Food Chemistry 138 (2013) 1062–1071

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Identification of phenolic constituents in red chicory salads (Cichorium intybus)

by high-performance liquid chromatography with diode array detection and
electrospray 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 a b s t r a c t

Article history: Phenolic acids and flavonoids extracted from several types of Cichorium intybus var. silvestre salads
Received 30 July 2012 (‘‘Chioggia’’, ‘‘Treviso’’, ‘‘Treviso tardivo’’, and ‘‘Verona’’) were characterised by high-performance liquid
Received in revised form 8 November 2012 chromatography–electrospray ionisation/mass spectrometry. Among the 64 compounds detected, several
Accepted 16 November 2012
hydroxycinnamic acid derivatives including 8 mono- and dicaffeoylquinic acids, 3 tartaric acid deriva-
Available online 23 November 2012
tives, 31 flavonol and 2 flavone glycosides, as well as 10 anthocyanins were characterised based on UV
spectra and MSn fragmentation patterns. Furthermore, several isomers of caffeic acid derivatives were
Keywords:
distinguished for the first time by their specific mass spectral data. This is the first study reporting the
HPLC-PDA–ESI/MSn
Flavonoids
glycosylation type and position of mono- and diglycosylated flavonoids in red salads.
Phenolic acids Ó 2012 Elsevier Ltd. All rights reserved.
Cichorium intybus var. silvestre

1. Introduction
High intakes of dietary fruit and vegetables are associated with
a reduced incidence of chronic diseases (Margett & Buttriss, 2003).
Their health-promoting factors, i.e. phytochemicals, have a wide
range of biological action, including antioxidant, anticancer, anti-
inflammatory, and a-glucosidase inhibition activities. The most
interesting 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 of
plant polyphenols (up to 1 g/day), which generally occur as glyco-
sides, because glycosylation makes them less reactive and more
water-soluble, hence easier to store in the cell vacuole. The flavo-
noids responsible for blue, red or purple leaf colours widespread
in the plant kingdom are anthocyanins. Their structures are based
on the C15 skeleton of anthocyanidins (consisting of a chromane
ring bearing a second aromatic ring B in position 2), which are
glycosylated and/or acylated at specific hydroxylated positions
(Delgado-Vargas & Paredes-Lopez, 2003). Cyanidin, delphinidin,
malvidin, pelargonidin, peonidin, and petunidin are the six most
common anthocyanin aglycones; glucose, rhamnose, galactose, xy-
lose, and arabinose are the most prevalent sugars, often acylated
with aromatic or aliphatic acids. Anthocyanins (of which US resi-
⇑ Corresponding author. Tel.: +39 0382 987863; fax: +39 0382 422975.
E-mail addresses: chiara.carazzone@unipv.it (C. Carazzone), dora.mascherpa@
unipv.it (D. Mascherpa), gabriella.gazzani@unipv.it (G. Gazzani), adele.papetti@
unipv.it (A. Papetti).
dents take up to 180–215 mg/day) have been found to afford arte-
rial protection, inhibit platelet aggregation, and protect endothelial
tissue, 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 among
the 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 (Cichorium
intybus L.) is a diploid plant species of the Asteraceae family; the
Cichorieae 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 northeastern
Italy, where due to their resistance to low temperatures they are
mostly 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 all
C. intybus var. silvestre. They have a distinctive, agreeable taste
and are popular on food markets including those of central Europe
and the United States. Radicchio ‘‘Treviso tardivo’’, a late winter
salad, has earned Protected Geographical Indication (PGI) and
Protected Designation of Origin (PDO) status.
A complete characterization of the polyphenols found in these
vegetables is not yet available. The aim of this work was a qualita-
tive investigation of the phenolic acid and flavonoid content of the
four cultivar of C. intybus var. silvestre. High-performance liquid
chromatography (HPLC) using a photodiode-array detector (PDA)

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.11.060
 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071
coupled with electrospray ionisation/mass spectrometry (ESI/MSn)
is the method of choice for such studies. MSn is the most effective
tool to elucidate polyphenol structures, since it provides informa-
tion on the molecular mass (MM) and fragmentation pattern of
analytes. Compared to the structural characterization of flavonoid
glycosides achieved with MS methods, HPLC–ESI/MSn provides
information 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 is
data-dependent acquisition, which employs user-specified criteria
to 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 dietary
intake.
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 standard
5-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 Acros
Organics (Geel, Belgium), cyanidin-3-O-glucoside and malvidin-3-
O-glucoside from Extrasynthese (Genay, France). HPLC-grade
water, used for sample preparation, was obtained with a Milli-Q
water purification system (Millipore, Billerica, MA). Filtration
membranes (0.45-lm cellulose acetate/cellulose nitrate mixed
esters) 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, suspended
in 12.5 mL of MeOH–HCOOH (99:1, v/v) and shaken for 1 h in an
ice bath in the dark. The mixture was then centrifuged for 5 min
at 8750g; the insoluble residue was re-extracted 3 times with a
fresh aliquot of the same mixture. The extracts obtained were
pooled, 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 Thermo
Finnigan 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 connected
to an LCQ Advantage Max ion trap mass spectrometer (all from
Thermo Fisher Scientific, Waltham, MA, USA) through an ESI
source.
Separation was achieved on a GeminiÒ 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) and
B (methanol) at a flow rate of 0.3 mL/min (injection volume
10 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, to
60% B in 10 min, and to 100% B in 10 min. An isocratic elution with
100% B was then carried out for 10 more minutes. The resulting to-
tal run time was 90 min, followed by column reconditioning
1063
(Papetti et al., 2008). The sample tray and column temperatures
were set at 4 °C.
The chromatogram was recorded at several wavelengths, char-
acteristic of different classes of polyphenols, such as 280 nm for
phenolic acids, 320 nm for hydroxycinnamic acids, 370 nm for
flavonols, 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 (100–1000
m/z), zoom scan, and MSn mode to obtain fragment ion m/z with
a collision energy of 35% and an isolation width of 3 m/z. Data-
dependent acquisition, where user-specified criteria are applied
to select the ion of interest for subsequent fragmentation, are
among the most useful approaches employed to identify unknown
compounds by MS. Using this approach single stage MS provides
the putative molecular mass that can be used in combination with
UV detection for a first tentative structure assignment; structure
elucidation and confirmation can then be obtained by tandem
MS analysis via the fragmentation pathway. When greater discrim-
ination was required additional targeted MS2 and MS3 experiments
were performed on selected pseudomolecular ions.
The negative and positive parameters of the ion mode ESI
source had previously been optimised by flow injection analysis,
using 5-CQA, kaempferol, and cyanidin-3-O-glucoside (5 ppm in
0.1% formic acid–methanol solution, 50:50, v/v) to a ionisation
voltage of 3.5 kV, a capillary temperature of 260 °C, a sheath gas
flow rate of 50 arbitrary units (AU), and an auxiliary gas flow rate
of 20 AU.
The Thermo Fisher Scientific Excalibur 2.0 software was used
for data acquisition and processing.
Three independent assays were performed to analyse each
methanolic 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 chromatographic
behaviour, UV–visible (UV–vis) spectra and mass spectra, and com-
parisons with the literature are addressed below and summarised
in Table 1.
Chromatographic peaks were preliminarily classified into
hydroxycinnamic acid, flavonol, and flavone derivatives, mainly
glycosides, according to PDA UV–vis spectra, when concentration
and resolution allowed to recover them. Phenolic compounds exhi-
bit absorbance maxima in the 275–285 nm wavelength region due
to the aromatic ring in their molecular structure. Phenolic acids
and flavonoids have characteristic UV–vis absorbances: hydroxy-
benzoic acids are detected at 280 nm, hydroxycinnamic acids at
320 nm, flavonols between 350 and 385 nm, and flavones in the
277–295 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, when
available, 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 glycosidic
linkage and concomitant H rearrangement leads to elimination of
the sugar residue, namely 162 amu (hexose; glucose or galactose),
146 amu (deoxyhexose; rhamnose), 132 amu (pentose; xylose or
arabinose), and 176 amu (glucuronic acid). The MS2 and MS3 prod-
uct ion spectra of flavonol 3,7-di-O-glycosides using negative ion
ESI-MS demonstrate that they can be differentiated from isomeric
mono-O-diglycosides and the glycosylation positions determined
(Ablajan et al., 2006).
1064 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

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

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

⁄,a 2
1 210 133 MS [133]: 115 (100), 89 (3) Malic acid
2⁄ 260 179 MS2[179]: 135 (100) Caffeic acid
3⁄,a 220, 272 191 MS2[191]: 173 (50), 127 (30), 111 (100) Quinic acid
4a 250, 323 353 MS2[353]: 191 (100), 179 (50), 135 (5) 3-Caffeoylquinic acid
303sh
5⁄ 246, 324 353 MS2[353]: 191 (100), 179 (3) 5-Caffeoylquinic acid
310sh
6a 252, 324 353 MS2[353]: 191 (15), 179 (60), 173 (100) 4-Caffeoylquinic acid
303sh
a,b,c 2
7 246, 324 353 MS [353]: 191 (100), 179 (2) cis-5-Caffeoylquinic acid
310sh
a,d 2
8 232, 277, 321 311 MS [311]: 179 (41), 149 (100), 135 (3) cis-Caftaric
9d 241, 327 311 MS2[311]: 179 (80), 149 (100), 135 (5) trans-Caftaric acid
310sh 623 MS2[623]: 491 (100), 311 (94)
10a,c,d 245, 320 335 MS2[335]: 179 (10), 161 (100), 135 (45) 5-Caffeoylshikimic acid
11d 241, 321 337 MS2[337]: 191 (100) 5-p-Coumaroylquinic acid
300sh
12a 256, 352 727+ MS2[727]: 479 (100), 303 (28) Quercetin-3-O-glucuronide-7-O-(600 -O-malonyl)-glucoside
MS3[479]: 303(100)
13d 264, 366 697+ MS2[697]: 535 (65), 449 (100), 287 (30) Kaempferol-3-O-glucosyl-7-O-(600 -O-malonyl)-glucoside
14 519 MS2[519]: 259 (100), 215 (25) Dimer of unknown acid
15a 234, 324 363 MS2[363]: 207 (30), 155 (100), 137 (46) Dimethoxycinnamoyl shikimic acid
16b,c,d 265, 366 463+ MS2[463]: 287 (100) Unknown kaempferol derivative
17b,c 263, 340 611+ MS2[611]: 449 (20), 431 (40), 287 (100) Kaempferol-3-O-sophoroside
18a,b,c 249, 268, 345 519 MS2[519]: 315 (100) Isorhamnetin-7-O-(600 -O-acetyl)-glucoside
MS3[315]: 300 (100)
19 242, 323 367 MS2[367]: 191 (100), 173 (10) 5-O-feruloyquinic acid
301sh
20 241, 327 473 MS2[473]: 311 (100), 293 (20), 179 (45), 149 (30) Dicaffeoyltartaric acid (chicoric acid)
305sh 947
a,d
21 265, 363 697+ 2
MS [697]: 535 (100), 449 (50), 287 (30) Kaempferol-7-O-glucosyl-3-O-(600 -malonyl)-glucoside
22d 279, 517 713+ MS2[713]: 465 (100), 551 (38), 303 (78) Delphinidin-3-O-(600 -O-malonyl)-glucoside-5-O-glucoside
23 280, 514 783+ MS2[783]: 535 (100), 287(40) Cyanidin-3,5-di-O-(600 -O-malonyl)-glucoside
24b,c,d 280, 515 535+ MS2[535]: 287 (100) Cyanidin-3-O-(600 -O-malonyl)-glucoside
25b,c,d 277, 525 565+ MS2[565]: 317 (100) Petunidin-3-O-(600 -O-malonyl)-glucoside
26 279, 516 287+ Cyanidin
27 280, 516 449+ MS2[449]: 287 (100) Cyanidin-3-O-galactoside
28⁄,d 280, 516 449+ MS2[449]: 287 (100) Cyanidin-3-O-glucoside
29 279, 514 491+ MS2[491]: 449 (12), 287 (100) Cyanidin-3-O-(600 -O-acetyl)-glucoside
30⁄,a,b,c 277, 527 493+ MS2[493]: 331 (100) Malvidin-3-O-glucoside
31 d 280, 513 447+ MS2[447]: 271 (100) Pelargonidin-3-O-monoglucuronide
32a 243, 322 367 MS2[367]: 191 (80), 173 (100) 4-O-feruloyquinic acid
301sh
33b,c 267, 366 431 MS2[431]: 269 (100), 268 (90) Apigenin-7-O-glucoside
34b,c,d 261, 350 463+ MS2[463]: 301(100) Chrysoeriol-3-O-glucoside
35a,b,c 251,357 491 MS2[491]: 329 (100), 311 (5), 293 (95) Tricin-3-O-glucoside
36 248, 324 515 MS2[515]: 353 (100), 335 (30) 1,3-Dicaffeoylquinic acid
301sh MS3[353]: 191 (100), 179 (50)
37 248, 325 515 MS2[515]: 353 (100), 299 (35), 317 (28), 255 (10), 203 (5) 1,4-Dicaffeoylquinic acid
302sh MS3[353]: 191 (24), 173 (100)
38 248, 323 515 MS2[515]: 353 (100), 299 (25), 255 (18), 203 (8) 3,4-Dicaffeoylquinic acid
302sh MS3[353]: 191 (35), 173 (100)
a,d
39 256, 351 463 MS2[463]: 301 (100), 300 (84) Quercetin-7-O-galactoside
40a,d 257, 368 551+ MS2[549]: 389 (54), 303 (100) Quercetin-3-O-(600 -O-malonyl)-glucoside
41 248,358 475 MS2[475]: 299 (100) Kaempferide glucuronide
42a,b,c 255, 351 465+ MS2[465]: 303 (100), 302 (68) Quercetin-7-O-glucoside
43b,c 255, 351 479+ MS2[479]: 303 (100) Quercetin-7-O-glucuronide
44 229, 325 515 MS2[515]: 353 (100), 191 (18) 3,5-Di-caffeoylquinic acid
302sh MS3[353]: 191 (100), 179 (50), 173 /2), 135 (10)
45 255, 349 505 MS2[505]: 301 (100) Quercetin-7-O-(600 -O-acetyl)-glucoside
46b,c,d 268, 366 447 MS2[447]: 327 (35), 285 (100), 284 (74), 257 (45), 255 (18) Kaempferol-7-O-glucoside
318sh
47d 262, 361 593 MS2[593]: 447 (43), 285 (100), 257 (25) Kaempferol-7-O-rutinoside
48 256, 351 609 MS2[609]: 463 (5), 301 (100), 300 (68) Quercetin-7-O-p-coumaroylglucoside
49d 248, 268, 341 623 MS2[623]:315 (100) Isorhamnetin-7-O-neohesperidoside
MS3[315]: 300 (100)
50 265, 365 535+ MS2[535]: 449 (35), 287 (100), 286 (38) Kaempferol-7-O-(600 -O-malonyl)-glucoside
51 261, 363 463+ MS2[463]: 286 (20), 287 (100) Kaempferol-7-O-glucuronide
52 253, 361 549+ MS2[549]: 463 (14), 301 (100) Kaempferide-3-O-(600 -O-malonyl)-glucoside
53a,d 261, 364 463+ MS2[463]: 286 (100), 287 (36) Kaempferol-3-O-glucuronide
54a,d 263,363 625+ MS2[625]: 449 (100), 287 (8) Kaempferol-3-O-glucuronide-7-O-glucoside
55a,d 249, 268, 342 565+ MS2[565]: 479 (4), 317 (100) Isorhamnetin-7-O-(600 -O-malonyl)-glucoside
MS3[317]: 302 (100)
56 264, 366 535+ MS2[535]: 449 (28), 287 (42), 286 (100) Kaempferol-3-O-(600 -O-malonyl)-glucoside
 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071 1065

Table 1 (continued)

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

57 ⁄
266, 318 447 MS2[447]: 285 (70), 284 (100), 257 (5), 255 (35) Kaempferol-3-O-glucoside
300sh
a,b,c 2
58 269, 374 565 MS [565]: 521 (40), 317 (100) Myricetin-7-O-(600 -O-malonyl)-glucoside
59d 264,360 593 MS2[593]: 447 (100), 285 (36), 257 (25) Kaempferol-7-O-neohesperidoside
60 263, 361 489 MS2[489]: 285 (100), 284 (15) Kaempferol-7-O-(600 -O-acetyl)-glucoside
61 263, 361 489 MS2[489]: 285 (10), 284 (100) Kaempferol-3-O-(600 -O-acetyl)-glucoside
62b,c 252, 269, 344 477 MS2[477]: 315 (100) Isorhamnetin-7-O-glucoside
MS3[315]: 300 (100)
63 251, 269, 342 491 MS2[491]: 315 (100) Isorahmnetin-7-O-glucuronide
MS3[315]: 300 (100)

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.
a
Not found in ‘‘Chioggia’’; bnot found in ‘‘Treviso’’; cnot found in ‘‘Treviso tardivo’’; dnot found in ‘‘Verona’’.

Ionisation was performed both in positive and in negative ion
mode. Combined use of ionisation in the two modes affords extra
certainty of the determination of the molecular mass. In the nega-
tive ionisation mode, hydroxybenzoic and hydroxycinnamic acids
deprotonated easily, whereas in the positive mode they formed ad-
ducts with the cations in the sample or mobile phase, i.e. sodium
ions. 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 the
range 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 their
UV–vis spectra and MS fragmentation patterns with standard com-
pounds. Peak 1, with an [MH] at m/z 133, produced the MS2 base
peak at m/z 115, corresponding to the loss of a water molecule
[MHH2O] and was assigned to malic acid (MM 134). Peak 3,
with an [MH] at m/z 191, with MS2 fragments at m/z 173
[MHH2O] and at m/z 127 [MHCO2H2O], was identified
as quinic acid (Gouveia & Castilho, 2009).
3.2. Characterisation of hydroxycinnamic acid derivatives
The presence of free and esterified phenolic acids in Cichorium
genus 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 base
peak at m/z 135, corresponding to the loss of a carboxyl group
[MHCO2]. We identified several caffeoyl and feruloyl deriva-
tives containing a quinic acid unit. Product ion scan experiments
of these compounds disclosed characteristic fragmentation involv-
ing the cleavage of one or two acyl moieties. The linkage position of
these 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-CQA
isomers, whose presence in nature is well known (Clifford, Zheng,
& Kuhnert, 2006; Ma, Dastmalchi, Whitaker, & Kennelly, 2011). The
literature for mono-CQA reports an ion at m/z 191 as the base peak
[MHcaffeoyl] when the acyl group is linked to the 3-OH or
5-OH position, and an ion at m/z 173 when the acyl group is linked

Fig. 1. HPLC-PDA chromatogram (200–600 nm) of the polyphenolic extract of Cichorium intybus var. silvestre ‘‘Treviso’’.
1066 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

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).

to the 4-OH position. This allowed differentiation of 3-CQA from
5-CQA solely on the basis of the relative intensity of m/z 179
[MHquinic acid], which is more significant for 3-OH com-
pounds (Schram, Miketiva, Slanina, Humpa, & Taborska, 2004;
Schütz, Kammerer, Carle, & Schieber, 2005). Based on these data
and on the comparison of retention times, UV–vis spectra, and
MS2 fragments with the commercial standard, compound 5 was
identified 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 fragmentation
patterns 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).
 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071
Fig. 3. Total ion chromatogram (TIC)of Cichorium intybus var. silvestre ‘‘Treviso’’ (a), ‘‘Treviso tardivo’’ (b), ‘‘Chioggia’’ (c), and ‘‘Verona’’ (d).
Four different isomers of diCQA were found in red chicories be-
sides monoCQA. Compounds 36, 37, 38, and 44, eluting later in the
chromatogram, gave [MH] at m/z 515, and MS2 fragment at m/z
353, corresponding to the loss of a caffeoyl moiety [MH162]
was the base peak (Fig. 4). Moreover, the presence and intensity
of MS2 secondary fragments allowed discriminating the different
diCQA isomers. Ion m/z 299 (intensity 35%) and the less intense
fragments at m/z 255 and 203 were unique to compounds 37 and
38 and are characteristic of 4-acyl diCQA. Furthermore, the pres-
ence of m/z 317 in the MS2 spectrum of compound 37 led to the
assignment to 1,4-diCQA. The MS3 spectra of the four compounds
were also quite different; compounds 36 and 44 showed a
fragment ion at m/z 179 (50% of the base peak), characteristic of
a 3-OH substituted quinic acid. This fragment was not found in
the spectra of compounds 37 and 38, which showed base peaks
at m/z 173, characteristic of a 4-OH substitution. Based on their
fragmentation 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). These
isomers 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) as
MS2 fragments and a low abundance signal at m/z 135 for the
decarboxylated 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 signal
at 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
1067
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 the
absorption maxima characteristic of hydroxycinnamic acid
(320 nm) was found in all extracts. It gave a pseudomolecular ion
at m/z 473 and a dimer at m/z 947, producing a base peak at m/z
311 by MS2 fragmentation due to the loss of 162 amu, correspond-
ing to a caffeoyl moiety. Other MS2 fragments were m/z 293
[MHcaffeoylH2O], m/z 179 [MHcaffeoyltartaric], and m/
z 149 [MHdicaffeoyl], leading to identification of di-caffeoyltar-
taric acid, also known as chicoric acid. The occurrence of this acid
has 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 the
same molecular ion at m/z 367. They shared similar UV–vis spectra
but different fragmentation patterns. Compound 19 gave the base
peak at m/z 191, due to the loss of a feruloyl moiety, and compound
32 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 comparison
with 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 179
for the loss of a caffeoyl moiety, m/z 161 (base peak), and m/z 135
(Jaiswal, Febi Matei, Ullrich, & Kuhnert, 2011b). The other shikimic
1068 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071

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).

acid derivative was identified as dimethoxycinnamoylshikimic acidHH2O]) and secondary peaks at m/z 207 [dimethoxycin-
acid. It produced the MS2 base peak at m/z 155 ([shikimic namic acidH] and m/z 137 [shikimic acidH2H2O].
 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071
The last hydroxycinnamic acid derivative identified in this work
was a 5-p-coumaroylquinic acid (compound 11), due to the loss of
146 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 derivatives
Compounds 46 (found only in the ‘‘Chioggia’’ extract) and 57
both had a molecular ion at m/z 447. They were characterised by
different retention times but led to the same fragmentation pat-
tern, albeit with different intensities. The relative abundance of
the radical aglycone ion correlated closely with the glycosylation
position: compound 46 showed the base peak at m/z 285 due to
the 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/z
284, 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 than
the ion at m/z 257, whereas the opposite was seen in kaempfer-
ol-7-O-glucoside. Furthermore, compound 46 produced a fragment
[MH120] at m/z 327 that was not found in the corresponding
spectrum of compound 57, indicating a different glycosidic linkage,
as reported by Ablajan et al. (2006). Only the generic presence of
kaempferol monoglucoside had been described in Cichorium vege-
tables (DuPont, Mondin, Williamson, & Price, 2000).
A kaempferol derivative (compound 13), found in all extracts
but ‘‘Verona’’, responded to positive ionisation as m/z 697. Its frag-
mentation led to m/z 287, corresponding to the aglycone, to m/z
449 due to the loss of a malonyl–glucose moiety, and to m/z 535
from the loss of a glucose residue, suggesting that the two sugar
residues are not linked to the same position. The relative abun-
dance of m/z 449 was higher than that of m/z 535, indicating that
neutral loss of the 7-O-(600 -O-malonyl)-glucoside residue is more
favourable 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 another
compound 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 spectrum
of m/z 287, corresponding to the kaempferol aglycone.
According to the literature, the MS2 fragmentation of compound
17, found only in the ‘‘Verona’’ and ‘‘Chioggia’’ extracts, is typical of
3-O-sophorosides. In MS2 this compound revealed the base peak
[M+H324]+ and fragment ions [M+H180]+ and [M+H162]+,
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 ion
at m/z 535 (compounds 50 and 56). Also in this case they yielded
the same fragmentation pattern (m/z 287, m/z 286, and m/z 449),
although with different intensities; the relative abundance of the
radical aglycone ion allowed identification of the glycosylation
position.
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 the
radical 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 different
intensities of m/z 286 and m/z 287 they were identified as kaempf-
1069
erol-7-O-glucuronide and kaempferol-3-O-glucuronide, respec-
tively. The latter was not present in ‘‘Treviso’’ and ‘‘Treviso
tardivo’’. The fragmentation of compound 54, found only in the
two ‘‘Treviso’’ extracts, is typical of 3,7-disubstituted kaempferol:
in fact, the base peak at m/z 449 indicated the loss of a glucuronic
acid 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 was
identified as kaempferol-3-O-glucuronil-7-O-glucoside.
Another two kaempferol derivatives with identical MM 594
were 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 between
the monosaccharides rhamnose and glucose. The m/z 447 and m/
z 285 ions correspond to the loss of rhamnose (146 amu) and
rhamnosyl-glucose (308 amu) residues, respectively, which are
characteristic 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 elimination
of 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 discriminate
between 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 [MHglu-
curonic acid].
Compound 52, producing a pseudomolecular ion at m/z 549,
was identified as kaempferide-3-O-(600 -O-malonyl)-glucoside
based on the above consideration, in all extracts.
3.3.2. Quercetin derivatives
Compound 12, not found in ‘‘Chioggia’’, produced the molecular
ion in positive ionisation mode at m/z 727. Its MS2 fragmentation
led 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 more
likely than that of the 7-O-residue, therefore the malonyl–glucose
residue should be linked at the 7-OH flavonol position. Subsequent
MS3 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 MS
fragmentation 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 and
the UV–vis spectrum led to its identification as quercimeritrin
(quercetin-7-O-glucoside). A compound with identical UV–vis data
and 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 salad
types.
Compound 40, found only in the two ‘‘Treviso’’ extracts, was as-
signed to quercetin-3-O-(600 -O-malonyl)-glucoside by comparison
of its MS2 fragmentation, retention time, and UV–vis spectrum
with those reported in the literature. Its fragmentation yielded a
base peak at m/z 303 [M+H248]+, corresponding to the loss of a
malonyl–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 the
MS2 spectrum and by the ion at m/z 463, due to the loss of a cou-
maroyl residue.
1070 C. Carazzone et al. / Food Chemistry 138 (2013) 1062–1071
3.3.3. Isorhamnetin derivatives
Assignment 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 their
different MS fragmentation profiles. While formation of a m/z
165 A-ring fragment as the most prominent ion is a peculiarity
of rhamnetin, isorhamnetin glycosides produce an intense m/z
300 fragment in the MS3 event. In this study all these compounds
showed m/z 300 fragments in the MS3 event and were therefore
identified as isorhamnetin.
Peak 18, found only in ‘‘Verona’’ extract, produced the molecu-
lar ion at m/z 519 in negative ionisation mode. The MS2 spectrum
showed 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 49
was 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 [MH308] on the same phenolic
position.
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 479
due 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 by
isorhamnetin-7-O-glucoside (62, m/z 477), which fragmented to
m/z 315. Isorhamnetin-7-O-glucuronide was identified (compound
63) 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 derivatives
Two 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/z
329 [MH162] and m/z 311 [MH180], suggesting a glucose
in 3-O position. Peak 58 gave the molecular ion at m/z 565 and
fragmented to m/z 317 for the loss of 248 amu (malonyl–glucose
residue) and a secondary ion at m/z 521 corresponding to the loss
of 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 269
and a less intense ion at m/z 268, corresponding to the radical agly-
cone. The ratio of the non-radical to the radical aglycone allowed
identification 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+H162]+,
leading to the aglycone ion at m/z 301.
3.5. Anthocyanidin derivatives
In all extracts the cyanidin aglycone (compound 26) was found
along 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 with
the external standard (Ha et al., 2010). Compound 29, showing m/z
491, was assigned to cyanidin-3-O-(600 -O-acetyl)-glucoside, and
compound 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 fragmented
to 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 the
exception of ‘‘Verona’’. It showed the molecular ion at m/z 713 that
fragmented to yield a m/z 551 [Mglucose]+, m/z 465 [Mmalo-
nylglucose]+ and m/z 303 (aglycone). Since acylated anthocyani-
din 3,5-di-O-glycosides usually have their larger glycosil
substituent 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-glucuronide
by the loss of 176 amu (corresponding to a glucuronic acid moiety)
from the [M]+ 447, yielding to the m/z 271 characteristic of
pelargonidin.
Finally, malvidin-3-O-glucoside (compound 30) was found only
in the ‘‘Verona’’ extract by comparison of its retention time, UV–vis
spectrum and MS data with the external standard.
In summary, among the 35 compounds considered for the pro-
filing, ‘‘Treviso’’ and ‘‘Treviso tardivo’’ show almost identical
contents in polyphenols, differing only in the presence of
5-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. On
the other hand ‘‘Verona’’ salad has 7, 30, 18, 35 and 58 as unique
compounds 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 separation
and characterization of more than 60 polyphenols from four types
of 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 spectra
with the standards or/and with earlier publications. Most of the
flavonol and flavone glycosides and of the hydroxycinnamic acid
derivatives has never been described in these salads. MSn of caf-
feoylquinic, feruloylquinic, and caffeoyltartaric acid pseudomolec-
ular ions allowed differentiating individual isomers by their
fragmentation patterns. MS2 and MS3 analysis of the flavonoid
components of interest provided data on glycosylation type and
position, 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 methanolic
extracts.
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