Polyphenolic profile of butterhead lettuce cultivar by ultrahigh performance liquid chromatography coupled online to UV–visible spectrophotometry and quadrupole time-of-flight mass spectrometry

Food Chemis y 260 : 239-273 (2018)
This documen is he Accep ed Manusc ip e sion o a
Published Wo k ha appea ed in inal o m in Food Chemis y
260 : 239-273(2018)
h ps://doi.o g/10.1016/j. oodchem.2018.03.151
© 2018. This manusc ip e sion is made a ailable unde he CC-BY-NC-
ND 4.0 license h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
DOI:10.1016/j. oodchem.2018.03.151
Polyphenolic p o ile o bu e head le uce cul i a by ul ahigh
pe o mance liquid ch oma og aphy coupled online o UV– isible
spec opho ome y and quad upole ime-o - ligh mass spec ome y
Gab iela E. Viaca a, Sa a I. Rou a, Diana M. López-Má quez,
Luis A. Be ue a, Blanca Gallo, Rosa M. Alonso-Salces
1
Polyphenolic p o ile o bu e head le uce cul i a by ul ahigh pe o mance liquid 1
ch oma og aphy coupled online o UV- isible spec opho ome y and quad upole ime-o -2
ligh mass spec ome y 3
4
Running i le: 5
Polyphenolic p o ile o bu e head le uce by UHPLC-DAD-ESI-QToF/MSE
6
7
Gab iela E. Viaca aa, Sa a I. Rou aa, Diana M. López-Má quezb, Luis A. Be ue ab, Blanca Gallob, 8
Rosa M. Alonso-Salcesc,*
9
10
a G upo de In es igación en Ingenie ía en Alimen os, CONICET, Depa amen o de Ingenie ía 11
Química y en Alimen os, Facul ad de Ingenie ía, Uni e sidad Nacional de Ma del Pla a, Juan B. 12
Jus o 4302, 7600 Ma del Pla a, A gen ina. E-mail add ess: [email p o ec ed]; 13
[email p o ec ed] 14
b Depa amen o de Química Analí ica, Facul ad de Ciencia y Tecnología, Uni e sidad del País 15
Vasco/Euskal He iko Unibe si a ea (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain. E-mail 16
add ess: [email p o ec ed]; luisangel.be ue [email protected]; [email p o ec ed] 17
c Depa amen o de Biología, CONICET, Facul ad de Ciencias Exac as y Na u ales, Uni e sidad 18
Nacional de Ma del Pla a, Funes 3350, 7600 Ma del Pla a, A gen ina. 19
20
* Co esponding au ho .21
E-mail add ess: osama [email protected]
23
*Manusc ip
2
Abs ac 24
In he p esen s udy, he bu e head le uce cul i a was analyzed by ul ahigh pe o mance liquid 25
ch oma og aphy (UHPLC) coupled online o diode a ay de ec ion (DAD), elec osp ay ioniza ion 26
(ESI) and quad upole ime-o - ligh mass spec ome y (QToF/MS) in he posi i e and nega i e ion 27
mode in o de o cha ac e ize i s polyphenolic p o ile o he i s ime. The ins umen acquisi ion 28
mode MSE was used o collec au oma ic and simul aneous in o ma ion o exac mass a high and 29
low collision ene gies o p ecu so ions as well as o he ions p oduced as a esul o hei 30
agmen a ion. One hund ed ele en phenolic compounds we e iden i ied in he acidi ied 31
hyd ome hanolic ex ac o eeze-d ied lea es o bu e head le uce cul i a : 40 hyd oxycinnamic 32
acid de i a i es, 21 hyd oxybenzoic acid de i a i es, 2 hyd oxyphenylace ic acid de i a i es, 18 33
la onols, 9 la ones, one la anone, 7 couma ins, one hyd olysable annin and 12 lignans. Fo y 34
se en o hese compounds ha e been en a i ely iden i ied o he i s ime in le uce. 35
36
Keywo ds: Lac uca sa i a, le uce, phenolic compounds, UHPLC-QToF, mass spec ome y, MSE
37
38
Chemical compounds s udied in his a icle: 39
5-Ca eoylquinic acid (PubChem CID: 12310830); ca eoylmalic acid (PubChem CID: 4484594); 40
4-hyd oxyphenylace ic acid (PubChem CID: 127); que ce in-3-O-galac oside (PubChem CID: 41
90657624); que ce in-3-O-glucu onide (PubChem CID: 5274585); kaemp e ol-3-O-glucu onide 42
(PubChem CID: 5318759); lu eolin 7-glucoside (PubChem CID: 5280637); lu eolin 7- u inoside 43
(PubChem CID: 44258082); escule in-6-O-glucoside (PubChem CID: 5281417); sy inga esinol 44
(PubChem CID: 100067). 45
46
3
1. In oduc ion 47
Phenolic compounds a e seconda y plan me aboli es ubiqui ous in he plan kingdom 48
in ol ed in p o ec ion mechanisms agains bio ic and abio ic s esses, in he egula ion o plan 49
g ow h and de elopmen , and in he o ganolep ic quali y o plan -based oods (Dai & Mumpe , 50
2010). Mo eo e , he in ake o phenolic compounds h ough ui s and ege ables ha e been p o ed 51
o p o ide bene icial e ec s a ibu ed o hei an ioxidan capaci y agains oxida i e s ess, cance 52
and ca dio ascula diseases, among o he s (Wa son, P eedy, & Zibadi, 2014). Le uce (Lac uca 53
sa i a L.) is one o he mos popula lea y ege ables. In pa icula , he bu e head le uce is one o 54
he mos commonly consumed a ie y wo ldwide (Agüe o, Viaca a, Ponce, & Rou a, 2013); 55
howe e , i s polyphenolic p o ile has no been cha ac e ized ye o he au ho s’ knowledge. The 56
main classes o phenolic compounds ound in di e en a ie ies o le uce a e phenolic acids and 57
la onols, ollowed by la ones and an hocyanins (only in ed a ie ies) (Ala cón-Flo es, Rome o-58
González, Ma ínez Vidal, & Ga ido F enich, 2016; Ma in, Fe e es, Ba be á, & Gil, 2015; Pepe, 59
Sommella, Man a, De Nisco, Teno e, Scopa e al., 2015). Mos analy ical me hods used o 60
de e mine polyphenols in le uce a e based on high o ul ahigh pe o mance liquid ch oma og aphy 61
(HPLC o UHPLC) coupled o diode a ay de ec ion (DAD) and/o mass spec ome y (MS and 62
MS/MS) (Abu-Reidah, Con e as, A áez-Román, Segu a-Ca e e o, & Fe nández-Gu ié ez, 2013; 63
Ala cón-Flo es e al., 2016; Al unkaya & Gökmen, 2009; Llo ach, Ma ínez-Sánchez, Tomás-64
Ba be án, Gil, & Fe e es, 2008; Pepe e al., 2015; Ribas-Agus í, G a acós-Cuba sí, Sá aga, 65
Ga cía-Reguei o, & Cas ella i, 2011). UHPLC achie es apid analysis and be e peak sepa a ion 66
han HPLC, and coupled o ToF o QToF ins umen s p o ides a highly a ac i e analy ical 67
echnique wi h e y high esolu ion and accu a e mass measu emen s o he p ecu so and agmen 68
ions (Rami ez-Amb osi, Abad-Ga cia, Vilo ia-Be nal, Ga mon-Loba o, Be ue a, & Gallo, 2013). 69
This echnique has been al eady used o cha ac e ize 95 phenolic compounds in h ee le uce 70
cul i a s (baby, omaine, and icebe g) (Abu-Reidah e al., 2013). Technological ad ances such as 71
he so called MSE da a acquisi ion mode has been success ully used o he s uc u al elucida ion o 72
4
phenolic compounds in complex plan ex ac s (Rami ez-Amb osi e al., 2013). MSE acquisi ion 73
me hod maximizes he QToF ins umen du y cycle pe o ming simul aneous collec ion o 74
p ecu so ions as well as o he ions p oduced as a esul o hei agmen a ion in exac mass mode 75
o e a single expe imen al un. Since many compounds s ill emain uniden i ied in le uce cul i a s 76
and he u iliza ion o analy ical edge echnology can p o ide new s uc u al in o ma ion and allow 77
he iden i ica ion o unknown polyphenols, he p esen s udy exploi s he use o UHPLC-DAD-ESI-78
QToF/MSE o he cha ac e iza ion o he polyphenolic p o ile o he bu e head le uce cul i a , 79
which is he e epo ed o he i s ime o he au ho s’ knowledge. 80
2. Ma e ials and Me hods 81
2.1. Reagen s, sol en s and s anda ds 82
Wa e , me hanol, ace oni ile, and o mic acid (Fishe Scien i ic, Fai Lawn, NJ, USA) we e 83
o Op ima® LC/MS g ade; asco bic acid (Pan eac, Ba celona, Spain), analy ical g ade; and glacial 84
ace ic acid (Me ck, Da ms ad , Ge many), Sup apu ® quali y. Leucine Enkephalin ace a e hyd a e 85
and sodium o ma e solu ion we e p o ided by Sigma-Ald ich Chemie (S einheim, Ge many). 86
Lu eolin-7-O-glucoside, kaemp e ol-3-O-glucoside, que ce in-3-O-galac oside, que ce in-3-O-87
hamnoside we e pu chased om Ex asyn hèse (Genay, F ance); ca eoyl a a ic acid and 88
que ce in-3-O-glucoside, om Ch omadex (I ine, CA, USA); 5-O-ca eoylquinic acid, p-couma ic 89
acid, 1,5-dica eoylquinic acid, 1,3-dica eoylquinic acid, and que ce in-3-O- u inoside, om 90
Sigma-Ald ich Chemie (S einheim, Ge many); and e ulic acid, ca eic acid, and 3,4-91
dihyd oxybenzoic acid, om Fluka Chemie (S einheim,Ge many). S anda d s ock solu ions o 92
phenolic compounds we e p epa ed in me hanol; and a e e ence solu ion o hese compounds 93
(5 g/mL), in me hanol-wa e -ace ic acid (30:65:5, / / ). 94
2.2. Plan ma e ial 95
Heads o bu e head le uce (Lac uca sa i a a . Lo es) we e ob ained om a local p oduce 96
in Sie a de los Pad es (Ma del Pla a, A gen ina). Le uce samples we e ozen wi h liquid ni ogen 97

5
and eeze-d ied, homogenized and c ushed o ob ain a homogeneous powde , which was s o ed a 98
oom empe a u e in da k in a desicca o un il analysis. 99
2.3. Ex ac ion o polyphenols in le uce 100
F eeze-d ied le uce (0.1 g) was ex ac ed wi h 5 mL o me hanol-wa e -ace ic acid (30:65:5, 101
/ / ) con aining asco bic acid (2 g/L) in an ul asonic ba h o 10 min. Then, he ex ac was 102
cen i uged a 6000 pm du ing 15 min a 4 ºC, and he supe na an was il e ed h ough a 0.45 µm 103
PTFE il e (Wa e s, Mil o d, CA, USA) p io o injec ion in o he UHPLC sys em. 104
2.4. UHPLC-DAD-ESI-QToF/MSE 105
Le uce ex ac was analyzed using an ACQUITY UPLCTM sys em om Wa e s (Mil o d, 106
MA, USA), equipped wi h a bina y sol en deli e y pump, an au osample , a column compa men 107
a PDA de ec o , and con olled by MassLynx 4.1 so wa e. A e e se phase Acqui y UPLC BEH 108
C18 column (2.1 mm × 100 mm, 1.7 µm) and a Acqui y UPLC BEH C18 VanGua dTM p e-column 109
(1.7 µm) om Wa e s (Mil o d, USA) we e used. Flow a e was 0.5 mL/min; injec ion olume, 5 110
µL; column and au osample empe a u es, 40ºC and 4 ºC espec i ely. Mobile phases consis ed o 111
0.1% ( / ) ace ic acid in wa e (A) and 0.1% ( / ) ace ic acid in me hanol (B). The elu ion 112
condi ions applied we e: 0–8.5 min, linea g adien 0–13% B; 8.5–11 min, 13% B isoc a ic; 11–113
12.3 min, linea g adien 13–15% B; 12.3–13.8 min, linea g adien 15–19% B; 13.8–17.3 min, 114
linea g adien 19–23% B; 17.3–19 min, 23% B isoc a ic; 19–24 min, linea g adien 23–30% B; 115
24–26 min, 30% B isoc a ic; 26–27 min, linea g adien 30–100% B; 27–28 min, 100% B isoc a ic; 116
and inally econdi ioning o he column wi h 100% A isoc a ic. UV- isible spec a we e eco ded 117
om 210 o 500 nm (20 Hz, 1.2 nm esolu ion). Hyd oxybenzoic acids we e moni o ed a 254 nm; 118
la anones a 280 nm; hyd oxycinnamic acids and couma ins a 320 nm; la onols and la ones a 119
370 nm. 120
All MS da a acquisi ions we e pe o med on a SYNAPTTM G2 HDMS wi h a quad upole 121
ime o ligh (QToF) con igu a ion (Wa e s, Mil o d, MA, USA) equipped wi h an elec osp ay 122
ioniza ion (ESI) sou ce ope a ing in bo h posi i e and nega i e modes. The capilla y ol age was se 123
6
o 0.7 kV (ESI+) o 0.5 kV (ESI−). Ni ogen was used as he desol a ion and cone gas a low a es 124
o 900 L/h and 10 L/h, espec i ely. The sou ce and desol a ion empe a u es we e 120 ºC and 125
400 ºC espec i ely. Leucine-enkephalin solu ion (2 ng/µL) in 0.1% ( / ) o mic acid in 126
ace oni ile-wa e (50:50, / ) was used o he lock mass co ec ion (m/z 556.2771 and 278.1141, 127
o m/z 554.2615 and 236.1035, depending on he ioniza ion mode, we e moni o ed a scan ime 0.2 128
s, in e al 10 s, scans o a e age 3, mass window ± 0.5 Da, cone ol age 30 V, a a low a e 129
10 µL/min). Da a acquisi ion was eco ded in he mass ange 50–1200 u in esolu ion mode 130
(FWHM ≈ 20,000) wi h a scan ime o 0.2 s and an in e scan delay o he 0.024 s, and au oma ically 131
co ec ed du ing acquisi ion based on he lock mass. Be o e analysis, he mass spec ome e was 132
mass calib a ed wi h he sodium o ma e solu ion. To pe o m MSE mode analysis, he cone ol age 133
was se o 20 V (ESI+) o 30 V (ESI−) and he quad upole ope a ed in a wide band RF mode only. 134
Two disc e e and independen in e lea ed acquisi ion unc ions we e au oma ically c ea ed. The 135
i s unc ion, ypically se a 6 eV in ap cell o he T-Wa e, collec s low ene gy o un agmen ed 136
da a while he second unc ion collec s high ene gy o agmen ed da a ypically using 6 eV in ap 137
cell and a collision amp 10–40 eV in ans e cell. In bo h cases, A gon gas was used o Collision 138
Induced Dissocia ion (CID). Da a we e eco ded in con inuous mode. Fo ins umen con ol, da a 139
acquisi ion and p ocessing MassLynxTM so wa e Ve sion 4.1 (Wa e s MS Technology, Mil o d, 140
USA) was used. 141
2.5. Iden i ica ion o phenolic compounds 142
The iden i ica ion o he phenolic compounds o which s anda ds we e a ailable was ca ied 143
ou by he compa ison o hei e en ion imes, hei UV– is spec a and MSE spec a eco ded in 144
posi i e and nega i e mode wi h hose ob ained by injec ing s anda ds in he same condi ions. The 145
iden i y o he es o compounds was elucida ed using he ollowing analy ical da a: i) he UV– is 146
spec um when i was a ailable o assign he phenolic class (Abad-Ga cía, Be ue a, Ga món-147
Loba o, Gallo, & Vicen e, 2009), since each class exhibi s a cha ac e is ic UV– is spec um 148
(Ma kham, 1982); ii) he low collision ene gy MSE spec um in posi i e and nega i e ion mode o 149
7
de e mine he molecula weigh ; and since only he p o ona ed/dep o ona ed molecules a e able o 150
o m in he elec osp ay ioniza ion sou ce adduc s, clus e s and/o molecula complexes wi h 151
mobile phase species (e.g. adduc s wi h sodium [M+Na]+ a 22 u abo e he p o ona ed molecule, 152
[2M+Na]+ o monoacyl hyd oxycinnamic acids, he dehyd a ed p o ona ed molecule ([M+H–153
H2O]+) o phenolic acids and diacyl hyd oxycinnamic acids in posi i e mode; and adduc s wi h 154
HSO4− (97 u) and AcO− (43 u) and he dep o ona ed dime ion [2M–H]− o monoacyl 155
hyd oxycinnamic acid in nega i e mode), hei p esence in he low collision ene gy spec a allows 156
he unequi ocal iden i ica ion o he [M+H]+ o [M−H]− ions; and iii) he high collision ene gy MSE 157
spec um p o ides he polyphenol agmen a ion pa e ns, which a o d s uc u al in o ma ion 158
ela ed o he ype o ca bohyd a es, he sequence o he glycan pa , in e glycosidic linkages and 159
he aglycone moie y, allowing o assign he p o ona ed aglycone [Y0]+ and/o he dep o ona ed 160
aglycone [Y0]−. The iden i ica ion o he aglycone was ca ied ou based on he obse a ion o i,jA+
161
and i,jB+ ions (Ma, Li, Van den Heu el, & Claeys, 1997). Fu he mo e, he ch oma og aphic elu ion 162
o de aided in some s uc u al assignmen s, as well as bibliog aphic e e ences. IUPAC 163
nomencla u e and ecommended numbe ing sys em (Lozac’h, 1975) we e used o chlo ogenic 164
acids and la onoids; and common names we e used o o he phenolic acid de i a i es, couma ins, 165
hyd olysable annins and lignan de i a i es. S uc u es o each amily o compounds s udied a e 166
p esen ed in Fig. 1. 167
3. Resul s and Discussion 168
A o al o 111 phenolic compounds we e en a i ely iden i ied in he bu e head le uce 169
cul i a by UHPLC-DAD-ESI-QToF/MSE. The UV- isible and MS spec al da a a e summa ized in 170
Table 1. DAD and MS ch oma og ams a e shown in Figs. 1S-5S (supplemen a y ma e ial). The 171
high and low ene gy unc ion MS spec a o compounds om he di e en phenolic amilies 172
de ec ed in his cul i a a e displayed in Figs. 2 and 3, and in Figs. 6S-9S (supplemen a y ma e ial). 173
3.1. Phenolic acid de i a i es 174
8
Fo he iden i ica ion o phenolic acid de i a i es, mainly nega i e ion mode mass spec a 175
we e aken in o accoun , al hough he posi i e ion mode was used o e i ica ion. In he high 176
collision ene gy MS spec a, losses o H2O, CO2 and CO we e egula ly obse ed, which ha e also 177
been desc ibed by o he au ho s using IT, QqQ, and QToF (Gómez-Rome o, M., Segu a-Ca e e o, 178
& Fe nandez-Gu ie ez, 2010; Rami ez-Amb osi e al., 2013). 179
3.1.1. Hyd oxycinnamic de i a i es 180
3.1.1.1. Ca eoylquinic acids 181
Th ee majo ch oma og aphic peaks (1, 3, 6), p esen ing he same UV spec a as he s anda d 182
ans-5-ca eoylquinic acid ( ans-5-CQA), we e de ec ed in he ch oma og ams ex ac ed om he 183
To al Ion Cu en (TIC) MS scan ch oma og am in nega i e and posi i e modes a m/z 353 and 355 184
espec i ely, which we e due o h ee ca eoylquinic acid (CQA) isome s (Fig. 2S in he 185
supplemen a y ma e ial). Compound 3 (R = 7.32 min, max= 300, 324 nm) was iden i ied 186
unambiguously as ans-5-ca eoylquinic acid by compa ison wi h i s s anda d: he dep o ona ed 187
molecule [M−H]− a m/z 353 yielded agmen ions a m/z 191, 173 and 135; and he p o ona ed 188
molecule [M+H]+, a m/z 163 and 145. Mo eo e , i s sodium adduc s, [M+Na]+ and [2M+Na]+ a 189
m/z 377 and 731 espec i ely, we e also obse ed (Fig. 6S in he supplemen a y ma e ial). 190
Compounds 1 (R = 4.74 min, max= 301, 323 nm) and 6 (R = 10.23 min, max= 301, 316 nm) had 191
he same agmen a ion pa e n as 5-CQA, and hei m/z alues o [M+H]+ and [M-H]− we e 192
con i med wi h he sodium adduc a m/z 377 in posi i e ioniza ion mode, and he [2M−H]− ion a 193
m/z 707 in nega i e mode. All h ee peaks (1, 3, 6) yielded he same base peak a m/z 191 due o he 194
dep o ona ed quinic moie y in he nega i e high ene gy unc ion. None o he peaks yielded an 195
in ense agmen ion a m/z 173 ([quinic acid–H–H2O]−). This dehyd a ed ion o quinic acid is 196
cha ac e is ically o med in he nega i e ion mode when he cinnamoyl g oup is bonded o he 197
quinic moie y a posi ion 4, as al eady no ed by o he au ho s using o he QqQ/MS (Alonso-Salces, 198
Guillou, & Be ue a, 2009) o IT/MS (Cli o d, Johns on, Knigh , & Kuhne , 2003). Peak 1 also 199
ga e in ense ions om he ca eoyl moie y ([ca eic acid–H–CO2]−) a m/z 135 (71% ela i e 200
15
Two ch oma og aphic peaks showed p o ona ed and dep o ona ed molecules ha 356
co esponded o p-couma oylca eoylquinic acids, a m/z 501 in he posi i e ion mode and a m/z 357
499 in he nega i e mode: peak 18 (R = 23.58 min, max= 312 nm) and peak 19 (R = 23.95 min, 358
max= 316 nm). In he posi i e high ene gy unc ion, he base peaks yielded by bo h isome s we e 359
he agmen ion a m/z 147 due o [p-couma oyl+H]+, disclosing ha he p-couma oyl moie y was 360
a ached o he quinic acid in a weake posi ion han he ca eoyl one. This was also suppo ed by 361
he agmen a ion pa e n obse ed o bo h peaks in he nega i e ion mode, which yielded he 362
dep o ona ed molecules, and agmen s a m/z 353 due o he loss o he p-couma oyl moie y (85-363
95% RA) (Fig. 2S in he supplemen a y ma e ial) and a m/z 337 due o he loss o he ca eoyl 364
moie y (40-50% RA) (Fig. 3S in he supplemen a y ma e ial) in he low ene gy unc ion, indica ing 365
ha he o me loss was a o ed. This agmen a ion pa e n was epo ed o 3-p-couma oyl-4-366
ca eoylquinic acid (3-pCo-4-CQA) and 4-ca eoyl-5-p-couma oylquinic acid (4-C-5-pCoQA) 367
(Cli o d, Ma ks, Knigh , & Kuhne , 2006). The dep o ona ed quinic acid ion a m/z 191 was he 368
base peak in he high ene gy unc ion; his agmen is a cha ac e is ic base peak o 5-CQA, 3-CQA 369
and 5-pCoQA, and is yielded by 4-CQA (Cli o d e al., 2003). Thus, aking also in o accoun ha 370
he elu ion o de on endcapped C18 packing is 3,4-isome s, 3,5-isome s and 4,5-isome s (Cli o d, 371
Ma ks, e al., 2006), compounds 18 and 19 we e en a i ely assigned o 3-pCo-4-CQA and 4-C-5-372
pCoQA espec i ely, o he i s ime in le uce cul i a s. p-Couma oylca eoylquinic acids ha e 373
been p e iously epo ed in le uce (Abu-Reidah e al., 2013; Jaiswal e al., 2011). 374
3.1.1.8. Dica eoyl a a ic acids 375
Two peaks (20, 21), p esen ing he same UV spec a as ca eic acid s anda d, we e de ec ed 376
in he ch oma og ams ex ac ed om he TIC MS scan ch oma og am in posi i e and nega i e 377
modes a m/z 475 and 473, espec i ely, which we e due o wo dica eoyl a a ic acid isome s 378
(diCTA). Compound 20 (R = 10.53 min, max= 301, 324 nm) and compound 21 (R = 12.54 min, 379
max= 301, 323 nm) p esen ed he same agmen a ion pa e n, and hei iden i y was con i med 380
wi h he sodium adduc a m/z 497 in posi i e ioniza ion mode and he [2M−H]− ion a m/z 947 in 381

16
nega i e mode o peak 20, and he p o ona ed and dep o ona ed molecules o peak 21. In he 382
nega i e ion mode, bo h peaks (20, 21) yielded he same base peak a m/z 293 due o he loss o 383
wa e o he dep o ona ed ca eoyl a a ic acid, and [CTA–H]− a m/z 311 due o he loss o one o 384
he ca eoyl moie ies (Fig. 3S in he supplemen a y ma e ial), as well as ions om he a a ic 385
moie y, [ a a ic acid–H]− a m/z 149 and [ a a ic acid–H–CO2]− a m/z 105; and ions om he 386
ca eoyl moie y, [ca eic acid–H]− a m/z 179 and [ca eic acid–H–CO2]− a m/z 135. Compound 20 387
was en a i ely iden i ied as di-O-ca eoyl a a ic (chico ic acid), and compound 21 as meso-di-O-388
ca eoyl a a ic acid, since hey we e de ec ed in le uce elsewhe e; he o me being epo ed as he 389
mos abundan as we obse ed (Abu-Reidah e al., 2013; Jeong e al., 2015; Lin e al., 2012; Mai & 390
Glomb, 2013; Pepe e al., 2015; Ribas-Agus í e al., 2011; Romani e al., 2002; San os e al., 2014). 391
3.1.1.9. O he hyd oxycinnamic acid de i a i es 392
Se e al cinnamoyl glycosides we e ound in he le uce ex ac s, such as ca eoyl-hexosides, 393
p-couma oyl-hexosides, sinapoyl-hexosides and dihyd oca eic acid-hexosides, whose 394
agmen a ion pa e ns we e cha ac e ized by he aglycone p oduc ion esul ed om he loss o a 395
hexose esidue (Abu-Reidah e al., 2013; Gómez-Rome o, Ma ía, Zu ek, Schneide , Baessmann, 396
Segu a-Ca e e o, & Fe nández-Gu ié ez, 2011). 397
Eigh peaks (22, R = 5.39 min; 23, R = 5.64 min; 24, R = 6.08 min, max= 301, 325 nm; 25, 398
R = 7.69 min; 26, R = 8.44 min; 27, R = 9.01 min; 28 R = 9.52 min; and 29 R = 9.64 min) we e 399
obse ed in he ch oma og am ex ac ed a m/z 343 and 341 in posi i e and nega i e ion modes 400
espec i ely (Fig. 2S in he supplemen a y ma e ial). All o hem (22-29) p oduced m/z 179 and 135 401
in nega i e ion mode, and m/z 163, 145, 135, 117 and 89 in posi i e ion mode, consis en wi h he 402
p esence o a ca eic acid esidue. Thus, hese compounds we e en a i ely assigned as isome ic 403
ca eic acid-hexosides, in ag eemen wi h Cli o d e al. (2007) (Cli o d e al., 2007). Mo eo e , 404
he iden i y o peaks 22-26 and 28 we e con i med by he p esence o hei sodium adduc s in he 405
posi i e low ene gy unc ion. As well, peak 30 (R = 8.01 min, max= 301, 325 nm) showed he 406
same agmen a ion pa e n as ca eic acid, yielding also a monoiso opic p o ona ed molecule a 407
17
m/z 359.0802 (C18H15O8) in he posi i e ion mode, and a monoiso opic dep o ona ed molecule a 408
m/z 357.0633 (C18H13O8) in he nega i e ion mode. Thus, i was en a i ely assigned as a ca eoyl 409
de i a i e, howe e he na u e o he non-phenolic esidue (196.0387 u) was no able o be 410
disclosed. Such ca eoyl de i a i e has no p e iously been epo ed in le uce so a we a e awa e. 411
Simila ly, ou isome s o synapic acid-hexosides (31, R = 6.03 min, max= 301, 326 nm; 412
32, R = 9.70 min; 33, R = 10.36 min; 34, R = 13.13 min) we e en a i ely iden i ied in he ex ac ed 413
aces a m/z 387 and 385 in he posi i e and he nega i e ion modes espec i ely (Fig. 2S in he 414
supplemen a y ma e ial). Ions co esponding o he dep o ona ed aglycone a m/z 223, and he 415
subsequen deca boxyla ions and losses o me hyl esidues a m/z 208, 179, 164, and 149 om he 416
synapoyl moie y we e de ec ed in he nega i e ion mode. In addi ion, he posi i e ion mode yielded 417
he sodium adduc a m/z 409 and ions due o he loss o he hexose esidue a m/z 225, and 418
subsequen losses o H2O a m/z 207, CH3OH a m/z 192, and CO a m/z 129. One isome o synapic 419
acid-hexoside has been p e iously epo ed in g een le uce cul i a s (Abu-Reidah e al., 2013). 420
Following his agmen a ion pa e ns, a p-couma ic acid-hexoside (35, R = 8.32 min) and 421
wo dihyd oca eic acid-hexosides (36, R = 3.70 min; 37, R = 3.83 min) we e also cha ac e ized. 422
All o hem yielded he p oduc ion due o he loss o he hexose esidue (m/z 163 o 35, m/z 181 423
o 36 and 37), wi h he subsequen losses o H2O, CO and CO2 in he nega i e ion mode; and he 424
sodium adduc in he posi i e ion mode (m/z 349 o 35, m/z 367 o 36 and 37). 425
Se en ca eic acid-hexosides, a synapic acid-hexosides, a dihyd oca eic acid-hexoside and a 426
p-couma ic acid-hexoside ha e been p e iously epo ed in g een le uce cul i a s (Abu-Reidah e 427
al., 2013). In he p esen wo k, one mo e ca eic acid-hexoside, a dihyd oca eic acid-hexoside and 428
h ee synapic acid-hexosides we e iden i ied in he bu e head le uce cul i a . 429
Peaks 38 (R = 11.81 min, max= 307 nm), 39 (R = 14.47 min) and 40 (R = 16.48 min) we e 430
en a i ely p oposed as isome s o e ulic acid me hyl es e s. Acco ding o p e ious da a (Abu-431
Reidah e al., 2013; Gómez-Rome o, Ma ía e al., 2011), hese compounds showed deme hyla ed 432
agmen ions a m/z 192 ([M–H–CH3]−) and m/z 177 ([M–H–2CH3]−), which is cha ac e is ic o he 433
18
me hoxyla ed cinnamic acids. Two o hese isome s o e ulic acid me hyl es e s ha e been 434
p e iously epo ed in g een le uce cul i a s. 435
3.1.2. Hyd oxybenzoic de i a i es 436
Hyd oxybenzoic de i a i es we e no de ec ed in he posi i e ion mode. Thus, no peaks we e 437
de ec ed in he ch oma og ams ex ac ed om he TIC MS scan ch oma og am a he p o ona ed 438
molecule o he sodium adduc masses o he hyd oxybenzoic de i a i es obse ed in he nega i e 439
ion mode. Only one o he wo p e iously epo ed in g een le uce cul i a s (Abu-Reidah e al., 440
2013) isome s o hyd oxybenzoic acid (41: R = 4.67 min) and dihyd oxybenzoic acid (42: R = 5.42 441
min) we e de ec ed a m/z 137 and m/z 153 espec i ely (Fig. 2S in he supplemen a y ma e ial). 442
Thei co esponding deca boxyla ed ions we e also obse ed a m/z 93 and m/z 109 espec i ely. 443
Se e al hyd oxybenzoic glycoside es e s we e cha ac e ized acco ding o hei MS da a and 444
agmen a ion pa e n by he neu al loss o he glycosidic moie y. Hyd oxybenzoic acid-hexosides 445
(43, R = 4.22 min; 44, R = 5.15 min) yielded he dep o ona ed ion a m/z 299 and he p oduc ions 446
due o losses o he hexose esidue (m/z 137) and CO2 (m/z 93). Dihyd oxybenzoic acid-hexosides 447
(45, R = 2.49 min; 46, R = 2.69 min; 47, R = 3.74 min; 48, R = 3.91 min; 49, R = 4.48 min; 50, R = 448
4.68 min) p oduced he dep o ona ed molecule a m/z 315 (base peak), an odd elec on p oduc ion 449
a m/z 152 co esponding o he loss o hexose plus H (163 u), an e en elec on ion a m/z 153 due 450
o he loss o hexose (Fig. 2S in he supplemen a y ma e ial), he dehyd a ed ion a m/z 135, and he 451
deca boxyla ed ion a m/z 109, in ag eemen wi h bibliog aphy (Abu-Reidah e al., 2013). Hence, 452
one mo e hyd oxybenzoic acid-hexoside and ou mo e dihyd oxybenzoic acid-hexosides a e he e 453
de ec ed in bu e head le uce han in p e ious s udies on di e en le uce cul i a s. The elease o 454
such unusual losses was also obse ed o gallic acid-hexoside isome s. Thus, peaks 51 (R =2.80 455
min), 52 (R =2.88 min) and 53 (R =6.61 min) we e en a i ely p oposed as gallic acid-hexosides, 456
since hey yielded he dep o ona ed molecule a m/z 331 (base peak) (Fig. 3S in he supplemen a y 457
ma e ial), and an odd elec on p oduc ion a m/z 168, co esponding o he loss o hexose plus H 458
(163 u), an e en elec on ion a m/z 169 due o he loss o hexose, and [gallic acid–H–CO2]− a m/z 459
19
125. Two isome s o gallic acid-hexoside ha e been de ec ed p e iously only in he le uce c . baby 460
(Abu-Reidah e al., 2013). 461
Aside om he loss o he hexose moie y, sy ingic acid-hexoside (54, R = 5.90 min, m/z 359) 462
showed subsequen losses o CH3 om he me hoxy g oups o he aglycone and CO2 (m/z 182, 153, 463
138 and 123), as p e iously obse ed in li e a u e (Abu-Reidah e al., 2013; Gómez-Rome o, Ma ía 464
e al., 2011). 465
In ag eemen wi h p e ious s udies (Abu-Reidah e al., 2013), compounds 55 (R = 17.09 466
min) and 56 (R = 24.83 min) showing a dep o ona ed molecule a m/z 451 we e en a i ely assigned 467
as hyd oxybenzoyl-gallic acid-hexosides (Fig. 3S in he supplemen a y ma e ial). The high ene gy 468
unc ion yielded he agmen ion co esponding o he dep o ona ed gallic acid-hexoside a m/z 469
331, a e he loss o he hyd oxybenzoyl moie y (120 u). As well, p oduc ions due o successi e 470
losses o H2O a m/z 313, hexose plus H a m/z 168 and CO2 a m/z 124 we e obse ed. A simila 471
pa e n was ound o he hyd oxybenzoyl-dihyd oxybenzoic acid-hexosides (57, R = 17.68 min; 472
58, R = 19.41 min; 59, R = 23.64 min; 60, R = 26.88 min, max= 256, 335 nm; 61, R = 27.09 min) 473
de ec ed in he ex ac ed ace a m/z 435 (Fig. 3S in he supplemen a y ma e ial). Fo peak 59, only 474
he dep o ona ed molecule was de ec ed due o i s low concen a ion in he ex ac . All o he 475
isome s yielded he agmen ions co esponding o [dihyd oxybenzoic acid-hexoside–H]− a m/z 476
315, and he subsequen losses o H2O a m/z 297 and hexose plus H a m/z 152 and CO2 a m/z 108. 477
Peaks 58 and 61 showed he p oduc ion [dihyd oxybenzoic acid–H]− due o an e en elec on ion a 478
m/z 153 (loss o hexose), ins ead o he odd elec on p oduc ion a m/z 152. Besides, peaks 57, 60 479
and 61, yielded he agmen ion [hyd oxybenzoic acid–H]− a m/z 137 and i s co esponding 480
deca boxyla ion ion a m/z 93. This beha iou ag ees wi h ha obse ed o hyd oxycinnamic acid 481
glycosides abo e and in li e a u e (Cli o d e al., 2007), which sugges ha bo h, he 482
hyd oxybenzoic acid moie y and he dihyd oxybenzoic acid moie y, a e a ached h ough hei 483
phenolic hyd oxyl o di e en posi ions o he same hexose molecule. Jus one isome o 484
20
hyd oxybenzoyl-gallic acid-hexoside and wo isome s o hyd oxybenzoyl-dihyd oxybenzoic acid-485
hexosides ha e been p e iously cha ac e ized only in c . baby le uce (Abu-Reidah e al., 2013). 486
3.1.3. Hyd oxyphenylace ic de i a i es 487
Taking in o accoun he MS da a, he agmen a ion pa e ns obse ed o hyd oxybenzoic 488
acid in he nega i e ion mode and bibliog aphy (Abu-Reidah e al., 2013; Gómez-Rome o, Ma ía e 489
al., 2011), 4-hyd oxyphenylace ic acid was en a i ely assigned o peak 62 (R = 5.60 min) (Fig. 4S 490
in he supplemen a y ma e ial), which yielded he dep o ona ed molecule a m/z 151 and agmen 491
ions due o he loss o CO a m/z 123 and CO2 a m/z 107, showing he ypical deca boxyla ion o 492
phenolic acids. Likewise, peak 63 (R = 5.20 min, max= 270, 276 nm) obse ed in he ex ac ed 493
ace a m/z 313, p oduced he same deca boxyla ion ions, and a agmen ion a m/z 151 due o 494
dep o ona ed 4-hyd oxyphenylace ic acid ob ained a e he loss o a hexose moie y (Fig. 4S in he 495
supplemen a y ma e ial). Thus, i was p oposed as 4-hyd oxyphenylace ic acid-hexoside. Bo h 496
compounds ha e been p e iously de ec ed in g een le uce cul i a s (Abu-Reidah e al., 2013). 497
3.2. Fla onoids 498
3.2.1. Fla onols 499
Thi een que ce in glycosides (64-76) and ou kaemp e ol glycosides (77-80) we e de ec ed 500
and iden i ied on he basis o hei mass spec al da a, compa ison wi h a ailable s anda ds, and 501
li e a u e. Fla onol monoglycoside mass spec a in he posi i e mode showed he p o ona ed 502
molecule [M+H]+, he sodium adduc ion [M+Na]+ and he p o ona ed aglycone ion [Y0]+ as a esul 503
o he loss o he suga o o ganic acid esidue (losses: 146 u, hamnosyl esidue; 162 u, hexosyl 504
esidue; 176 u, glucu onic esidue; 178 u, gluconic esidue; 248 u, malonyl-hexosyl esidue; 324 u, 505
di-hexosyl esidue; 338 u, glucu onic + hexosyl esidue; 410 u, hexosyl + malonyl-hexosyl esidue; 506
424 u, glucu onic + malonyl-hexosyl esidue). In he mass spec um o la onol diglycosides, a 507
agmen [Y1]+ due o he loss o he i s suga o o ganic acid uni was also obse ed. In he 508
nega i e mode, he high ene gy unc ion p oduc ions co esponding o que ce in a m/z 300 (odd 509
elec on ion) and/o 301 (e en elec on ion) we e de ec ed (Fig. 4S in he supplemen a y ma e ial), 510

21
as obse ed in MS/MS elsewhe e (Abu-Reidah e al., 2013). Rega ding his, compounds 64 (R = 511
17.16 min, max= 279, 344 nm), 65 (R = 18.03 min, max= 252, 367 nm) and 66 (R = 20.25 min, 512
max= 252, 330 nm) we e iden i ied as que ce in-3-O-hexosides on he basis o hei p o ona ed 513
molecule a m/z 465 and a high ene gy unc ion p oduc ion a m/z 303, which indica es clea age o 514
a hexosyl g oup. This agmen a ion pa e n and ch oma og aphic e en ion ime o he e e ence 515
s anda d con i med ha compound 66 was que ce in-3-O-galac oside. Two isome s o que ce in 516
hexose ha e been p e iously desc ibed in le uce (Abu-Reidah e al., 2013; Becke , Klae ing, 517
Sch eine , K oh, & K umbein, 2014; Jeong e al., 2015; Lin e al., 2012; Llo ach e al., 2008; Mai & 518
Glomb, 2013; Ma in e al., 2015; Pepe e al., 2015; Romani e al., 2002; San os e al., 2014; So o, 519
Lundegå dh, Må ensson, Man a, Pepe, Sommella e al., 2016). 520
Compound 67 (R = 18.44 min, max= 254, 349 nm) was iden i ied as que ce in-3-O-521
glucu onide because o [M+H]+ a m/z 479, [M+Na]+ a m/z 501 and [Y0]+ a m/z 303, which 522
indica ed he loss o a glucu onic esidue in he posi i e mode (Fig. 2). Simila ly, in he nega i e 523
mode, he molecule [M−H]− a m/z 477 yielded [Y0]− a m/z 301; he loss o 176 u poin ed ou he 524
p esence o a glucu onic esidue (Fig. 2). The p esence o que ce in-3-O-glucu onide in le uce had 525
been p e iously con i med by nuclea magne ic esonance analysis (DuPon , Mondin, Williamson, 526
& P ice, 2000; Mai & Glomb, 2013). The glucu onic g oup was also obse ed in compound 68 (R = 527
9.50 min, max= 256, 352 nm) and compound 69 (R = 10.58 min), which ga e [M+H]+ a m/z 641, 528
[M+Na]+ a m/z 663, and [Y0]+ a m/z 303 in posi i e mode, and peak 69, also [Y1]+ a m/z 465. In 529
he nega i e mode, bo h compounds p esen ed simila ioniza ion and agmen a ion pa e n: [M−H]− 530
a m/z 639, [Y1]− a m/z 463 and [Y0]− a m/z 300 (odd elec on ion) and/o 301 (e en elec on ion). 531
Mo eo e , he loss o 162 u e ealed he clea age o a hexoxyl g oup, he e o e hese la onols 532
we e assigned o que ce in hexose-glucu onide isome s, which had been al eady desc ibed in baby, 533
omaine and icebe g cul i a s (Abu-Reidah e al., 2013). 534
Compounds 70 (R = 21.52 min, max= 255, 352 nm), 71 (R = 22.03 min, max= 252, 364 535
nm) and 72 (R = 23.69 min) we e iden i ied as que ce in malonylhexoside isome s since hey 536
22
p esen ed [M+H]+ a m/z 551, [M+Na]+ a m/z 573, and [Y0]+ a m/z 303 due o he loss o he 537
malonylhexosyl moie y in he posi i e ion mode; and [M−H]−a m/z 549, [Y0]− a m/z 301 (Fig. 4S 538
in he supplemen a y ma e ial), [M−H−CO2]− a m/z 505 (base peak) in he nega i e ion mode. The 539
neu al loss o CO2 is cha ac e is ic o compounds p esen ing he malonyl g oup, as p e iously 540
epo ed (Abu-Reidah e al., 2013). This ac is due o in-sou ce agmen a ion, which can a ec he 541
co ec iden i ica ion o he dep o ona ed molecule o in e es , because he ela i e abundance o 542
[M−H]− ion could be lowe han he p oduc ion [M−H−CO2]− as occu ed wi h hese peaks. This 543
pa icula ly labile g oup could be pa ially los du ing ion ans e om a highe -p essu e egion o 544
he sou ce o a lowe -p essu e egion (Ka a, Chowdhu y, & Chai , 1991), as obse ed o peak 70 545
(0.4 % RA), peak 71 (11 % RA) and peak 72 (0.4 % RA). The iden i ica ion o compound 70 was 546
also con i med by he p esence o [2M−H]− ion. Que ce in-3-O-(6’’-O-malonyl)-glucoside has been 547
epo ed in le uce in se e al publica ions (Becke e al., 2014; DuPon e al., 2000; Fe e es, Gil, 548
Cas añe , & Tomás-Ba be án, 1997; Heimle , Isolani, Vignolini, Tombelli, & Romani, 2007; 549
Llo ach e al., 2008; Mai & Glomb, 2013; Ma in e al., 2015; Ribas-Agus í e al., 2011; Romani e 550
al., 2002; San os e al., 2014), and con i med by NMR analysis (DuPon e al., 2000; Fe e es e al., 551
1997). Two isome s o que ce in malonylglucoside we e al eady desc ibed in di e en le uce 552
a ie ies (Abu-Reidah e al., 2013; Lin e al., 2012). The p esence o h ee que ce in 553
malonylhexoside isome s in le uce is desc ibed o he i s ime in he p esen s udy. 554
Compound 73 (R = 11.51 min, max= 253, 355 nm) was iden i ied as que ce in-3-O-(6''-O-555
malonyl)-glucoside-7-O-glucu onide, which has been p e iously desc ibed in le uce (Abu-Reidah 556
e al., 2013; Llo ach e al., 2008; San os e al., 2014). In he posi i e ion mode, [M+H]+ a m/z 727, 557
[M+Na]+ a m/z 749, and he agmen ions [Y1]+ a m/z 479 and [Y0]+ a m/z 303 indica ed he loss 558
o a malonyl-glucosyl g oup ollowed by a glucu onic g oup. In he nega i e ion mode, he neu al 559
loss o CO2 yielding [M−H−CO2]− a m/z 681 con i med he p esence o a malonyl esidue in he 560
molecula s uc u e; as well as he high ene gy unc ion p oduc ions a m/z 300 (odd elec on ion) 561
and/o 301 (e en elec on ion) (Fig. 4S in he supplemen a y ma e ial), he p esence o que ce in. 562
23
Simila ly, compound 74 (R = 13.82 min, max= 253, 350 nm) also con ained a malonyl esidue 563
since i s base peak in he nega i e mode was [M−H−CO2]− a m/z 667. The dep o ona ed molecule
564
a m/z 711 was also p esen and [Y0]− a m/z 300 (odd elec on ion) and/o 301 (e en elec on ion) 565
(Fig. 4S in he supplemen a y ma e ial) indica ed ha he aglycone was que ce in. The posi i e ion 566
mode yielding [M+H]+ a m/z 713, [M+Na]+ a m/z 735, and he agmen ions [Y1]+ a m/z 465 and 567
[Y0]+ a m/z 303 con i med he clea age o malonylhexosyl g oup ollowed by a hexosyl g oup. 568
Thus, compound 74 was en a i ely assigned o que ce in-3-O-(6''-O-malonyl)-glucoside-7-O-569
glucoside, which has been p e iously epo ed in le uce (Abu-Reidah e al., 2013; Llo ach e al., 570
2008; San os e al., 2014), and con i med by NMR analysis (Fe e es e al., 1997). 571
Compounds 75 (R = 12.18 min) and 76 (R = 16.07 min) p esen ed he same monoiso opic 572
molecula mass o [M+H]+ a m/z 627.1580 (C27H31O17) and [M−H]−a m/z 625.1405 (C27H29O17), 573
and [M+Na]+ a m/z 649.1381 (C27H30O17Na). The p esence o [Y0]+ a m/z 303 and [Y0]− a m/z 301 574
(Fig. 4S in he supplemen a y ma e ial) in he posi i e and nega i e ion modes, espec i ely, 575
disclosed ha he aglycone was que ce in. Howe e , hese compounds ollowed di e en 576
agmen a ion pa e ns. Peak 75 yielded [Y1]− a m/z 463 due o he loss o a hexosyl moie y (162 577
u), and e ealing ha [Y0]− was ob ained om he loss o a second hexosyl esidue. Thus, 578
compound 75 was assigned as a que ce in-O-di-hexoside. Ins ead, peak 76 yielded [Y1]− a m/z 447 579
due o he loss o a gluconic moie y (178 u), and disclosing a subsequen loss o a hamnosyl moie y 580
(146 u) o achie e [Y0]−. Peak 75 was en a i ely iden i ied as que ce in-di-glucoside, which has 581
been p e iously epo ed in g een le uce (San os e al., 2014). Peak 76 was en a i ely p oposed as 582
que ce in-O- hamnosyl-gluconide, which is he e epo ed o he i s ime o he au ho ’s 583
knowledge. 584
Rega ding kaemp e ol conjuga es, compound 77 (R = 25.27 min, max= 265, 347 nm) was 585
iden i ied as kaemp e ol-3-O-(6’’-O-malonyl)-glucoside, which has been al eady ound in di e en 586
le uce cul i a s (Heimle e al., 2007). In he posi i e mode, [M+H]+ a m/z 535, [M+Na]+ a m/z 587
557, and he agmen ions and [Y0]+ a m/z 287 e ealed he clea age o a malonyl-glucosyl g oup. 588
24
In he nega i e mode, [M−H]−a m/z 533, [Y0]− a m/z 285, [M−H−CO2]− a m/z 489 con i med he 589
p esence o he malonyl glucosyl moie y in he molecule (Fig. 4S in he supplemen a y ma e ial). 590
Rega ding he aglycone, kaemp e ol and he la one lu eolin a e isoba ic, bu hei conjuga es can 591
be dis inguished on he basis o hei MS and MS/MS da a. In he posi i e low ene gy unc ion, 592
kaemp e ol de i a i es yield [Y0]+ as he base peak o [M+H]+ as he base peak plus an in ense 593
[Y0]+, whe eas lu eolin de i a i es gi e as he base peak [M+H]+ o [M+H−H2O]+, and [Y0]+ does 594
no appea o p esen low ela i e abundance. In he nega i e low ene gy unc ion, bo h compounds 595
yield [M−H]− o [M−H−CO2]− (in he case o malonylglycosides) as he base peak, bu in he 596
nega i e high ene gy unc ion, kaemp e ol conjuga es gi e he base peak [Y0]−, whe eas lu eolin 597
compounds yield he base peak [M−H]− o [M−H−CO2]− and an in ense [Y0]−, o [Y0]− as he base 598
peak and an in ense [M−H]− wi h ela i e abundance highe han 50% RA. Mo eo e , se e al mino 599
monoiso opic p oduc ions a m/z 217.0501 (C12H9O4), 199.0395 (C12H7O3), 175.0395 (C10H7O3) 600
and 133.0290 (C8H5O2) a e cha ac e is ic o lu eolin, and helps o dis inguish i om i s kaemp e ol 601
isome s (Abu-Reidah e al., 2013; Gómez-Rome o, Ma ía e al., 2011). In his sense, hese agmen 602
ions did no appea in he nega i e high ene gy MS spec a o peak 77, sugges ing ha i is a 603
kaemp e ol de i a i e. Mo eo e , his iden i ica ion was also suppo ed by he base peaks yielded in 604
he posi i e low ene gy and he nega i e high ene gy unc ions, [Y0]+ and [Y0]− espec i ely, as well 605
as i s UV- isible spec a, and elu ion o de since kaemp e ol isome s elu e la e han lu eolin 606
isome s on endcapped C18 packings. 607
Two isome s (78: R = 23.90 min; 79: R = 26.43 min) we e de ec ed in he ex ac ed MS 608
ch oma og am a m/z 449 and 447 in he posi i e and nega i e ion modes espec i ely, which 609
yielded he p o ona ed ion, [M+Na]+ a m/z 471 and [Y0]+ a m/z 287 in he posi i e ion mode, and 610
he dep o ona ed molecule and [Y0]− a m/z 285 in he nega i e ion mode (Fig. 4S in he 611
supplemen a y ma e ial); e ealing he loss o a hexosyl esidue and he p esence o kaemp e ol o 612
lu eolin aglycone. The base peaks yielded in he posi i e low ene gy and he nega i e high ene gy 613
unc ions we e [Y0]+ and [Y0]− espec i ely, and no cha ac e is ic mino p oduc ions o lu eolin 614
31
suppo . Gab iela Elena Viaca a hanks CONICET and Asociación Uni e si a ia Ibe oame icana de 770
Pos g ado (AUIP) o he Ph.D. g an s. 771
Re e ences 772
Abad-Ga cía, B., Be ue a, L. A., Ga món-Loba o, S., Gallo, B., & Vicen e, F. (2009). A gene al 773
analy ical s a egy o he cha ac e iza ion o phenolic compounds in ui juices by high-774
pe o mance liquid ch oma og aphy wi h diode a ay de ec ion coupled o elec osp ay 775
ioniza ion and iple quad upole mass spec ome y. Jou nal o Ch oma og aphy A, 1216, 776
5398-5415. 777
Abu-Reidah, I. M., Con e as, M. M., A áez-Román, D., Segu a-Ca e e o, A., & Fe nández-778
Gu ié ez, A. (2013). Re e sed-phase ul a-high-pe o mance liquid ch oma og aphy 779
coupled o elec osp ay ioniza ion-quad upole- ime-o - ligh mass spec ome y as a 780
powe ul ool o me abolic p o iling o ege ables: Lac uca sa i a as an example o i s 781
applica ion. Jou nal o Ch oma og aphy A, 1313, 212-227. 782
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Mul iclass de e mina ion o phenolic compounds in di e en a ie ies o oma o and le uce 787
by ul a high pe o mance liquid ch oma og aphy coupled o andem mass spec ome y. 788
In e na ional Jou nal o Food P ope ies, 19, 494-507. 789
Alonso-Salces, R. M., Guillou, C., & Be ue a, L. A. (2009). Liquid ch oma og aphy coupled wi h 790
ul a iole abso bance de ec ion, elec osp ay ioniza ion, collision-induced dissocia ion and 791
andem mass spec ome y on a iple quad upole o he on-line cha ac e iza ion o 792
polyphenols and me hylxan hines in g een co ee beans. Rapid Communica ions in Mass 793
Spec ome y, 23, 363-383. 794

32
Al unkaya, A., & Gökmen, V. (2009). E ec o a ious an i-b owning agen s on phenolic 795
compounds p o ile o esh le uce (L. sa i a). Food Chemis y, 117, 122-126. 796
Becke , C., Klae ing, H. P., Sch eine , M., K oh, L. W., & K umbein, A. (2014). Unlike que ce in 797
glycosides, cyanidin glycoside in ed lea le uce esponds mo e sensi i ely o inc easing 798
low adia ion in ensi y be o e han a e head o ma ion has s a ed. Jou nal o Ag icul u al 799
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MSn iden i ica ion o chlo ogenic acids. Jou nal o Ag icul u al and Food Chemis y, 51, 802
2900-2911. 803
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Cli o d, M. N., Knigh , S., & Kuhne , N. (2005). Disc imina ing be ween he six isome s o 806
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Cli o d, M. N., Knigh , S., Su ucu, B., & Kuhne , N. (2006). Cha ac e iza ion by LC-MSn o ou 809
new classes o chlo ogenic acids in g een co ee beans: Dime hoxycinnamoylquinic acids, 810
di e uloylquinic acids, ca eoyl-dime hoxycinnamoylquinic acids, and e uloyl-811
dime hoxycinnamoylquinic acids. Jou nal o Ag icul u al and Food Chemis y, 54, 1957-812
1969. 813
Cli o d, M. N., Ma ks, S., Knigh , S., & Kuhne , N. (2006). Cha ac e iza ion by LC-MSn o ou 814
new classes o p-couma ic acid-con aining diacyl chlo ogenic acids in g een co ee beans. 815
Jou nal o Ag icul u al and Food Chemis y, 54, 4095-4101. 816
Cli o d, M. N., Wu, W., Ki kpa ick, J., & Kuhne , N. (2007). P o iling he chlo ogenic acids and 817
o he ca eic acid de i a i es o he bal ch ysan hemum by LC-MSn. Jou nal o Ag icul u al 818
and Food Chemis y, 55, 929-936. 819
33
Dai, J., & Mumpe , R. J. (2010). Plan phenolics: ex ac ion, analysis and hei an ioxidan and 820
an icance p ope ies. Molecules, 15, 7313-7352. 821
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ca eoylquinic acids in aqueous solu ions. Eu opean Food Resea ch and Technology, 233, 823
223-232. 824
DuPon , M. S., Mondin, Z., Williamson, G., & P ice, K. R. (2000). E ec o a ie y, p ocessing, 825
and s o age on he la onoid glycoside con en and composi ion o le uce endi e. Jou nal o 826
Ag icul u al and Food Chemis y, 48, 3957-3964. 827
Fe e es, F., Gil, M. I., Cas añe , M., & Tomás-Ba be án, F. A. (1997). Phenolic Me aboli es in Red 828
Pigmen ed Le uce (Lac uca sa i a). Changes wi h Minimal P ocessing and Cold S o age. 829
Jou nal o Ag icul u al and Food Chemis y, 45, 4249-4254. 830
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Gómez-Rome o, M., Zu ek, G., Schneide , B., Baessmann, C., Segu a-Ca e e o, A., & Fe nández-834
Gu ié ez, A. (2011). Au oma ed iden i ica ion o phenolics in plan -de i ed oods by using 835
lib a y sea ch app oach. Food Chemis y, 124, 379-386. 836
Heimle , D., Isolani, L., Vignolini, P., Tombelli, S., & Romani, A. (2007). Polyphenol con en and 837
an ioxida i e ac i i y in some species o eshly consumed salads. Jou nal o Ag icul u al 838
and Food Chemis y, 55, 1724-1729. 839
Jaiswal, R., Kip o ich, J., & Kuhne , N. (2011). De e mina ion o he hyd oxycinnama e p o ile o 840
12 membe s o he As e aceae amily. Phy ochemis y, 72, 781-790. 841
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empe a u es and cul i a ion du a ions on he polyphenolic con en s o le uce: Applica ion 844
o p incipal componen analysis. Jou nal o Ad anced Resea ch, 6, 493-499. 845
34
Ka a, V., Chowdhu y, S. K., & Chai , B. T. (1991). Use o a single-quad upole mass spec ome e 846
o collision-induced dissocia ion s udies o mul iply cha ged pep ide ions p oduced by 847
elec osp ay ioniza ion. Analy ical Chemis y, 63, 174-178. 848
Lin, L. Z., Ha nly, J., Zhang, R. W., Fan, X. E., & Chen, H. J. (2012). Quan i a ion o he 849
hyd oxycinnamic acid de i a i es and he glycosides o la onols and la ones by UV 850
abso bance a e iden i ica ion by LC-MS. Jou nal o Ag icul u al and Food Chemis y, 60, 851
544-553. 852
Lozac’h, N. (1975). Nomencla u e o Cycli ols. Eu opean Jou nal o Biochemis y, 57, 1-7. 853
Llo ach, R., Ma ínez-Sánchez, A., Tomás-Ba be án, F. A., Gil, M. I., & Fe e es, F. (2008). 854
Cha ac e isa ion o polyphenols and an ioxidan p ope ies o i e le uce a ie ies and 855
esca ole. Food Chemis y, 108, 1028-1038. 856
Ma, Y. L., Li, Q. M., Van den Heu el, H., & Claeys, M. (1997). Cha ac e iza ion o la one and 857
la onol aglycones by collision-induced dissocia ion andem mass spec ome y. Rapid 858
Communica ions in Mass Spec ome y, 11, 1357-1364. 859
Mai, F., & Glomb, M. A. (2013). Isola ion o phenolic compounds om icebe g le uce and impac 860
on enzyma ic b owning. Jou nal o Ag icul u al and Food Chemis y, 61, 2868-2874. 861
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phenolic con en o pigmen ed baby lea le uces h oughou he season. Jou nal o 863
Ag icul u al and Food Chemis y, 63, 1673-1681. 864
Ma kham, K. R. (1982). Techniques o Fla onoid Iden i ica ion. London: AcademicP ess Inc. 865
Pepe, G., Sommella, E., Man a, M., De Nisco, M., Teno e, G. C., Scopa, A., So o, A., Ma zocco, 866
S., Adesso, S., No ellino, T., & Campiglia, P. (2015). E alua ion o an i-in lamma o y 867
ac i i y and as UHPLC-DAD-IT-TOF p o iling o polyphenolic compounds ex ac ed 868
om g een le uce (Lac uca sa i a L.; Va . Ma a illa de Ve ano). Food Chemis y, 167, 869
153-161. 870
35
Rami ez-Amb osi, M., Abad-Ga cia, B., Vilo ia-Be nal, M., Ga mon-Loba o, S., Be ue a, L. A., & 871
Gallo, B. (2013). A new ul ahigh pe o mance liquid ch oma og aphy wi h diode a ay 872
de ec ion coupled o elec osp ay ioniza ion and quad upole ime-o - ligh mass 873
spec ome y analy ical s a egy o as analysis and imp o ed cha ac e iza ion o phenolic 874
compounds in apple p oduc s. Jou nal o Ch oma og aphy A, 1316, 78-91. 875
Ribas-Agus í, A., G a acós-Cuba sí, M., Sá aga, C., Ga cía-Reguei o, J. A., & Cas ella i, M. 876
(2011). Analysis o ele en phenolic compounds including no el p-couma oyl de i a i es in 877
le uce (Lac uca sa i a L.) by ul a-high-pe o mance liquid ch oma og aphy wi h 878
pho odiode a ay and mass spec ome y de ec ion. Phy ochemical Analysis, 22, 555-563. 879
Romani, A., Pinelli, P., Gala di, C., Sani, G., Cima o, A., & Heimle , D. (2002). Polyphenols in 880
g eenhouse and open-ai -g own le uce. Food Chemis y, 79, 337-342. 881
San os, J., Oli ei a, M. B. P. P., Ibáñez, E., & He e o, M. (2014). Phenolic p o ile e olu ion o 882
di e en eady- o-ea baby-lea ege ables du ing s o age. Jou nal o Ch oma og aphy A, 883
1327, 118-131. 884
Schü z, K., Ca le, R., & Schiebe , A. (2006). Ta axacumA e iew on i s phy ochemical and 885
pha macological p o ile. Jou nal o E hnopha macology, 107, 313-323. 886
Sobole , A. P., B osio, E., Gian e i, R., & Seg e, A. L. (2005). Me abolic p o ile o le uce lea es 887
by high- ield NMR spec a. Magne ic Resonance in Chemis y, 43, 625-638. 888
So o, A., Lundegå dh, B., Må ensson, A., Man a, M., Pepe, G., Sommella, E., De Nisco, M., 889
Teno e, G. C., Campiglia, P., & Scopa, A. (2016). Di e en ag onomic and e iliza ion 890
sys ems a ec polyphenolic p o ile, an ioxidan capaci y and mine al composi ion o le uce. 891
Scien ia Ho icul u ae, 204, 106-115. 892
Wa son, R. R., P eedy, V. R., & Zibadi, S. (2014). Polyphenols in human heal h and disease (1s 893
ed.). San Diego: Academic P ess. 894
895
36
Figu e cap ions 896
Fig. 1. Chemical s uc u es o phenolic compounds ound in bu e head le uce cul i a . 897
Abb e ia ions o he phenolic moie ies: C, ca eoyl; pCo, p-couma oyl; F, e uloyl; 898
dhC, dihyd oca eoyl; Sp, sinapoyl; 4-OH-Bz, 4-hyd oxybenzoyl; 3,4-diOH-Bz, 3,4-899
dihyd oxybenzoyl; Gal, galloyl; Sy , sy ingoyl; 4-OH-PhAc, 4-hyd oxyphenylace oyl; 900
Que, que ce in (Z1=OH, Z2=OH); Kaemp, kaemp e ol (Z1=H, Z2=OH); Lu , lu eolin 901
(Z1=OH, Z2=H); Api, apigenin (Z1=H, Z2=H); 6,7-diOH-Cou, 6,7-dihid oxycouma in. 902
Abb e ia ions o he non-phenolic moie ies: Q, quinic acid; Ta , a a ic acid, Mal, 903
malic acid; Mln, malonic acid; Glc , glucu onic acid; Glcn, gluconic acid; Hex, hexose; 904
Rha, hamnose; Ru , u inose ( hamnosylglucose). R, R1, R2, R3, R4 and R5 in non-905
phenolic moie ies can be es e i ied in posi ion X o phenolic acids o e he i ied wi h 906
phenolic OH g oups. 907
Fig. 2. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 908
mode o que ce in-3-O-glucu onide. ESI, elec osp ay ioniza ion. 909
Fig. 3. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 910
mode o e iodic yol-O-glucu onide. ESI, elec osp ay ioniza ion. 911
912

37
Supplemen a y ma e ial 913
Fig. 1S. Low ene gy unc ion (F1) base peak ch oma og ams in posi i e and nega i e ion modes 914
and DAD ch oma og ams a 280 and 370 nm o he bu e head le uce cul i a . 915
Fig. 2S. Bu e head le uce cul i a ch oma og ams ex ac ed om he TIC-MS scan 916
ch oma og am in nega i e ion mode a m/z 353, 341, 385 and 153 o he low ene gy 917
unc ion (F1). Ch oma og aphic peaks a e numbe ed as in Tables 1. 918
Fig. 3S. Bu e head le uce cul i a ch oma og ams ex ac ed om he TIC-MS scan 919
ch oma og am in nega i e ion mode a m/z 337, 311, 331, 451 and 435 o he low ene gy 920
unc ion (F1). Ch oma og aphic peaks a e numbe ed as in Tables 1. 921
Fig. 4S. Bu e head le uce cul i a ch oma og ams ex ac ed om he TIC-MS scan 922
ch oma og am in nega i e ion mode a m/z 151 o he low ene gy unc ion (F1), and a 923
m/z 301, 285 and 269 o he high ene gy unc ion (F2). Ch oma og aphic peaks a e 924
numbe ed as in Tables 1. 925
Fig. 5S. Bu e head le uce cul i a ch oma og ams ex ac ed om he TIC-MS scan 926
ch oma og am in nega i e ion mode a m/z 287, 177 and 581 o he low ene gy unc ion 927
(F1), and a m/z 417 o he high ene gy unc ion (F2). Ch oma og aphic peaks a e 928
numbe ed as in Tables 1. 929
Fig. 6S. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 930
mode o 5- ans-O-ca oylquinic acid. ESI, elec osp ay ioniza ion. 931
Fig. 7S. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 932
mode o lu eolin-7-O-glucu onide. ESI, elec osp ay ioniza ion. 933
Fig. 8S. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 934
mode o escule in-6-O-glucoside. ESI, elec osp ay ioniza ion. 935
Fig. 9S. Low (F1) and high (F2) ene gy unc ion MS spec a in he nega i e and posi i e ion 936
mode o sy inga esinol-ace ylhexose. ESI, elec osp ay ioniza ion. 937
1
Table 1 1
Re en ion imes, UV- isible maxima and MSE da a o polyphenols iden i ied by UHPLC-DAD-ESI-Q-ToF/MS in he bu e head le uce cul i a .a, b, c 2
LC
DAD
ESI(+)QToF/MS
ESI()QToF/MS
Assignmen
Nº
R
(min)
UV
bands
(nm)
Exp. Acc.
Mass
[M+H]+
E o
(mDa)
Fo mula
[M+H]+
Adduc s & agmen ions o [M+H]+
m/z
Exp. Acc.
Mass
[MH]
E o
(mDa)
Fo mula
[MH]
Adduc s & agmen ions o [MH]
m/z
Ten a i e iden i ica ion
Phenolic acids
Hyd oxycinnamic de i a i es
Ca eoylquinic acids
1
4.74
301 sh,
355.1068
3.9
C16H19O9
377.0858
[M+Na]+
353.0872
0.1
C16H17O9
191.0556
[QuinH] (100)
3 ansOCa eoylquinic acid
323
163.0398
[Ca eoyl+H]+
179.0348
[Ca eicH] (32)
145.0279
[Ca eoyl+HH2O]+
173.0437
[QuinHH2O] (4)
135.0448
[Ca eoyl+HCO]+
135.0446
[Ca eicHCO2] (71)
117.0343
[Ca eoyl+HCOH2O]+
89.0397
[Ca eoyl+HH2O2CO]+
2
6.65
-
355.1026
0.3
C16H19O9
731.1791
[2M+Na]+
353.0869
0.4
C16H17O9
707.1821
[2MH]
1 ansOCa eoylquinic acid
551.1234
[2M+Naca eic]+
191.0561
[QuinH] (100)
377.0846
[M+Na]+
163.0421
[Ca eoyl+H]+
145.0279
[Ca eoyl+HH2O]+
135.0433
[Ca eoyl+HCO]+
117.0342
[Ca eoyl+HCOH2O]+
89.0396
[Ca eoyl+HH2O2CO]+
3
7.32
300 sh,
355.1026
0.3
C16H19O9
731.1791
[2M+Na]+
353.0869
0.4
C16H17O9
707.1821
[2MH]
5 ansOCa eoylquinic acid
324
551.1234
[2M+Naca eic]+
191.0556
[QuinH] (100)
377.0846
[M+Na]+
179.0343
[Ca eicH] (1)
163.0421
[Ca eoyl+H]+
173.0449
[QuinHH2O] (3)
145.0279
[Ca eoyl+HH2O]+
135.0443
[Ca eicHCO2](2)
135.0433
[Ca eoyl+HCO]+
117.0342
[Ca eoyl+HCOH2O]+
89.0396
[Ca eoyl+HH2O2CO]+
4
8.12

355.1068
3.9
C16H19O9
731.1739
[2M+Na]+
353.0861
1.2
C16H17O9
707.1796
[2MH]
3cisOCa eoylquinic acid
709.1981
[2M+H]+
191.0557
[QuinH] (100)
163.0397
[Ca eoyl+H]+
179.0344
[Ca eicH] (12)
145.0128
[Ca eoyl+HH2O]+
135.0441
[Ca eicHCO2] (21)
135.0463
[Ca eoyl+HCO]+
117.0333
[Ca eoyl+HCOH2O]+
89.0383
[Ca eoyl+HH2O2CO]+
5
8.36

355.1068
3.9
C16H19O9
377.0844
[M+Na]+
353.0865
0.8
C16H17O9
191.0554
[QuinH] (100)
4 ansOCa eoylquinic acid
163.0445
[Ca eoyl+H]+
173.0458
[QuinHH2O] (13)
145.0325
[Ca eoyl+HH2O]+
135.0408
[Ca eoyl+HCO]+
117.0364
[Ca eoyl+HCOH2O]+
6
10.23
301 sh,
355.1068
3.9
C16H19O9
731.1746
[2M+Na]+
353.0867
0.6
C16H17O9
707.1816
[2MH]
5cisOCa eoylquinic acid
316
551.1199
[2M+Naca eic]+
191.0557
[QuinH] (100)
377.0841
[M+Na]+
173.0449
[QuinHH2O] (3)
163.0400
[Ca eoyl+H]+
145.0284
[Ca eoyl+HH2O]+
135.0443
[Ca eoyl+HCO]+
117.0346
[Ca eoyl+HCOH2O]+
89.0396
[Ca eoyl+HH2O2CO]+
7
15.06

C16H19O9
163.0399
[Ca eoyl+H]+
353.0876
0.3
C16H17O9
191.0578
[QuinH] (100)
4cisOCa eoylquinic acid
145.0287
[Ca eoyl+HH2O]+
179.0314
[Ca eicH] (5)
135.0446
[Ca eoyl+HCO]+
173.0455
[QuinHH2O] (2)
117.0278
[Ca eoyl+HCOH2O]+
Table1
2
LC
DAD
ESI(+)QToF/MS
ESI()QToF/MS
Assignmen
Nº
R
(min)
UV
bands
(nm)
Exp. Acc.
Mass
[M+H]+
E o
(mDa)
Fo mula
[M+H]+
Adduc s & agmen ions o [M+H]+
m/z
Exp. Acc.
Mass
[MH]
E o
(mDa)
Fo mula
[MH]
Adduc s & agmen ions o [MH]
m/z
Ten a i e iden i ica ion
p-Couma oylquinic acids
8
9.82
312
339.1075
0.5
C16H19O8
699.1888
[2M+Na]+
337.0921
0.2
C16H17O8
675.1904
[2MH]
3pCouma oylquinic acid
361.0892
[M+Na]+
191.0467
[QuinH]
147.0451
[pCouma oyl+H]+
163.0393
[pCouma icH]
119.0500
[pCouma oyl+HCO]+
119.0496
[pCouma icHCO2]
91.0556
[pCouma oyl+H2CO]+
9
13.74
308
339.1133
5.3
C16H19O8
699.1916
[2M+Na]+
337.0919
0.4
C16H17O8
191.0553
[QuinH]
5pCouma oylquinic acid
361.0907
[M+Na]+
173.0449
[QuinHH2O]
147.0453
[pCouma oyl+HH2O]+
163.0390
[pCouma icH]
119.0500
[pCouma oyl+HH2OCO]+
119.0491
[pCouma icHCO2]
91.0561
[pCouma oyl+HH2O2CO]+
Ca eoyl a a ic acid
10
9.06
301 sh,
C13H13O9
311.0526
12.3
C13H11O9
293.0287
[Ca a HH2O]
Ca eoyl a a ic acid
323
179.0349
[Ca eicH]
149.0227
[Ta a icH]
135.0432
[Ca eicHCO2]
p-Couma oyl a a ic acid
11
15.63
310
C13H13O8
295.0457
0.3
C13H11O8
163.0393
[pCouma icH]
pCouma oyl a a ic acid
149.0104
[Ta a icH]
119.0481
[pCouma icHCO2]
Ca eoylmalic acid
12
9.05
301 sh,
297.0585
2.5
C13H13O8
319.0429
[M+Na]+
295.0448
0.6
C13H11O8
591.0983
[2MH]
Ca eoylmalic acid
323
163.0404
[Ca eoyl+H]+
179.0345
[Ca eicH]
145.0297
[Ca eoyl+HH2O]+
135.0446
[Ca eicHCO2]
135.0447
[Ca eoyl+HCO]+
133.0275
[MalicH]
117.0348
[Ca eoyl+HCOH2O]+
115.0032
[MalicHH2O]
89.0397
[Ca eoyl+HH2O2CO]+
105.0342
[MalicHCO]
Dica eoylquinic acids and ca eoylquinic acid glycosides
13
5.86

517.1548
0.9
C22H29O14
539.1364
[M+Na]+
515.1402
0.1
C22H27O14
353.0869
[Ca quinH]
Ca eoylquinic acidhexoside
355.1038
[Mhexosyl]+
191.0548
[QuinH]
163.0415
[Ca eoyl+H]+
145.0310
[Ca eoyl+HH2O]+
135.0449
[Ca eoyl+HCO]+
117.0385
[Ca eoyl+HCOH2O]+
89.0399
[Ca eoyl+HH2O2CO]+
14
7.56

C22H29O14
539.1367
[M+Na]+
515.1402
0.1
C22H27O14
Ca eoylquinic acidhexoside
15
20.20
321
517.1423
7.7
C25H25O12
539.1155
[M+Na]+
515.1194
0.4
C25H23O12
353.0871
[Ca quinH]
1,5diOCa eoylquinic acid
499.1237
[M+HH2O]+
335.0771
[Ca quinHH2O]
355.0985
[Ca quin+H]+
191.0558
[QuinH]
163.0403
[Ca eoyl+H]+
179.0349
[Ca eicH]
145.0159
[Ca eoyl+HH2O]+
135.0448
[Ca eicHCO2]
135.0451
[Ca eoyl+HCO]+
117.0350
[Ca eoyl+HCOH2O]+
89.0404
[Ca eoyl+HH2O2CO]+
16
20.63
326
517.1332
1.4
C25H25O12
539.1155
[M+Na]+
515.1186
0.4
C25H23O12
353.0866
[Ca quinH]
3,5diOCa eoylquinic acid
499.1230
[M+HH2O]+
335.0761
[Ca quinHH2O]
355.1016
[Ca quin+H]+
191.0556
[QuinH]
163.0401
[Ca eoyl+H]+
179.0347
[Ca eicH]
145.0291
[Ca eoyl+HH2O]+
135.0446
[Ca eicHCO2]
135.0450
[Ca eoyl+HCO]+
117.0346
[Ca eoyl+HCOH2O]+
89.0401
[Ca eoyl+HH2O2CO]+
3
LC
DAD
ESI(+)QToF/MS
ESI()QToF/MS
Assignmen
Nº
R
(min)
UV
bands
(nm)
Exp. Acc.
Mass
[M+H]+
E o
(mDa)
Fo mula
[M+H]+
Adduc s & agmen ions o [M+H]+
m/z
Exp. Acc.
Mass
[MH]
E o
(mDa)
Fo mula
[MH]
Adduc s & agmen ions o [MH]
m/z
Ten a i e iden i ica ion
17
24.17
331
517.1423
7.7
C25H25O12
539.1165
[M+Na]+
515.1190
0.0
C25H23O12
353.0860
[Ca quinH]
4,5diOCa eoylquinic acid
499.1228
[M+HH2O]+
335.0802
[Ca quinHH2O]
473.2006
[M+HCO2]+
179.0347
[Ca eicH]
355.0161
[Ca quin+H]+
173.0449
[QuinHH2O]
163.0395
[Ca eoyl+H]+
135.0441
[Ca eicHCO2]
135.0447
[Ca eoyl+HCO]+
117.0347
[Ca eoyl+HCOH2O]+
89.0400
[Ca eoyl+HH2O2CO]+
p-Couma oylca eoylquinic acids
18
23.58
312
501.1384
1.3
C25H25O11
523.1219
[M+Na]+
499.1233
0.7
C25H23O11
353.0868
[MHcouma oyl]
3pCouma oyl4ca eoylquinic acid
483.1295
[M+HH2O]+
337.0916
[MHca eoyl]
163.0399
[Ca eoyl+HH2O]+
191.0560
[QuinH]
147.0446
[pCouma oyl+H]+
179.0353
[Ca eicH]
145.0279
[Ca eoyl+H2H2O]+
163.0398
[pCouma icH]
135.0455
[Ca eoyl+HH2OCO]+
135.0452
[Ca eicHCO2]
119.0497
[pCouma oyl+HH2OCO]+
119.0503
[pCouma icHCO2]
117.0335
[Ca eoyl+H2H2OCO]+
91.0550
[pCouma oyl+HH2O2CO]+
89.0398
[Ca eoyl+H2H2O2CO]+
19
23.95
316
501.1377
2.0
C25H25O11
523.1216
[M+Na]+
499.1241
0.1
C25H23O11
353.0852
[MHcouma oyl]
4Ca eoyl5pcouma oylquinic acid
483.1281
[M+HH2O]+
337.0928
[MHca eoyl]
147.0445
[pCouma oyl+H]+
191.0553
[QuinH]
119.0493
[pCouma oyl+HCO]+
179.0342
[Ca eicH]
91.0550
[pCouma oyl+H2CO]+
163.0390
[pCouma icH]
135.0448
[Ca eicHCO2]
119.0490
[pCouma icHCO2]
Dica eoyl a a ic acids
20
10.53
301 sh,
C22H19O12
497.0677
[M+Na]+
473.0719
0.1
C22H17O12
947.1354
[2MH]
diOCa eoyl a a ic acid
324
457.0698
[M+HH2O]+
311.0402
[Ca a H]
295.0577
[Ca a HH2O]+
293.0296
[Ca a HH2O]
163.0397
[Ca eoyl+H]+
179.0345
[Ca eicH]
145.0292
[Ca eoyl+HH2O]+
149.0091
[Ta a icH]
135.0448
[Ca eoyl+HCO]+
135.0443
[Ca eicHCO2]
117.0343
[Ca eoyl+HCOH2O]+
105.0339
[Ta a icHCO2]
89.0396
[Ca eoyl+HH2O2CO]+
21
12.54
301 sh,
C22H19O12
295.0563
[Ca a HH2O]+
473.0713
0.7
C22H17O12
311.0387
[Ca a H]
mesodiOCa eoyl a a ic acid
323
163.0398
[Ca eoyl+H]+
293.0297
[Ca a HH2O]
145.0288
[Ca eoyl+HH2O]+
179.0346
[Ca eicH]
135.0446
[Ca eoyl+HCO]+
149.0126
[Ta a icH]
117.0341
[Ca eoyl+HCOH2O]+
135.0448
[Ca eicHCO2]
89.0398
[Ca eoyl+HH2O2CO]+
105.0343
[Ta a icHCO2]
O he hyd oxycinnamic acid de i a i es
22
5.39

343.1098
6.9
C15H19O9
365.0878
[M+Na]+
341.0905
3.2
C15H17O9
Ca eic acidhexoside
163.0394
[Ca eoyl+H]+
145.0104
[Ca eoyl+HH2O]+
135.0497
[Ca eoyl+HCO]+
89.0401
[Ca eoyl+HH2O2CO]+
23
5.64

C15H19O9
365.0833
[M+Na]+
341.0854
1.9
C15H17O9
179.0330
[Ca eicH]
Ca eic acidhexoside
163.0389
[Ca eoyl+H]+
135.0435
[Ca eicHCO2]
145.0289
[Ca eoyl+HH2O]+
135.0473
[Ca eoyl+HCO]+
117.0309
[Ca eoyl+HCOH2O]+
24
6.08
301 sh,
C15H19O9
365.0844
[M+Na]+
341.0873
0.0
C15H17O9
179.0348
[Ca eicH]
Ca eic acidhexoside
325
135.0452
[Ca eicHCO2]
10
LC
DAD
ESI(+)QToF/MS
ESI()QToF/MS
Assignmen
Nº
R
(min)
UV
bands
(nm)
Exp. Acc.
Mass
[M+H]+
E o
(mDa)
Fo mula
[M+H]+
Adduc s & agmen ions o [M+H]+
m/z
Exp. Acc.
Mass
[MH]
E o
(mDa)
Fo mula
[MH]
Adduc s & agmen ions o [MH]
m/z
Ten a i e iden i ica ion
106
24.50

C30H39O14
621.2183
0.0
C30H37O14
417.1548
[MHace ylhexosyl]
Sy inga esinolace ylhexose
402.1313
[MHace ylhexosylCH3]
387.1078
[MHace ylhexosyl2CH3]
359.1111
[MHace ylhexosyl2CH3CO]
181.0503
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)]
166.0268
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)CH3]
151.0044
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)2CH3]
123.0065
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)2CH3CO]
107
24.63

C30H39O14
621.2181
0.2
C30H37O14
417.1546
[MHace ylhexosyl]
Sy inga esinolace ylhexose
402.1313
[MHace ylhexosylCH3]
387.1074
[MHace ylhexosyl2CH3]
359.1084
[MHace ylhexosyl2CH3CO]
181.0503
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)]
166.0269
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)CH3]
151.0041
[MHace ylhexosyl2CH3OOHC6
H2CHO2(CH2CH)2CH3]
108
19.22

C28H39O13
581.2239
0.5
C28H37O13
341.1392
[MHhexosylCH3COOHH2O]
Dime hoxyhexosylla ici esinol
329.1390
[MHhexosylCH3COOH2CH3]
109
19.39

C28H39O13
581.2238
0.4
C28H37O13
359.1494
[MHhexosylCH3COOH]
Dime hoxyhexosylla ici esinol
341.1383
[MHhexosylCH3COOHH2O]
329.1392
[MHhexosylCH3COOH2CH3]
110
19.82

C28H39O13
581.2201
3.3
C28H37O13
359.1445
[MHhexosylCH3COOH]
Dime hoxyhexosylla ici esinol
329.1392
[MHhexosylCH3COOH2CH3]
111
16.37

C34H49O18
743.2742
2.0
C34H47O18
581.2249
[MHhexosyl]
Dime hoxydihexosylla ici esinol
359.1494
[MH2hexosylCH3COOH]
341.1383
[MH2hexosylCH3COOHH2O]
329.1392
[MH2hexosylCH3COOH2CH3]
3
a F agmen ions p oduced in MS we e named acco ding o Ma e al. (1997)(Ma, Li, Van den Heu el, & Claeys, 1997). 4
b Abb e ia ions: Ca eic, ca eic acid; Ca quin, ca eoylquinic acid; Ca a , ca eoyl a a ic acid; DiHBZ, dihyd oxybenzoic acid; DiHBZhex, 5
dihyd oxybenzoic acid-hexoside; Dihyd oCa , dihyd oca eic acid; Gallic, gallic acid; HBZ, hyd oxybenzoic acid; hex, hexose; 4-hyd oxyphenylace ic, 4-6
hyd oxyphenylace ic acid; 4-hyd oxyphenylace ichex, 4-hyd oxyphenylace ic acid-hexoside ; Malic, malic acid; pCouma ic, p-couma ic acid; Quin, quinic 7
acid; Ta a ic, a a ic acid; sh, shoulde . 8
c Abundances o he agmen ions o ca eoylquinic acids in he nega i e mode a e gi en in pa en hesis. 9

OR1
HOOC
OR5
OR3
OR4
Q
OR2
OR1
HO
OH
O
O
Ta
OR
OH
HO
O
O
Mal
OR
OO
HO
Mln
O
HO
HO
OH
OH
OR
OH
Glcn
O
OH
OH
OH
CH3
OR
Rha
Hex
O
HO
HO
OR
OH
OH
O
HO
HO
OH
OR
OH
O
Glc
O
HO
HO
OH
OR
O
HO
H3C
HO
O
OH
Ru
Non-phenolic
Phenolic acids
OH
OH
XO
C
OH
XO
pCo
OCH3
OH
XO
F
OH
OH
XO
dhC
OCH3
H3CO
OH
XO
Sp
OH
HO OH
XO
Gal
HO
OH
XO
3,4-diOH-Bz
OH
XO
4-OH-Bz
OH
X
O
4-OH-PhAc
Sy
H3CO OCH3
OH
XO
OO
H3CO
OCH3
OCH3
H3CO
HO
OH
Sy inga esinol
O
HO
OCH3
H3CO
OH
OH
La ici esinol
Lignans
O
HO
HO O
6,7-diOH-Cou
Couma ins
Que / Kaemp / Lu / Api
O
Z2
OH O
HO
Z1
OH
O
O
OH
HO
OH
OH
E iodic yol
Fla onoids
1
2
3
4
5
6
7
8
2‘
3‘
4‘
5‘
6‘
AC
B
Phenolic
Figu e 1 e ised
Click he e o download Figu e(s): FoodChem_Fig1_Alonso-Salces_ e ised.eps
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100 477.0675
299.0200
301.0347
Scan ESI- F1: 3.32e6
[M−H]−
[Y0−2H]−
[Y0]−
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100 303.0507
479.0826
501.0644
Scan ESI+ F1: 2.602e6
[M+H]+
[M+Na]+
[Y0]+
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100 301.0347
299.0200
151.0036
477.0675
Scan ESI- F2: 1.79e6
255.0293
[Y0−2H]−
[Y0]−
[M−H]−
[Y0−CHO−OH]−
[1,3A]−
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100 303.0507
257.0443
501.0644
479.0826
Scan ESI+ F2: 3.19e6
153.0186
[Y0−CHO−OH]+
[1,3A]+[M+H]+
[M+Na]+
[Y0]+
1,3A
1,3B
0,2A
0,2B
Y0
O
O
OH O
HO
OH
OH
O
HO
OH
HO
COOH
Figu e 2
Click he e o download Figu e(s): FoodChem_Fig2_Alonso-Salces.eps
m/z
50 100 150 200 250 300 350 400 450 500 550
%
0
100 463.0882
151.0037
135.0452
285.0407
287.0555
107.0133
Scan ESI- F2: 2.69e4
[Y0−2H]−
[M−H]−
[1,3B]−
[Y0]−
[1,3A]−
[0,4A]−
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100 289.0715
487.0830
465.1026
153.0187
Scan ESI+ F2: 4.61e4
[1,3A]+
[M+H]+
[M+Na]+
[Y0]+
m/z
50 100 150 200 250 300 350 400 450 500 550
%
0
100 463.0882
285.0407
151.0037
287.0555
Scan ESI- F1: 1.38e5
[M−H]−
[Y0−2H]−
[Y0]−
[1,3A]−
m/z
50 100 150 200 250 300 350 400 450 500
%
0
100
465.1026
487.0830
Scan ESI+ F1: 5.33e4
[M+H]+
[M+Na]+
[Y0]+289.0715
O
O
OH
O
OH
OH
O
HO
OH
HO
COOH
1,3A
1,3B
0,2A
0,2B
Y0
0,4B
0,4A
Figu e 3
Click he e o download Figu e(s): FoodChem_Fig3_Alonso-Salces.eps