquinic acid

20 241, 327305sh

473947

MS2[473]: 311 (100), 293 (20), 179 (45), 149 (30) Dicaffeoyltartaric acid (chicoric acid)

21a,d 265, 363 697+ MS2[697]: 535 (100), 449 (50), 287 (30) Kaempferol-7-O-glucosyl-3-O-(600-malonyl)-glucoside22d 279, 517 713+ MS2[713]: 465 (100), 551 (38), 303 (78) Delphinidin-3-O-(600-O-malonyl)-glucoside-5-O-glucoside23 280, 514 783+ MS2[783]: 535 (100), 287(40) Cyanidin-3,5-di-O-(600-O-malonyl)-glucoside24b,c,d 280, 515 535+ MS2[535]: 287 (100) Cyanidin-3-O-(600-O-malonyl)-glucoside25b,c,d 277, 525 565+ MS2[565]: 317 (100) Petunidin-3-O-(600-O-malonyl)-glucoside26 279, 516 287+ Cyanidin27 280, 516 449+ MS2[449]: 287 (100) Cyanidin-3-O-galactoside28⁄,d 280, 516 449+ MS2[449]: 287 (100) Cyanidin-3-O-glucoside29 279, 514 491+ MS2[491]: 449 (12), 287 (100) Cyanidin-3-O-(600-O-acetyl)-glucoside30⁄,a,b,c 277, 527 493+ MS2[493]: 331 (100) Malvidin-3-O-glucoside31 d 280, 513 447+ MS2[447]: 271 (100) Pelargonidin-3-O-monoglucuronide32a 243, 322

301sh367 MS2[367]: 191 (80), 173 (100) 4-O-feruloyquinic acid

33b,c 267, 366 431 MS2[431]: 269 (100), 268 (90) Apigenin-7-O-glucoside34b,c,d 261, 350 463+ MS2[463]: 301(100) Chrysoeriol-3-O-glucoside35a,b,c 251,357 491 MS2[491]: 329 (100), 311 (5), 293 (95) Tricin-3-O-glucoside36 248, 324

301sh515 MS2[515]: 353 (100), 335 (30)

MS3[353]: 191 (100), 179 (50)1,3-Dicaffeoylquinic acid

37 248, 325302sh

515 MS2[515]: 353 (100), 299 (35), 317 (28), 255 (10), 203 (5)MS3[353]: 191 (24), 173 (100)

1,4-Dicaffeoylquinic acid

38 248, 323302sh

515 MS2[515]: 353 (100), 299 (25), 255 (18), 203 (8)MS3[353]: 191 (35), 173 (100)

3,4-Dicaffeoylquinic acid

39a,d 256, 351 463 MS2[463]: 301 (100), 300 (84) Quercetin-7-O-galactoside40a,d 257, 368 551+ MS2[549]: 389 (54), 303 (100) Quercetin-3-O-(600-O-malonyl)-glucoside41 248,358 475 MS2[475]: 299 (100) Kaempferide glucuronide42a,b,c 255, 351 465+ MS2[465]: 303 (100), 302 (68) Quercetin-7-O-glucoside43b,c 255, 351 479+ MS2[479]: 303 (100) Quercetin-7-O-glucuronide44 229, 325

302sh515 MS2[515]: 353 (100), 191 (18)

MS3[353]: 191 (100), 179 (50), 173 /2), 135 (10)3,5-Di-caffeoylquinic acid

45 255, 349 505 MS2[505]: 301 (100) Quercetin-7-O-(600-O-acetyl)-glucoside46b,c,d 268, 366

318sh447 MS2[447]: 327 (35), 285 (100), 284 (74), 257 (45), 255 (18) Kaempferol-7-O-glucoside

47d 262, 361 593 MS2[593]: 447 (43), 285 (100), 257 (25) Kaempferol-7-O-rutinoside48 256, 351 609 MS2[609]: 463 (5), 301 (100), 300 (68) Quercetin-7-O-p-coumaroylglucoside49d 248, 268, 341 623 MS2[623]:315 (100)

MS3[315]: 300 (100)Isorhamnetin-7-O-neohesperidoside

50 265, 365 535+ MS2[535]: 449 (35), 287 (100), 286 (38) Kaempferol-7-O-(600-O-malonyl)-glucoside51 261, 363 463+ MS2[463]: 286 (20), 287 (100) Kaempferol-7-O-glucuronide52 253, 361 549+ MS2[549]: 463 (14), 301 (100) Kaempferide-3-O-(600-O-malonyl)-glucoside53a,d 261, 364 463+ MS2[463]: 286 (100), 287 (36) Kaempferol-3-O-glucuronide54a,d 263,363 625+ MS2[625]: 449 (100), 287 (8) Kaempferol-3-O-glucuronide-7-O-glucoside55a,d 249, 268, 342 565+ MS2[565]: 479 (4), 317 (100)

MS3[317]: 302 (100)Isorhamnetin-7-O-(600-O-malonyl)-glucoside

56 264, 366 535+ MS2[535]: 449 (28), 287 (42), 286 (100) Kaempferol-3-O-(600-O-malonyl)-glucoside

1064 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

Table 1 (continued)

No UV kmax (nm) m/z MSn m/z Identification

57⁄ 266, 318300sh

447 MS2[447]: 285 (70), 284 (100), 257 (5), 255 (35) Kaempferol-3-O-glucoside

58a,b,c 269, 374 565 MS2[565]: 521 (40), 317 (100) Myricetin-7-O-(600-O-malonyl)-glucoside59d 264,360 593 MS2[593]: 447 (100), 285 (36), 257 (25) Kaempferol-7-O-neohesperidoside60 263, 361 489 MS2[489]: 285 (100), 284 (15) Kaempferol-7-O-(600-O-acetyl)-glucoside61 263, 361 489 MS2[489]: 285 (10), 284 (100) Kaempferol-3-O-(600-O-acetyl)-glucoside62b,c 252, 269, 344 477 MS2[477]: 315 (100)

MS3[315]: 300 (100)Isorhamnetin-7-O-glucoside

63 251, 269, 342 491 MS2[491]: 315 (100)MS3[315]: 300 (100)

Isorahmnetin-7-O-glucuronide

Compounds are reported in order of elution.⁄Compared with standard compound; sh indicates a shoulder in the UV–vis spectrum; + indicates positive ionisation.Numbers in brackets indicate the relative intensity of fragment ions.aNot found in ‘‘Chioggia’’; bnot found in ‘‘Treviso’’; cnot found in ‘‘Treviso tardivo’’; dnot found in ‘‘Verona’’.

C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071 1065

Ionisation was performed both in positive and in negative ionmode. Combined use of ionisation in the two modes affords extracertainty of the determination of the molecular mass. In the nega-tive ionisation mode, hydroxybenzoic and hydroxycinnamic acidsdeprotonated easily, whereas in the positive mode they formed ad-ducts with the cations in the sample or mobile phase, i.e. sodiumions. Flavonol and flavone glycosides showed response in both ion-isation modes, whereas anthocyanidins did so exclusively in posi-tive ionisation mode (Olsen et al., 2009; Schmidt et al., 2010).

Figs. 1 and 2 show the PDA chromatograms acquired in therange 200–600 nm of ‘‘Treviso’’ extract and the comparison of‘‘Treviso tardivo’’, ‘‘Chioggia’’ and ‘‘Verona’’ extracts, respectively.Fig. 3 show the TIC plots for the four methanolic extracts analysed.

3.1. Characterisation of free small organic acids

Two free small organic acids were identified by comparing theirUV–vis spectra and MS fragmentation patterns with standard com-pounds. Peak 1, with an [M�H]� at m/z 133, produced the MS2 basepeak at m/z 115, corresponding to the loss of a water molecule[M�H�H2O]� and was assigned to malic acid (MM 134). Peak 3,with an [M�H]� at m/z 191, with MS2 fragments at m/z 173

Fig. 1. HPLC-PDA chromatogram (200–600 nm) of the polyphe

[M�H�H2O]� and at m/z 127 [M�H�CO�2H2O]�, was identifiedas quinic acid (Gouveia & Castilho, 2009).

3.2. Characterisation of hydroxycinnamic acid derivatives

The presence of free and esterified phenolic acids in Cichoriumgenus vegetables is not surprising (Heimler, Isolani, Vignolini,Tombelli, & Roman, 2007; Innocenti et al., 2005; Rossetto et al.,2005). Peak 2 was identified as caffeic acid (MM 180) due to a basepeak at m/z 135, corresponding to the loss of a carboxyl group[M�H�CO2]�. We identified several caffeoyl and feruloyl deriva-tives containing a quinic acid unit. Product ion scan experimentsof these compounds disclosed characteristic fragmentation involv-ing the cleavage of one or two acyl moieties. The linkage position ofthese groups in quinic acid was determined based on the MS2 frag-mentation behaviour of pseudomolecular ions.

The MM of compounds 4, 5, and 6 was 354. Based on their sim-ilar UV and MS spectra we concluded that they are all mono-CQAisomers, whose presence in nature is well known (Clifford, Zheng,& Kuhnert, 2006; Ma, Dastmalchi, Whitaker, & Kennelly, 2011). Theliterature for mono-CQA reports an ion at m/z 191 as the base peak[M�H�caffeoyl]� when the acyl group is linked to the 3-OH or5-OH position, and an ion at m/z 173 when the acyl group is linked

nolic extract of Cichorium intybus var. silvestre ‘‘Treviso’’.

Fig. 2. HPLC-PDA chromatograms (200–600 nm) of the polyphenolic extracts of Cichorium intybus var. silvestre ‘‘Treviso tardivo’’ (a), ‘‘Chioggia’’ (b), and ‘‘Verona’’ (c).

1066 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

to the 4-OH position. This allowed differentiation of 3-CQA from5-CQA solely on the basis of the relative intensity of m/z 179[M�H�quinic acid]�, which is more significant for 3-OH com-pounds (Schram, Miketiva, Slanina, Humpa, & Taborska, 2004;Schütz, Kammerer, Carle, & Schieber, 2005). Based on these dataand on the comparison of retention times, UV–vis spectra, andMS2 fragments with the commercial standard, compound 5 wasidentified as 5-CQA, commonly known as chlorogenic acid,

whereas compounds 4 and 6, not found in the ‘‘Chioggia’’ extract,were recognised as 3-CQA and 4-CQA, respectively (Slimestad,Vangdal, & Brede, 2009). Another CQA was found in the ‘‘Verona’’extract (compound 7); it displayed identical 5-CQA fragmentationpatterns and was therefore assigned to the cis isomer, which orig-inates from trans–cis isomerization when the plant tissue is ex-posed to UV light (Clifford, Kirkpatrick, Kuhnert, Roozendaal, &Roderigues Salgado, 2008; Jaiswal, Kiprotich, & Kuhnert, 2011a).

Fig. 3. Total ion chromatogram (TIC)of Cichorium intybus var. silvestre ‘‘Treviso’’ (a), ‘‘Treviso tardivo’’ (b), ‘‘Chioggia’’ (c), and ‘‘Verona’’ (d).

C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071 1067

Four different isomers of diCQA were found in red chicories be-sides monoCQA. Compounds 36, 37, 38, and 44, eluting later in thechromatogram, gave [M�H]� at m/z 515, and MS2 fragment at m/z353, corresponding to the loss of a caffeoyl moiety [M�H�162]�

was the base peak (Fig. 4). Moreover, the presence and intensityof MS2 secondary fragments allowed discriminating the differentdiCQA isomers. Ion m/z 299 (intensity 35%) and the less intensefragments at m/z 255 and 203 were unique to compounds 37 and38 and are characteristic of 4-acyl diCQA. Furthermore, the pres-ence of m/z 317 in the MS2 spectrum of compound 37 led to theassignment to 1,4-diCQA. The MS3 spectra of the four compoundswere also quite different; compounds 36 and 44 showed afragment ion at m/z 179 (50% of the base peak), characteristic ofa 3-OH substituted quinic acid. This fragment was not found inthe spectra of compounds 37 and 38, which showed base peaksat m/z 173, characteristic of a 4-OH substitution. Based on theirfragmentation pattern, the intensity of MSn ions, and their hydro-philicity, compounds 36, 38 and 44 were identified as 1,3-diCQA,3,4-diCQA, and 3,5-diCQA, respectively (Clifford et al., 2008). Theseisomers have never previously been identified in C. intybus.

Compound 8, found only in ‘‘Treviso’’ and ‘‘Treviso tardivo’’ ex-tracts, gave both tartaric (m/z 149) and caffeic acids (m/z 179) asMS2 fragments and a low abundance signal at m/z 135 for thedecarboxylated caffeic acid. It was therefore identified as caffeoyl-tartaric acid (cis-caftaric acid). In addition, another compound (9)found in all extracts but ‘‘Verona’’, displayed a similar MS/MS frag-mentation but showed in the mass spectrum a predominant signalat m/z 623, resulting from adduct formation of two individual mol-ecules of caftaric acid. Based on the presence of m/z 623 in the MS

spectrum and on UV data, compound 9 was assigned to trans-caf-feoyltartaric acid (trans-caftaric acid) (Jaiswal, Kiprotich, et al.,2011a; Schütz et al., 2005).

A very intense peak eluting as compound 20 and exhibiting theabsorption maxima characteristic of hydroxycinnamic acid(320 nm) was found in all extracts. It gave a pseudomolecular ionat m/z 473 and a dimer at m/z 947, producing a base peak at m/z311 by MS2 fragmentation due to the loss of 162 amu, correspond-ing to a caffeoyl moiety. Other MS2 fragments were m/z 293[M�H�caffeoyl�H2O]�, m/z 179 [M�H�caffeoyltartaric]�, and m/z 149 [M�H�dicaffeoyl]�, leading to identification of di-caffeoyltar-taric acid, also known as chicoric acid. The occurrence of this acidhas been reported in different members of the Asteraceae family(Jaiswal, Kiprotich, et al., 2011), in particular in Chicorium genus plants(Heimler et al., 2007; Innocenti et al., 2005; Rossetto et al., 2005).

Two peaks (19 and 32) eluting at different times produced thesame molecular ion at m/z 367. They shared similar UV–vis spectrabut different fragmentation patterns. Compound 19 gave the basepeak at m/z 191, due to the loss of a feruloyl moiety, and compound32 at m/z 173. These data are consistent respectively with 5-feru-loyquinc acid and 4-feruloyquinc acid (not found in the ‘‘Chioggia’’extract).

Two derivatives of shikimic acid were identified by comparisonwith the literature: compound 10 was found only in the ‘‘Treviso’’extract and compound 15 in all but the ‘‘Chioggia’’ extract. The for-mer was identified as 5-caffeoylshikimic acid; it exhibited absorp-tion maxima at 245 and 320 nm and produced fragments m/z 179for the loss of a caffeoyl moiety, m/z 161 (base peak), and m/z 135(Jaiswal, Febi Matei, Ullrich, & Kuhnert, 2011b). The other shikimic

Fig. 4. MS2 and MS3 spectra of 1,3-dicaffeoylquinic acid (36), 1,4-dicaffeoylquinic acid (37), 3,4-dicaffeoylquinic acid (38), and 3,5-dicaffeoylquinic acid (44).

1068 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

acid derivative was identified as dimethoxycinnamoylshikimicacid. It produced the MS2 base peak at m/z 155 ([shikimic

acid�H�H2O]�) and secondary peaks at m/z 207 [dimethoxycin-namic acid�H]� and m/z 137 [shikimic acid�H�2H2O]�.

C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071 1069

The last hydroxycinnamic acid derivative identified in this workwas a 5-p-coumaroylquinic acid (compound 11), due to the loss of146 amu, corresponding to a coumaroyl residue in MS2 spectrum,as reported in the literature (Jaiswal, Sovdat, Vivan, & Kuhnert,2010).

3.3. Characterisation of flavonol derivatives

3.3.1. Kaempferol derivativesCompounds 46 (found only in the ‘‘Chioggia’’ extract) and 57

both had a molecular ion at m/z 447. They were characterised bydifferent retention times but led to the same fragmentation pat-tern, albeit with different intensities. The relative abundance ofthe radical aglycone ion correlated closely with the glycosylationposition: compound 46 showed the base peak at m/z 285 due tothe loss of 162 amu, deriving from heterolytic cleavage of deproto-nated flavonoid glycosides (Ablajan et al., 2006; Lu et al., 2010),whereas compound 57 produced the radical aglycone ion at m/z284, deriving from homolytic cleavage, as the base peak. These fea-tures are characteristic of 7-O-glycosylation and 3-O-glycosylation,respectively. In addition, the MS2 spectrum of kaempferol-3-O-glu-coside disclosed an ion at m/z 255 that was more abundant thanthe ion at m/z 257, whereas the opposite was seen in kaempfer-ol-7-O-glucoside. Furthermore, compound 46 produced a fragment[M�H�120]� at m/z 327 that was not found in the correspondingspectrum of compound 57, indicating a different glycosidic linkage,as reported by Ablajan et al. (2006). Only the generic presence ofkaempferol monoglucoside had been described in Cichorium vege-tables (DuPont, Mondin, Williamson, & Price, 2000).

A kaempferol derivative (compound 13), found in all extractsbut ‘‘Verona’’, responded to positive ionisation as m/z 697. Its frag-mentation led to m/z 287, corresponding to the aglycone, to m/z449 due to the loss of a malonyl–glucose moiety, and to m/z 535from the loss of a glucose residue, suggesting that the two sugarresidues are not linked to the same position. The relative abun-dance of m/z 449 was higher than that of m/z 535, indicating thatneutral loss of the 7-O-(600-O-malonyl)-glucoside residue is morefavourable than that of 3-O-glucoside. The compound was there-fore identified as kaempferol-3-O-glucosyl-7-O-(600-O-malonyl)-glucoside (Ablajan et al., 2006). In both ‘‘Treviso’’ extracts anothercompound with the same UV–vis spectra and fragmentation pat-terns, but different intensities, was assigned to kaempferol-7-O-glucosyl-3-O-(600-O-malonyl)-glucoside (compound 21).

In ‘‘Chioggia’’ salad the presence of a kaempferol derivative(compound 16) was attested by formation in the MS2 spectrumof m/z 287, corresponding to the kaempferol aglycone.

According to the literature, the MS2 fragmentation of compound17, found only in the ‘‘Verona’’ and ‘‘Chioggia’’ extracts, is typical of3-O-sophorosides. In MS2 this compound revealed the base peak[M+H�324]+ and fragment ions [M+H�180]+ and [M+H�162]+,suggesting a sophoroside (1 ? 2 glycosidic linkage) in position 3-O of kaempferol (Schmidt et al., 2010).

Two peaks corresponding to kaempferol-7-O-(600-O-malonyl)-glucoside and kaempferol-3-O-(600-O-malonyl)-glucoside, respec-tively, were detected in positive ionisation mode and gave an ionat m/z 535 (compounds 50 and 56). Also in this case they yieldedthe same fragmentation pattern (m/z 287, m/z 286, and m/z 449),although with different intensities; the relative abundance of theradical aglycone ion allowed identification of the glycosylationposition.

Two different isomeric forms of kaempferol–monoglucuronide,compounds 51 and 53, were observed at different retention times.They both fragmented in positive ionisation mode to produce theradical aglycone ion at m/z 286 and the aglycone ion at m/z 287,due to the loss of a glucuronic acid residue. Based on the differentintensities of m/z 286 and m/z 287 they were identified as kaempf-

erol-7-O-glucuronide and kaempferol-3-O-glucuronide, respec-tively. The latter was not present in ‘‘Treviso’’ and ‘‘Trevisotardivo’’. The fragmentation of compound 54, found only in thetwo ‘‘Treviso’’ extracts, is typical of 3,7-disubstituted kaempferol:in fact, the base peak at m/z 449 indicated the loss of a glucuronicacid residue linked to the 3-O position, and the ion at m/z 287 indi-cated the subsequent loss of a glucose moiety. The compound wasidentified as kaempferol-3-O-glucuronil-7-O-glucoside.

Another two kaempferol derivatives with identical MM 594were detected in all extracts except ‘‘Verona’’. Kaempferol-7-O-neohesperidoside (compound 59) and kaempferol-7-O-rutinoside(compound 47) only differ by the interglycosidic linkage betweenthe monosaccharides rhamnose and glucose. The m/z 447 and m/z 285 ions correspond to the loss of rhamnose (146 amu) andrhamnosyl-glucose (308 amu) residues, respectively, which arecharacteristic of the glycan sequence. However, the relative abun-dances of the two ions were strikingly different, with a 1 ? 2 link-age between the monosaccharides (59) favouring the eliminationof the disaccharide residue to yield a deprotonated aglycone ion(Ma, Cuyckens, Van de Heuvel, & Claeys, 2001).

All extracts contained kaempferol-7-O-(600-O-acetyl)-glucoside(compound 60) and kaempferol-3-O-(600-O-acetyl)-glucoside (com-pound 61). The criteria described above were used to discriminatebetween the two isomers.

Compound 41 was a kaempferide–glucuronide with the molec-ular ion at m/z 475, yielding a MS2 fragment at m/z 299 [M�H�glu-curonic acid]�.

Compound 52, producing a pseudomolecular ion at m/z 549,was identified as kaempferide-3-O-(600-O-malonyl)-glucosidebased on the above consideration, in all extracts.

3.3.2. Quercetin derivativesCompound 12, not found in ‘‘Chioggia’’, produced the molecular

ion in positive ionisation mode at m/z 727. Its MS2 fragmentationled to an ion at m/z 479 (base peak); as generally happens in flavo-nol protonated 3,7-di-O-glycosides, loss of the 3-O-residue is morelikely than that of the 7-O-residue, therefore the malonyl–glucoseresidue should be linked at the 7-OH flavonol position. SubsequentMS3 experiments found a glucuronic acid moiety linked at 3-OH,allowing attribution of compound 12 to quercetin-3-O-glucuro-nyl-7-O-(600-O-malonyl)-glucoside.

A quercetin derivative (compound 42), found only in the ‘‘Vero-na’’ extract, was detected as a monoglycoside derivative; its MSfragmentation was characteristic of flavonol, with a hexoside at po-sition 7. The base peak was at m/z 303 and a secondary peak, deriv-ing from homolytic cleavage, was at m/z 302. These findings andthe UV–vis spectrum led to its identification as quercimeritrin(quercetin-7-O-glucoside). A compound with identical UV–vis dataand fragmentation pattern but eluting earlier, found in the two‘‘Treviso’’ extracts, was assigned to quercetin 7-O-galactoside(compound 39).

Quercetin-7-O-glucuronide (compound 43) was identified in‘‘Verona’’ and ‘‘Chioggia’’ extracts, while quercetin-7-O-acetylg-lucoside (m/z 505, compound 45) was observed in all four saladtypes.

Compound 40, found only in the two ‘‘Treviso’’ extracts, was as-signed to quercetin-3-O-(600-O-malonyl)-glucoside by comparisonof its MS2 fragmentation, retention time, and UV–vis spectrumwith those reported in the literature. Its fragmentation yielded abase peak at m/z 303 [M+H�248]+, corresponding to the loss of amalonyl–glucose residue, and a secondary ion at m/z 389 corre-sponding to the loss of a glucose residue.

Finally, quercetin-7-O-p-coumaroylglucoside (48) was identi-fied in all extracts by the very abundant ion at m/z 301 in theMS2 spectrum and by the ion at m/z 463, due to the loss of a cou-maroyl residue.

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3.3.3. Isorhamnetin derivativesAssignment of the aglycones to isorhamnetin was based on MS3

fragmentation. According to the literature (Stintzinga et al., 2004),methoxylated flavonoid aglycones can be distinguished by theirdifferent MS fragmentation profiles. While formation of a m/z165 A-ring fragment as the most prominent ion is a peculiarityof rhamnetin, isorhamnetin glycosides produce an intense m/z300 fragment in the MS3 event. In this study all these compoundsshowed m/z 300 fragments in the MS3 event and were thereforeidentified as isorhamnetin.

Peak 18, found only in ‘‘Verona’’ extract, produced the molecu-lar ion at m/z 519 in negative ionisation mode. The MS2 spectrumshowed the base peak at m/z 315 due to the loss of 204 amu (acet-yl-glucose residue); the compound was identified as isorhamentin-7-O-acetylglucoside.

In the ‘‘Chioggia’’ and the two ‘‘Treviso’’ extracts compound 49was identified as isorhamentin-7-O-neohesperidose. The MS2 spec-trum showed a base peak at m/z 315 formed by the loss of two su-gar moieties 1 ? 2 linked [M�H�308]� on the same phenolicposition.

Compound 55 yielded the base peak at m/z 317 in positive ion-isation mode and a secondary peak with low intensity at m/z 479due to the loss of a malonyl residue. It was thus identified as isorh-amentin-7-O-(600-O-malonyl)-glucoside.

The ‘‘Verona’’ and ‘‘Chioggia’’ extracts were characterised byisorhamnetin-7-O-glucoside (62, m/z 477), which fragmented tom/z 315. Isorhamnetin-7-O-glucuronide was identified (compound63) by the loss of a glucuronide moiety from the ion at m/z 491,leading to m/z 315 as the base peak in the MS2 spectrum.

3.3.4. Other flavonol derivativesTwo further flavonol derivatives were found in ‘‘Verona’’ ex-

tract. Peak 35, eluting earlier and characterised by m/z 491, was as-signed to tricin-3-O-glucoside. Its MS2 fragmentation led to m/z329 [M�H�162]� and m/z 311 [M�H�180]�, suggesting a glucosein 3-O position. Peak 58 gave the molecular ion at m/z 565 andfragmented to m/z 317 for the loss of 248 amu (malonyl–glucoseresidue) and a secondary ion at m/z 521 corresponding to the lossof a carboxylic group. It was therefore assigned to myricetin-7-O-(600-O-malonyl)-glucoside.

3.4. Flavone derivatives

One flavone derivative was identified in ‘‘Chioggia’’ and ‘‘Vero-na’’ as apigenin-7-O-glucoside. Peak 33 produced a pseudomolecu-lar ion at m/z 431 that fragmented to a very intense ion at m/z 269and a less intense ion at m/z 268, corresponding to the radical agly-cone. The ratio of the non-radical to the radical aglycone allowedidentification of a 7-OH substitution.

A chrysoeriol-3-O-glucoside (34) was identified, only in the‘‘Chioggia’’ extract, by the loss of a glucose moiety [M+H�162]+,leading to the aglycone ion at m/z 301.

3.5. Anthocyanidin derivatives

In all extracts the cyanidin aglycone (compound 26) was foundalong with some of its derivatives, identified by the loss of the gly-cosyl moiety on MS2. Compounds 27 and 28 with m/z 449 [M]+

were further discriminated as cyanidin-3-O-glucoside and cyani-din-3-O-galactoside by their elution order and by comparison withthe external standard (Ha et al., 2010). Compound 29, showing m/z491, was assigned to cyanidin-3-O-(600-O-acetyl)-glucoside, andcompound 24 (m/z 535) to cyanidin-3-O-(600-O-malonyl)-glucoside.This compound together with petunidin-3-O-(600-O-malonyl)-glu-coside (peak 25, m/z 565) were found only in the ‘‘Chioggia’’extract.

The fragmentation pattern of compound 23 with m/z 783,clearly showed the loss of 248 amu, corresponding to a malonyl–glucoside moiety, leading to m/z 535, which further fragmentedto m/z 287. This compound was therefore identified as cyanidin-3,5-di-O-(600-O-malonyl)-glucoside.

A delphinidin derivative was identified in all extracts with theexception of ‘‘Verona’’. It showed the molecular ion at m/z 713 thatfragmented to yield a m/z 551 [M�glucose]+, m/z 465 [M�malo-nyl�glucose]+ and m/z 303 (aglycone). Since acylated anthocyani-din 3,5-di-O-glycosides usually have their larger glycosilsubstituent in position 3, delphinidin 3-O-(600-O-malonyl)-gluco-side-5-O-glucoside was assigned to compound 22 (Lin, Sun, Chen,& Harnly, 2011).

Compound 31 was identified as pelargonidin-3-O-glucuronideby the loss of 176 amu (corresponding to a glucuronic acid moiety)from the [M]+ 447, yielding to the m/z 271 characteristic ofpelargonidin.

Finally, malvidin-3-O-glucoside (compound 30) was found onlyin the ‘‘Verona’’ extract by comparison of its retention time, UV–visspectrum and MS data with the external standard.

In summary, among the 35 compounds considered for the pro-filing, ‘‘Treviso’’ and ‘‘Treviso tardivo’’ show almost identicalcontents in polyphenols, differing only in the presence of5-caffeoylshikimic acid, unique to ‘‘Treviso’’ extract. ‘‘Chioggia’’ sal-ad shows a few unique compounds (24, 25, 16, 46, 34) but charac-teristic for this sample is also the lack of compounds 4, 6, 15, 32,12. It is noticeable the lack of 3- and 4-CQA in this cultivar. Onthe other hand ‘‘Verona’’ salad has 7, 30, 18, 35 and 58 as uniquecompounds and lacks of 22, 31, 9, 13, 59, 47, 49, 28. Furthermore‘‘Chioggia’’ and ‘‘Verona’’ share the presence of compounds 17,53, 43, 62 and 33, which are absent in the two ‘‘Treviso’’ extracts.

4. Conclusion

An HPLC-PDA–ESI-MSn method was applied to the separationand characterization of more than 60 polyphenols from four typesof Cichorium intybus var. silvestre (‘‘Treviso’’, ‘‘Treviso tardivo’’,‘‘Chioggia’’ and ‘‘Verona’’). Identification and evaluation were per-formed by comparing retention times and UV–vis and mass spectrawith the standards or/and with earlier publications. Most of theflavonol and flavone glycosides and of the hydroxycinnamic acidderivatives has never been described in these salads. MSn of caf-feoylquinic, feruloylquinic, and caffeoyltartaric acid pseudomolec-ular ions allowed differentiating individual isomers by theirfragmentation patterns. MS2 and MS3 analysis of the flavonoidcomponents of interest provided data on glycosylation type andposition, nature of the aglycones, and structure/linkage informa-tion of their glycan moieties. In addition chicoric acid was identi-fied as the major compound in C. intybus var. silvestre methanolicextracts.

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