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 Chemistry 260 : 239-273 (2018) This document is the Accepted Manuscript version of a Published Work that appeared in final form in Food Chemistry 260 : 239-273(2018) https://doi.org/10.1016/j.foodchem.2018.03.151 © 2018. This manuscript version is made available under the CC-BY-NCND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/ DOI:10.1016/j.foodchem.2018.03.151 Polyphenolic profile of butterhead lettuce cultivar by ultrahigh performance liquid chromatography coupled online to UV–visible spectrophotometry and quadrupole time-of-flight mass spectrometry Gabriela E. Viacava, Sara I. Roura, Diana M. López-Márquez, Luis A. Berrueta, Blanca Gallo, Rosa M. Alonso-Salces 1 Polyphenolic profile of butterhead lettuce cultivar by ultrahigh performance liquid 1 chromatography coupled online to UV-visible spectrophotometry and quadrupole time-of-2 flight mass spectrometry 3 4 Running title: 5 Polyphenolic profile of butterhead lettuce by UHPLC-DAD-ESI-QToF/MSE 6 7 Gabriela E. Viacavaa, Sara I. Rouraa, Diana M. López-Márquezb, Luis A. Berruetab, Blanca Gallob, 8 Rosa M. Alonso-Salcesc,* 9 10 a Grupo de Investigación en Ingeniería en Alimentos, CONICET, Departamento de Ingeniería 11 Química y en Alimentos, Facultad de Ingeniería, Universidad Nacional de Mar del Plata, Juan B. 12 Justo 4302, 7600 Mar del Plata, Argentina. E-mail address: [email protected]; 13 [email protected] 14 b Departamento de Química Analítica, Facultad de Ciencia y Tecnología, Universidad del País 15 Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain. E-mail 16 address: [email protected]; [email protected]; [email protected] 17 c Departamento de Biología, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad 18 Nacional de Mar del Plata, Funes 3350, 7600 Mar del Plata, Argentina. 19 20 * Corresponding author.21 E-mail address: [email protected] 23 *Manuscript 2 Abstract 24 In the present study, the butterhead lettuce cultivar was analyzed by ultrahigh performance liquid 25 chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization 26 (ESI) and quadrupole time-of-flight mass spectrometry (QToF/MS) in the positive and negative ion 27 mode in order to characterize its polyphenolic profile for the first time. The instrument acquisition 28 mode MSE was used to collect automatic and simultaneous information of exact mass at high and 29 low collision energies of precursor ions as well as other ions produced as a result of their 30 fragmentation. One hundred eleven phenolic compounds were identified in the acidified 31 hydromethanolic extract of freeze-dried leaves of butterhead lettuce cultivar: 40 hydroxycinnamic 32 acid derivatives, 21 hydroxybenzoic acid derivatives, 2 hydroxyphenylacetic acid derivatives, 18 33 flavonols, 9 flavones, one flavanone, 7 coumarins, one hydrolysable tannin and 12 lignans. Forty 34 seven of these compounds have been tentatively identified for the first time in lettuce. 35 36 Keywords: Lactuca sativa, lettuce, phenolic compounds, UHPLC-QToF, mass spectrometry, MSE 37 38 Chemical compounds studied in this article: 39 5-Caffeoylquinic acid (PubChem CID: 12310830); caffeoylmalic acid (PubChem CID: 4484594); 40 4-hydroxyphenylacetic acid (PubChem CID: 127); quercetin-3-O-galactoside (PubChem CID: 41 90657624); quercetin-3-O-glucuronide (PubChem CID: 5274585); kaempferol-3-O-glucuronide 42 (PubChem CID: 5318759); luteolin 7-glucoside (PubChem CID: 5280637); luteolin 7-rutinoside 43 (PubChem CID: 44258082); esculetin-6-O-glucoside (PubChem CID: 5281417); syringaresinol 44 (PubChem CID: 100067). 45 46 3 1. Introduction 47 Phenolic compounds are secondary plant metabolites ubiquitous in the plant kingdom 48 involved in protection mechanisms against biotic and abiotic stresses, in the regulation of plant 49 growth and development, and in the organoleptic quality of plant-based foods (Dai & Mumper, 50 2010). Moreover, the intake of phenolic compounds through fruits and vegetables have been proved 51 to provide beneficial effects attributed to their antioxidant capacity against oxidative stress, cancer 52 and cardiovascular diseases, among others (Watson, Preedy, & Zibadi, 2014). Lettuce (Lactuca 53 sativa L.) is one of the most popular leafy vegetables. In particular, the butterhead lettuce is one of 54 the most commonly consumed variety worldwide (Agüero, Viacava, Ponce, & Roura, 2013); 55 however, its polyphenolic profile has not been characterized yet to the authors’ knowledge. The 56 main classes of phenolic compounds found in different varieties of lettuce are phenolic acids and 57 flavonols, followed by flavones and anthocyanins (only in red varieties) (Alarcón-Flores, Romero-58 González, Martínez Vidal, & Garrido Frenich, 2016; Marin, Ferreres, Barberá, & Gil, 2015; Pepe, 59 Sommella, Manfra, De Nisco, Tenore, Scopa et al., 2015). Most analytical methods used to 60 determine polyphenols in lettuce are based on high or ultrahigh performance liquid chromatography 61 (HPLC or UHPLC) coupled to diode array detection (DAD) and/or mass spectrometry (MS and 62 MS/MS) (Abu-Reidah, Contreras, Arráez-Román, Segura-Carretero, & Fernández-Gutiérrez, 2013; 63 Alarcón-Flores et al., 2016; Altunkaya & Gökmen, 2009; Llorach, Martínez-Sánchez, Tomás-64 Barberán, Gil, & Ferreres, 2008; Pepe et al., 2015; Ribas-Agustí, Gratacós-Cubarsí, Sárraga, 65 García-Regueiro, & Castellari, 2011). UHPLC achieves rapid analysis and better peak separation 66 than HPLC, and coupled to ToF or QToF instruments provides a highly attractive analytical 67 technique with very high resolution and accurate mass measurements of the precursor and fragment 68 ions (Ramirez-Ambrosi, Abad-Garcia, Viloria-Bernal, Garmon-Lobato, Berrueta, & Gallo, 2013). 69 This technique has been already used to characterize 95 phenolic compounds in three lettuce 70 cultivars (baby, romaine, and iceberg) (Abu-Reidah et al., 2013). Technological advances such as 71 the so called MSE data acquisition mode has been successfully used for the structural elucidation of 72 4 phenolic compounds in complex plant extracts (Ramirez-Ambrosi et al., 2013). MSE acquisition 73 method maximizes the QToF instrument duty cycle performing simultaneous collection of 74 precursor ions as well as other ions produced as a result of their fragmentation in exact mass mode 75 over a single experimental run. Since many compounds still remain unidentified in lettuce cultivars 76 and the utilization of analytical edge technology can provide new structural information and allow 77 the identification of unknown polyphenols, the present study exploits the use of UHPLC-DAD-ESI-78 QToF/MSE for the characterization of the polyphenolic profile of the butterhead lettuce cultivar, 79 which is here reported for the first time to the authors’ knowledge. 80 2. Materials and Methods 81 2.1. Reagents, solvents and standards 82 Water, methanol, acetonitrile, and formic acid (Fisher Scientific, Fair Lawn, NJ, USA) were 83 of Optima® LC/MS grade; ascorbic acid (Panreac, Barcelona, Spain), analytical grade; and glacial 84 acetic acid (Merck, Darmstadt, Germany), Suprapur® quality. Leucine Enkephalin acetate hydrate 85 and sodium formate solution were provided by Sigma-Aldrich Chemie (Steinheim, Germany). 86 Luteolin-7-O-glucoside, kaempferol-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-87 rhamnoside were purchased from Extrasynthèse (Genay, France); caffeoyltartaric acid and 88 quercetin-3-O-glucoside, from Chromadex (Irvine, CA, USA); 5-O-caffeoylquinic acid, p-coumaric 89 acid, 1,5-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, and quercetin-3-O-rutinoside, from 90 Sigma-Aldrich Chemie (Steinheim, Germany); and ferulic acid, caffeic acid, and 3,4-91 dihydroxybenzoic acid, from Fluka Chemie (Steinheim,Germany). Standard stock solutions of 92 phenolic compounds were prepared in methanol; and a reference solution of these compounds 93 (5 g/mL), in methanol-water-acetic acid (30:65:5, v/v/v). 94 2.2. Plant material 95 Heads of butterhead lettuce (Lactuca sativa var. Lores) were obtained from a local producer 96 in Sierra de los Padres (Mar del Plata, Argentina). Lettuce samples were frozen with liquid nitrogen 97 5 and freeze-dried, homogenized and crushed to obtain a homogeneous powder, which was stored at 98 room temperature in dark in a desiccator until analysis. 99 2.3. Extraction of polyphenols in lettuce 100 Freeze-dried lettuce (0.1 g) was extracted with 5 mL of methanol-water-acetic acid (30:65:5, 101 v/v/v) containing ascorbic acid (2 g/L) in an ultrasonic bath for 10 min. Then, the extract was 102 centrifuged at 6000 rpm during 15 min at 4 ºC, and the supernatant was filtered through a 0.45 µm 103 PTFE filter (Waters, Milford, CA, USA) prior to injection into the UHPLC system. 104 2.4. UHPLC-DAD-ESI-QToF/MSE 105 Lettuce extract was analyzed using an ACQUITY UPLCTM system from Waters (Milford, 106 MA, USA), equipped with a binary solvent delivery pump, an autosampler, a column compartment 107 a PDA detector, and controlled by MassLynx v4.1 software. A reverse phase Acquity UPLC BEH 108 C18 column (2.1 mm × 100 mm, 1.7 µm) and a Acquity UPLC BEH C18 VanGuardTM pre-column 109 (1.7 µm) from Waters (Milford, USA) were used. Flow rate was 0.5 mL/min; injection volume, 5 110 µL; column and autosampler temperatures, 40ºC and 4 ºC respectively. Mobile phases consisted of 111 0.1% (v/v) acetic acid in water (A) and 0.1% (v/v) acetic acid in methanol (B). The elution 112 conditions applied were: 0–8.5 min, linear gradient 0–13% B; 8.5–11 min, 13% B isocratic; 11–113 12.3 min, linear gradient 13–15% B; 12.3–13.8 min, linear gradient 15–19% B; 13.8–17.3 min, 114 linear gradient 19–23% B; 17.3–19 min, 23% B isocratic; 19–24 min, linear gradient 23–30% B; 115 24–26 min, 30% B isocratic; 26–27 min, linear gradient 30–100% B; 27–28 min, 100% B isocratic; 116 and finally reconditioning of the column with 100% A isocratic. UV-visible spectra were recorded 117 from 210 to 500 nm (20 Hz, 1.2 nm resolution). Hydroxybenzoic acids were monitored at 254 nm; 118 flavanones at 280 nm; hydroxycinnamic acids and coumarins at 320 nm; flavonols and flavones at 119 370 nm. 120 All MS data acquisitions were performed on a SYNAPTTM G2 HDMS with a quadrupole 121 time of flight (QToF) configuration (Waters, Milford, MA, USA) equipped with an electrospray 122 ionization (ESI) source operating in both positive and negative modes. The capillary voltage was set 123 6 to 0.7 kV (ESI+) or 0.5 kV (ESI−). Nitrogen was used as the desolvation and cone gas at flow rates 124 of 900 L/h and 10 L/h, respectively. The source and desolvation temperatures were 120 ºC and 125 400 ºC respectively. Leucine-enkephalin solution (2 ng/µL) in 0.1% (v/v) formic acid in 126 acetonitrile-water (50:50, v/v) was used for the lock mass correction (m/z 556.2771 and 278.1141, 127 or m/z 554.2615 and 236.1035, depending on the ionization mode, were monitored at scan time 0.2 128 s, interval 10 s, scans to average 3, mass window ± 0.5 Da, cone voltage 30 V, at a flow rate 129 10 µL/min). Data acquisition was recorded in the mass range 50–1200 u in resolution mode 130 (FWHM ≈ 20,000) with a scan time of 0.2 s and an interscan delay of the 0.024 s, and automatically 131 corrected during acquisition based on the lock mass. Before analysis, the mass spectrometer was 132 mass calibrated with the sodium formate solution. To perform MSE mode analysis, the cone voltage 133 was set to 20 V (ESI+) or 30 V (ESI−) and the quadrupole operated in a wide band RF mode only. 134 Two discrete and independent interleaved acquisition functions were automatically created. The 135 first function, typically set at 6 eV in trap cell of the T-Wave, collects low energy or unfragmented 136 data while the second function collects high energy or fragmented data typically using 6 eV in trap 137 cell and a collision ramp 10–40 eV in transfer cell. In both cases, Argon gas was used for Collision 138 Induced Dissociation (CID). Data were recorded in continuous mode. For instrument control, data 139 acquisition and processing MassLynxTM software Version 4.1 (Waters MS Technology, Milford, 140 USA) was used. 141 2.5. Identification of phenolic compounds 142 The identification of the phenolic compounds for which standards were available was carried 143 out by the comparison of their retention times, their UV–vis spectra and MSE spectra recorded in 144 positive and negative mode with those obtained by injecting standards in the same conditions. The 145 identity of the rest of compounds was elucidated using the following analytical data: i) the UV–vis 146 spectrum when it was available to assign the phenolic class (Abad-García, Berrueta, Garmón-147 Lobato, Gallo, & Vicente, 2009), since each class exhibits a characteristic UV–vis spectrum 148 (Markham, 1982); ii) the low collision energy MSE spectrum in positive and negative ion mode to 149 7 determine the molecular weight; and since only the protonated/deprotonated molecules are able to 150 form in the electrospray ionization source adducts, clusters and/or molecular complexes with 151 mobile phase species (e.g. adducts with sodium [M+Na]+ at 22 u above the protonated molecule, 152 [2M+Na]+ of monoacyl hydroxycinnamic acids, the dehydrated protonated molecule ([M+H–153 H2O]+) of phenolic acids and diacyl hydroxycinnamic acids in positive mode; and adducts with 154 HSO4− (97 u) and AcO− (43 u) and the deprotonated dimer ion [2M–H]− of monoacyl 155 hydroxycinnamic acid in negative mode), their presence in the low collision energy spectra allows 156 the unequivocal identification of the [M+H]+ or [M−H]− ions; and iii) the high collision energy MSE 157 spectrum provides the polyphenol fragmentation patterns, which afford structural information 158 related to the type of carbohydrates, the sequence of the glycan part, interglycosidic linkages and 159 the aglycone moiety, allowing to assign the protonated aglycone [Y0]+ and/or the deprotonated 160 aglycone [Y0]−. The identification of the aglycone was carried out based on the observation of i,jA+ 161 and i,jB+ ions (Ma, Li, Van den Heuvel, & Claeys, 1997). Furthermore, the chromatographic elution 162 order aided in some structural assignments, as well as bibliographic references. IUPAC 163 nomenclature and recommended numbering system (Lozac’h, 1975) were used for chlorogenic 164 acids and flavonoids; and common names were used for other phenolic acid derivatives, coumarins, 165 hydrolysable tannins and lignan derivatives. Structures of each family of compounds studied are 166 presented in Fig. 1. 167 3. Results and Discussion 168 A total of 111 phenolic compounds were tentatively identified in the butterhead lettuce 169 cultivar by UHPLC-DAD-ESI-QToF/MSE. The UV-visible and MS spectral data are summarized in 170 Table 1. DAD and MS chromatograms are shown in Figs. 1S-5S (supplementary material). The 171 high and low energy function MS spectra of compounds from the different phenolic families 172 detected in this cultivar are displayed in Figs. 2 and 3, and in Figs. 6S-9S (supplementary material). 173 3.1. Phenolic acid derivatives 174 8 For the identification of phenolic acid derivatives, mainly negative ion mode mass spectra 175 were taken into account, although the positive ion mode was used for verification. In the high 176 collision energy MS spectra, losses of H2O, CO2 and CO were regularly observed, which have also 177 been described by other authors using IT, QqQ, and QToF (Gómez-Romero, M., Segura-Carretero, 178 & Fernandez-Gutierrez, 2010; Ramirez-Ambrosi et al., 2013). 179 3.1.1. Hydroxycinnamic derivatives 180 3.1.1.1. Caffeoylquinic acids 181 Three major chromatographic peaks (1, 3, 6), presenting the same UV spectra as the standard 182 trans-5-caffeoylquinic acid (trans-5-CQA), were detected in the chromatograms extracted from the 183 Total Ion Current (TIC) MS scan chromatogram in negative and positive modes at m/z 353 and 355 184 respectively, which were due to three caffeoylquinic acid (CQA) isomers (Fig. 2S in the 185 supplementary material). Compound 3 (Rt= 7.32 min, max= 300, 324 nm) was identified 186 unambiguously as trans-5-caffeoylquinic acid by comparison with its standard: the deprotonated 187 molecule [M−H]− at m/z 353 yielded fragment ions at m/z 191, 173 and 135; and the protonated 188 molecule [M+H]+, at m/z 163 and 145. Moreover, its sodium adducts, [M+Na]+ and [2M+Na]+ at 189 m/z 377 and 731 respectively, were also observed (Fig. 6S in the supplementary material). 190 Compounds 1 (Rt= 4.74 min, max= 301, 323 nm) and 6 (Rt= 10.23 min, max= 301, 316 nm) had 191 the same fragmentation pattern as 5-CQA, and their m/z values for [M+H]+ and [M-H]− were 192 confirmed with the sodium adduct at m/z 377 in positive ionization mode, and the [2M−H]− ion at 193 m/z 707 in negative mode. All three peaks (1, 3, 6) yielded the same base peak at m/z 191 due to the 194 deprotonated quinic moiety in the negative high energy function. None of the peaks yielded an 195 intense fragment ion at m/z 173 ([quinic acid–H–H2O]−). This dehydrated ion of quinic acid is 196 characteristically formed in the negative ion mode when the cinnamoyl group is bonded to the 197 quinic moiety at position 4, as already noted by other authors using other QqQ/MS (Alonso-Salces, 198 Guillou, & Berrueta, 2009) or IT/MS (Clifford, Johnston, Knight, & Kuhnert, 2003). Peak 1 also 199 gave intense ions from the caffeoyl moiety ([caffeic acid–H–CO2]−) at m/z 135 (71% relative 200 15 Two chromatographic peaks showed protonated and deprotonated molecules that 356 corresponded to p-coumaroylcaffeoylquinic acids, at m/z 501 in the positive ion mode and at m/z 357 499 in the negative mode: peak 18 (Rt= 23.58 min, max= 312 nm) and peak 19 (Rt= 23.95 min, 358 max= 316 nm). In the positive high energy function, the base peaks yielded by both isomers were 359 the fragment ion at m/z 147 due to [p-coumaroyl+H]+, disclosing that the p-coumaroyl moiety was 360 attached to the quinic acid in a weaker position than the caffeoyl one. This was also supported by 361 the fragmentation pattern observed for both peaks in the negative ion mode, which yielded the 362 deprotonated molecules, and fragments at m/z 353 due to the loss of the p-coumaroyl moiety (85-363 95% RA) (Fig. 2S in the supplementary material) and at m/z 337 due to the loss of the caffeoyl 364 moiety (40-50% RA) (Fig. 3S in the supplementary material) in the low energy function, indicating 365 that the former loss was favored. This fragmentation pattern was reported for 3-p-coumaroyl-4-366 caffeoylquinic acid (3-pCo-4-CQA) and 4-caffeoyl-5-p-coumaroylquinic acid (4-C-5-pCoQA) 367 (Clifford, Marks, Knight, & Kuhnert, 2006). The deprotonated quinic acid ion at m/z 191 was the 368 base peak in the high energy function; this fragment is a characteristic base peak of 5-CQA, 3-CQA 369 and 5-pCoQA, and is yielded by 4-CQA (Clifford et al., 2003). Thus, taking also into account that 370 the elution order on endcapped C18 packing is 3,4-isomers, 3,5-isomers and 4,5-isomers (Clifford, 371 Marks, et al., 2006), compounds 18 and 19 were tentatively assigned to 3-pCo-4-CQA and 4-C-5-372 pCoQA respectively, for the first time in lettuce cultivars. p-Coumaroylcaffeoylquinic acids have 373 been previously reported in lettuce (Abu-Reidah et al., 2013; Jaiswal et al., 2011). 374 3.1.1.8. Dicaffeoyltartaric acids 375 Two peaks (20, 21), presenting the same UV spectra as caffeic acid standard, were detected 376 in the chromatograms extracted from the TIC MS scan chromatogram in positive and negative 377 modes at m/z 475 and 473, respectively, which were due to two dicaffeoyltartaric acid isomers 378 (diCTA). Compound 20 (Rt= 10.53 min, max= 301, 324 nm) and compound 21 (Rt= 12.54 min, 379 max= 301, 323 nm) presented the same fragmentation pattern, and their identity was confirmed 380 with the sodium adduct at m/z 497 in positive ionization mode and the [2M−H]− ion at m/z 947 in 381 16 negative mode for peak 20, and the protonated and deprotonated molecules for peak 21. In the 382 negative ion mode, both peaks (20, 21) yielded the same base peak at m/z 293 due to the loss of 383 water of the deprotonated caffeoyltartaric acid, and [CTA–H]− at m/z 311 due to the loss of one of 384 the caffeoyl moieties (Fig. 3S in the supplementary material), as well as ions from the tartaric 385 moiety, [tartaric acid–H]− at m/z 149 and [tartaric acid–H–CO2]− at m/z 105; and ions from the 386 caffeoyl moiety, [caffeic acid–H]− at m/z 179 and [caffeic acid–H–CO2]− at m/z 135. Compound 20 387 was tentatively identified as di-O-caffeoyltartaric (chicoric acid), and compound 21 as meso-di-O-388 caffeoyltartaric acid, since they were detected in lettuce elsewhere; the former being reported as the 389 most abundant as we observed (Abu-Reidah et al., 2013; Jeong et al., 2015; Lin et al., 2012; Mai & 390 Glomb, 2013; Pepe et al., 2015; Ribas-Agustí et al., 2011; Romani et al., 2002; Santos et al., 2014). 391 3.1.1.9. Other hydroxycinnamic acid derivatives 392 Several cinnamoyl glycosides were found in the lettuce extracts, such as caffeoyl-hexosides, 393 p-coumaroyl-hexosides, sinapoyl-hexosides and dihydrocaffeic acid-hexosides, whose 394 fragmentation patterns were characterized by the aglycone product ion resulted from the loss of a 395 hexose residue (Abu-Reidah et al., 2013; Gómez-Romero, María, Zurek, Schneider, Baessmann, 396 Segura-Carretero, & Fernández-Gutiérrez, 2011). 397 Eight peaks (22, Rt= 5.39 min; 23, Rt= 5.64 min; 24, Rt= 6.08 min, max= 301, 325 nm; 25, 398 Rt= 7.69 min; 26, Rt= 8.44 min; 27, Rt= 9.01 min; 28 Rt= 9.52 min; and 29 Rt= 9.64 min) were 399 observed in the chromatogram extracted at m/z 343 and 341 in positive and negative ion modes 400 respectively (Fig. 2S in the supplementary material). All of them (22-29) produced m/z 179 and 135 401 in negative ion mode, and m/z 163, 145, 135, 117 and 89 in positive ion mode, consistent with the 402 presence of a caffeic acid residue. Thus, these compounds were tentatively assigned as isomeric 403 caffeic acid-hexosides, in agreement with Clifford et al. (2007) (Clifford et al., 2007). Moreover, 404 the identity of peaks 22-26 and 28 were confirmed by the presence of their sodium adducts in the 405 positive low energy function. As well, peak 30 (Rt= 8.01 min, max= 301, 325 nm) showed the 406 same fragmentation pattern as caffeic acid, yielding also a monoisotopic protonated molecule at 407 17 m/z 359.0802 (C18H15O8) in the positive ion mode, and a monoisotopic deprotonated molecule at 408 m/z 357.0633 (C18H13O8) in the negative ion mode. Thus, it was tentatively assigned as a caffeoyl 409 derivative, however the nature of the non-phenolic residue (196.0387 u) was not able to be 410 disclosed. Such caffeoyl derivative has not previously been reported in lettuce so far we are aware. 411 Similarly, four isomers of synapic acid-hexosides (31, Rt= 6.03 min, max= 301, 326 nm; 412 32, Rt= 9.70 min; 33, Rt= 10.36 min; 34, Rt= 13.13 min) were tentatively identified in the extracted 413 traces at m/z 387 and 385 in the positive and the negative ion modes respectively (Fig. 2S in the 414 supplementary material). Ions corresponding to the deprotonated aglycone at m/z 223, and the 415 subsequent decarboxylations and losses of methyl residues at m/z 208, 179, 164, and 149 from the 416 synapoyl moiety were detected in the negative ion mode. In addition, the positive ion mode yielded 417 the sodium adduct at m/z 409 and ions due to the loss of the hexose residue at m/z 225, and 418 subsequent losses of H2O at m/z 207, CH3OH at m/z 192, and CO at m/z 129. One isomer of synapic 419 acid-hexoside has been previously reported in green lettuce cultivars (Abu-Reidah et al., 2013). 420 Following this fragmentation patterns, a p-coumaric acid-hexoside (35, Rt= 8.32 min) and 421 two dihydrocaffeic acid-hexosides (36, Rt= 3.70 min; 37, Rt= 3.83 min) were also characterized. 422 All of them yielded the product ion due to the loss of the hexose residue (m/z 163 for 35, m/z 181 423 for 36 and 37), with the subsequent losses of H2O, CO and CO2 in the negative ion mode; and the 424 sodium adduct in the positive ion mode (m/z 349 for 35, m/z 367 for 36 and 37). 425 Seven caffeic acid-hexosides, a synapic acid-hexosides, a dihydrocaffeic acid-hexoside and a 426 p-coumaric acid-hexoside have been previously reported in green lettuce cultivars (Abu-Reidah et 427 al., 2013). In the present work, one more caffeic acid-hexoside, a dihydrocaffeic acid-hexoside and 428 three synapic acid-hexosides were identified in the butterhead lettuce cultivar. 429 Peaks 38 (Rt= 11.81 min, max= 307 nm), 39 (Rt= 14.47 min) and 40 (Rt= 16.48 min) were 430 tentatively proposed as isomers of ferulic acid methyl esters. According to previous data (Abu-431 Reidah et al., 2013; Gómez-Romero, María et al., 2011), these compounds showed demethylated 432 fragment ions at m/z 192 ([M–H–CH3]−) and m/z 177 ([M–H–2CH3]−), which is characteristic of the 433 18 methoxylated cinnamic acids. Two of these isomers of ferulic acid methyl esters have been 434 previously reported in green lettuce cultivars. 435 3.1.2. Hydroxybenzoic derivatives 436 Hydroxybenzoic derivatives were not detected in the positive ion mode. Thus, no peaks were 437 detected in the chromatograms extracted from the TIC MS scan chromatogram at the protonated 438 molecule or the sodium adduct masses of the hydroxybenzoic derivatives observed in the negative 439 ion mode. Only one of the two previously reported in green lettuce cultivars (Abu-Reidah et al., 440 2013) isomers of hydroxybenzoic acid (41: Rt= 4.67 min) and dihydroxybenzoic acid (42: Rt= 5.42 441 min) were detected at m/z 137 and m/z 153 respectively (Fig. 2S in the supplementary material). 442 Their corresponding decarboxylated ions were also observed at m/z 93 and m/z 109 respectively. 443 Several hydroxybenzoic glycoside esters were characterized according to their MS data and 444 fragmentation pattern by the neutral loss of the glycosidic moiety. Hydroxybenzoic acid-hexosides 445 (43, Rt= 4.22 min; 44, Rt= 5.15 min) yielded the deprotonated ion at m/z 299 and the product ions 446 due to losses of the hexose residue (m/z 137) and CO2 (m/z 93). Dihydroxybenzoic acid-hexosides 447 (45, Rt= 2.49 min; 46, Rt= 2.69 min; 47, Rt= 3.74 min; 48, Rt= 3.91 min; 49, Rt= 4.48 min; 50, Rt= 448 4.68 min) produced the deprotonated molecule at m/z 315 (base peak), an odd electron product ion 449 at m/z 152 corresponding to the loss of hexose plus H (163 u), an even electron ion at m/z 153 due 450 to the loss of hexose (Fig. 2S in the supplementary material), the dehydrated ion at m/z 135, and the 451 decarboxylated ion at m/z 109, in agreement with bibliography (Abu-Reidah et al., 2013). Hence, 452 one more hydroxybenzoic acid-hexoside and four more dihydroxybenzoic acid-hexosides are here 453 detected in butterhead lettuce than in previous studies on different lettuce cultivars. The release of 454 such unusual losses was also observed for gallic acid-hexoside isomers. Thus, peaks 51 (Rt=2.80 455 min), 52 (Rt=2.88 min) and 53 (Rt=6.61 min) were tentatively proposed as gallic acid-hexosides, 456 since they yielded the deprotonated molecule at m/z 331 (base peak) (Fig. 3S in the supplementary 457 material), and an odd electron product ion at m/z 168, corresponding to the loss of hexose plus H 458 (163 u), an even electron ion at m/z 169 due to the loss of hexose, and [gallic acid–H–CO2]− at m/z 459 19 125. Two isomers of gallic acid-hexoside have been detected previously only in the lettuce cv. baby 460 (Abu-Reidah et al., 2013). 461 Aside from the loss of the hexose moiety, syringic acid-hexoside (54, Rt= 5.90 min, m/z 359) 462 showed subsequent losses of CH3 from the methoxy groups of the aglycone and CO2 (m/z 182, 153, 463 138 and 123), as previously observed in literature (Abu-Reidah et al., 2013; Gómez-Romero, María 464 et al., 2011). 465 In agreement with previous studies (Abu-Reidah et al., 2013), compounds 55 (Rt= 17.09 466 min) and 56 (Rt= 24.83 min) showing a deprotonated molecule at m/z 451 were tentatively assigned 467 as hydroxybenzoyl-gallic acid-hexosides (Fig. 3S in the supplementary material). The high energy 468 function yielded the fragment ion corresponding to the deprotonated gallic acid-hexoside at m/z 469 331, after the loss of the hydroxybenzoyl moiety (120 u). As well, product ions due to successive 470 losses of H2O at m/z 313, hexose plus H at m/z 168 and CO2 at m/z 124 were observed. A similar 471 pattern was found for the hydroxybenzoyl-dihydroxybenzoic acid-hexosides (57, Rt= 17.68 min; 472 58, Rt= 19.41 min; 59, Rt= 23.64 min; 60, Rt= 26.88 min, max= 256, 335 nm; 61, Rt= 27.09 min) 473 detected in the extracted trace at m/z 435 (Fig. 3S in the supplementary material). For peak 59, only 474 the deprotonated molecule was detected due to its low concentration in the extract. All other 475 isomers yielded the fragment ions corresponding to [dihydroxybenzoic acid-hexoside–H]− at m/z 476 315, and the subsequent losses of H2O at m/z 297 and hexose plus H at m/z 152 and CO2 at m/z 108. 477 Peaks 58 and 61 showed the product ion [dihydroxybenzoic acid–H]− due to an even electron ion at 478 m/z 153 (loss of hexose), instead of the odd electron product ion at m/z 152. Besides, peaks 57, 60 479 and 61, yielded the fragment ion [hydroxybenzoic acid–H]− at m/z 137 and its corresponding 480 decarboxylation ion at m/z 93. This behaviour agrees with that observed for hydroxycinnamic acid 481 glycosides above and in literature (Clifford et al., 2007), which suggest that both, the 482 hydroxybenzoic acid moiety and the dihydroxybenzoic acid moiety, are attached through their 483 phenolic hydroxyl to different positions of the same hexose molecule. Just one isomer of 484 20 hydroxybenzoyl-gallic acid-hexoside and two isomers of hydroxybenzoyl-dihydroxybenzoic acid-485 hexosides have been previously characterized only in cv. baby lettuce (Abu-Reidah et al., 2013). 486 3.1.3. Hydroxyphenylacetic derivatives 487 Taking into account the MS data, the fragmentation patterns observed for hydroxybenzoic 488 acid in the negative ion mode and bibliography (Abu-Reidah et al., 2013; Gómez-Romero, María et 489 al., 2011), 4-hydroxyphenylacetic acid was tentatively assigned to peak 62 (Rt= 5.60 min) (Fig. 4S 490 in the supplementary material), which yielded the deprotonated molecule at m/z 151 and fragment 491 ions due to the loss of CO at m/z 123 and CO2 at m/z 107, showing the typical decarboxylation of 492 phenolic acids. Likewise, peak 63 (Rt= 5.20 min, max= 270, 276 nm) observed in the extracted 493 trace at m/z 313, produced the same decarboxylation ions, and a fragment ion at m/z 151 due to 494 deprotonated 4-hydroxyphenylacetic acid obtained after the loss of a hexose moiety (Fig. 4S in the 495 supplementary material). Thus, it was proposed as 4-hydroxyphenylacetic acid-hexoside. Both 496 compounds have been previously detected in green lettuce cultivars (Abu-Reidah et al., 2013). 497 3.2. Flavonoids 498 3.2.1. Flavonols 499 Thirteen quercetin glycosides (64-76) and four kaempferol glycosides (77-80) were detected 500 and identified on the basis of their mass spectral data, comparison with available standards, and 501 literature. Flavonol monoglycoside mass spectra in the positive mode showed the protonated 502 molecule [M+H]+, the sodium adduct ion [M+Na]+ and the protonated aglycone ion [Y0]+ as a result 503 of the loss of the sugar or organic acid residue (losses: 146 u, rhamnosyl residue; 162 u, hexosyl 504 residue; 176 u, glucuronic residue; 178 u, gluconic residue; 248 u, malonyl-hexosyl residue; 324 u, 505 di-hexosyl residue; 338 u, glucuronic + hexosyl residue; 410 u, hexosyl + malonyl-hexosyl residue; 506 424 u, glucuronic + malonyl-hexosyl residue). In the mass spectrum of flavonol diglycosides, a 507 fragment [Y1]+ due to the loss of the first sugar or organic acid unit was also observed. In the 508 negative mode, the high energy function product ions corresponding to quercetin at m/z 300 (odd 509 electron ion) and/or 301 (even electron ion) were detected (Fig. 4S in the supplementary material), 510 21 as observed in MS/MS elsewhere (Abu-Reidah et al., 2013). Regarding this, compounds 64 (Rt= 511 17.16 min, max= 279, 344 nm), 65 (Rt= 18.03 min, max= 252, 367 nm) and 66 (Rt= 20.25 min, 512 max= 252, 330 nm) were identified as quercetin-3-O-hexosides on the basis of their protonated 513 molecule at m/z 465 and a high energy function product ion at m/z 303, which indicates cleavage of 514 a hexosyl group. This fragmentation pattern and chromatographic retention time of the reference 515 standard confirmed that compound 66 was quercetin-3-O-galactoside. Two isomers of quercetin 516 hexose have been previously described in lettuce (Abu-Reidah et al., 2013; Becker, Klaering, 517 Schreiner, Kroh, & Krumbein, 2014; Jeong et al., 2015; Lin et al., 2012; Llorach et al., 2008; Mai & 518 Glomb, 2013; Marin et al., 2015; Pepe et al., 2015; Romani et al., 2002; Santos et al., 2014; Sofo, 519 Lundegårdh, Mårtensson, Manfra, Pepe, Sommella et al., 2016). 520 Compound 67 (Rt= 18.44 min, max= 254, 349 nm) was identified as quercetin-3-O-521 glucuronide because of [M+H]+ at m/z 479, [M+Na]+ at m/z 501 and [Y0]+ at m/z 303, which 522 indicated the loss of a glucuronic residue in the positive mode (Fig. 2). Similarly, in the negative 523 mode, the molecule [M−H]− at m/z 477 yielded [Y0]− at m/z 301; the loss of 176 u pointed out the 524 presence of a glucuronic residue (Fig. 2). The presence of quercetin-3-O-glucuronide in lettuce had 525 been previously confirmed by nuclear magnetic resonance analysis (DuPont, Mondin, Williamson, 526 & Price, 2000; Mai & Glomb, 2013). The glucuronic group was also observed in compound 68 (Rt= 527 9.50 min, max= 256, 352 nm) and compound 69 (Rt= 10.58 min), which gave [M+H]+ at m/z 641, 528 [M+Na]+ at m/z 663, and [Y0]+ at m/z 303 in positive mode, and peak 69, also [Y1]+ at m/z 465. In 529 the negative mode, both compounds presented similar ionization and fragmentation pattern: [M−H]− 530 at m/z 639, [Y1]− at m/z 463 and [Y0]− at m/z 300 (odd electron ion) and/or 301 (even electron ion). 531 Moreover, the loss of 162 u revealed the cleavage of a hexoxyl group, therefore these flavonols 532 were assigned to quercetin hexose-glucuronide isomers, which had been already described in baby, 533 romaine and iceberg cultivars (Abu-Reidah et al., 2013). 534 Compounds 70 (Rt= 21.52 min, max= 255, 352 nm), 71 (Rt= 22.03 min, max= 252, 364 535 nm) and 72 (Rt= 23.69 min) were identified as quercetin malonylhexoside isomers since they 536 22 presented [M+H]+ at m/z 551, [M+Na]+ at m/z 573, and [Y0]+ at m/z 303 due to the loss of the 537 malonylhexosyl moiety in the positive ion mode; and [M−H]−at m/z 549, [Y0]− at m/z 301 (Fig. 4S 538 in the supplementary material), [M−H−CO2]− at m/z 505 (base peak) in the negative ion mode. The 539 neutral loss of CO2 is characteristic of compounds presenting the malonyl group, as previously 540 reported (Abu-Reidah et al., 2013). This fact is due to in-source fragmentation, which can affect the 541 correct identification of the deprotonated molecule of interest, because the relative abundance of 542 [M−H]− ion could be lower than the product ion [M−H−CO2]− as occurred with these peaks. This 543 particularly labile group could be partially lost during ion transfer from a higher-pressure region of 544 the source to a lower-pressure region (Katta, Chowdhury, & Chait, 1991), as observed for peak 70 545 (0.4 % RA), peak 71 (11 % RA) and peak 72 (0.4 % RA). The identification of compound 70 was 546 also confirmed by the presence of [2M−H]− ion. Quercetin-3-O-(6’’-O-malonyl)-glucoside has been 547 reported in lettuce in several publications (Becker et al., 2014; DuPont et al., 2000; Ferreres, Gil, 548 Castañer, & Tomás-Barberán, 1997; Heimler, Isolani, Vignolini, Tombelli, & Romani, 2007; 549 Llorach et al., 2008; Mai & Glomb, 2013; Marin et al., 2015; Ribas-Agustí et al., 2011; Romani et 550 al., 2002; Santos et al., 2014), and confirmed by NMR analysis (DuPont et al., 2000; Ferreres et al., 551 1997). Two isomers of quercetin malonylglucoside were already described in different lettuce 552 varieties (Abu-Reidah et al., 2013; Lin et al., 2012). The presence of three quercetin 553 malonylhexoside isomers in lettuce is described for the first time in the present study. 554 Compound 73 (Rt= 11.51 min, max= 253, 355 nm) was identified as quercetin-3-O-(6''-O-555 malonyl)-glucoside-7-O-glucuronide, which has been previously described in lettuce (Abu-Reidah 556 et al., 2013; Llorach et al., 2008; Santos et al., 2014). In the positive ion mode, [M+H]+ at m/z 727, 557 [M+Na]+ at m/z 749, and the fragment ions [Y1]+ at m/z 479 and [Y0]+ at m/z 303 indicated the loss 558 of a malonyl-glucosyl group followed by a glucuronic group. In the negative ion mode, the neutral 559 loss of CO2 yielding [M−H−CO2]− at m/z 681 confirmed the presence of a malonyl residue in the 560 molecular structure; as well as the high energy function product ions at m/z 300 (odd electron ion) 561 and/or 301 (even electron ion) (Fig. 4S in the supplementary material), the presence of quercetin. 562 23 Similarly, compound 74 (Rt= 13.82 min, max= 253, 350 nm) also contained a malonyl residue 563 since its base peak in the negative mode was [M−H−CO2]− at m/z 667. The deprotonated molecule 564 at m/z 711 was also present and [Y0]− at m/z 300 (odd electron ion) and/or 301 (even electron ion) 565 (Fig. 4S in the supplementary material) indicated that the aglycone was quercetin. The positive ion 566 mode yielding [M+H]+ at m/z 713, [M+Na]+ at m/z 735, and the fragment ions [Y1]+ at m/z 465 and 567 [Y0]+ at m/z 303 confirmed the cleavage of malonylhexosyl group followed by a hexosyl group. 568 Thus, compound 74 was tentatively assigned to quercetin-3-O-(6''-O-malonyl)-glucoside-7-O-569 glucoside, which has been previously reported in lettuce (Abu-Reidah et al., 2013; Llorach et al., 570 2008; Santos et al., 2014), and confirmed by NMR analysis (Ferreres et al., 1997). 571 Compounds 75 (Rt= 12.18 min) and 76 (Rt= 16.07 min) presented the same monoisotopic 572 molecular mass for [M+H]+ at m/z 627.1580 (C27H31O17) and [M−H]−at m/z 625.1405 (C27H29O17), 573 and [M+Na]+ at m/z 649.1381 (C27H30O17Na). The presence of [Y0]+ at m/z 303 and [Y0]− at m/z 301 574 (Fig. 4S in the supplementary material) in the positive and negative ion modes, respectively, 575 disclosed that the aglycone was quercetin. However, these compounds followed different 576 fragmentation patterns. Peak 75 yielded [Y1]− at m/z 463 due to the loss of a hexosyl moiety (162 577 u), and revealing that [Y0]− was obtained from the loss of a second hexosyl residue. Thus, 578 compound 75 was assigned as a quercertin-O-di-hexoside. Instead, peak 76 yielded [Y1]− at m/z 447 579 due to the loss of a gluconic moiety (178 u), and disclosing a subsequent loss of a rhamnosyl moiety 580 (146 u) to achieve [Y0]−. Peak 75 was tentatively identified as quercetin-di-glucoside, which has 581 been previously reported in green lettuce (Santos et al., 2014). Peak 76 was tentatively proposed as 582 quercertin-O-rhamnosyl-gluconide, which is here reported for the first time to the author’s 583 knowledge. 584 Regarding kaempferol conjugates, compound 77 (Rt= 25.27 min, max= 265, 347 nm) was 585 identified as kaempferol-3-O-(6’’-O-malonyl)-glucoside, which has been already found in different 586 lettuce cultivars (Heimler et al., 2007). In the positive mode, [M+H]+ at m/z 535, [M+Na]+ at m/z 587 557, and the fragment ions and [Y0]+ at m/z 287 revealed the cleavage of a malonyl-glucosyl group. 588 24 In the negative mode, [M−H]−at m/z 533, [Y0]− at m/z 285, [M−H−CO2]− at m/z 489 confirmed the 589 presence of the malonyl glucosyl moiety in the molecule (Fig. 4S in the supplementary material). 590 Regarding the aglycone, kaempferol and the flavone luteolin are isobaric, but their conjugates can 591 be distinguished on the basis of their MS and MS/MS data. In the positive low energy function, 592 kaempferol derivatives yield [Y0]+ as the base peak or [M+H]+ as the base peak plus an intense 593 [Y0]+, whereas luteolin derivatives give as the base peak [M+H]+ or [M+H−H2O]+, and [Y0]+ does 594 not appear or present low relative abundance. In the negative low energy function, both compounds 595 yield [M−H]− or [M−H−CO2]− (in the case of malonylglycosides) as the base peak, but in the 596 negative high energy function, kaempferol conjugates give the base peak [Y0]−, whereas luteolin 597 compounds yield the base peak [M−H]− or [M−H−CO2]− and an intense [Y0]−, or [Y0]− as the base 598 peak and an intense [M−H]− with relative abundance higher than 50% RA. Moreover, several minor 599 monoisotopic product ions at m/z 217.0501 (C12H9O4), 199.0395 (C12H7O3), 175.0395 (C10H7O3) 600 and 133.0290 (C8H5O2) are characteristic of luteolin, and helps to distinguish it from its kaempferol 601 isomers (Abu-Reidah et al., 2013; Gómez-Romero, María et al., 2011). In this sense, these fragment 602 ions did not appear in the negative high energy MS spectra of peak 77, suggesting that it is a 603 kaempferol derivative. Moreover, this identification was also supported by the base peaks yielded in 604 the positive low energy and the negative high energy functions, [Y0]+ and [Y0]− respectively, as well 605 as its UV-visible spectra, and elution order since kaempferol isomers elute later than luteolin 606 isomers on endcapped C18 packings. 607 Two isomers (78: Rt= 23.90 min; 79: Rt= 26.43 min) were detected in the extracted MS 608 chromatogram at m/z 449 and 447 in the positive and negative ion modes respectively, which 609 yielded the protonated ion, [M+Na]+ at m/z 471 and [Y0]+ at m/z 287 in the positive ion mode, and 610 the deprotonated molecule and [Y0]− at m/z 285 in the negative ion mode (Fig. 4S in the 611 supplementary material); revealing the loss of a hexosyl residue and the presence of kaempferol or 612 luteolin aglycone. The base peaks yielded in the positive low energy and the negative high energy 613 functions were [Y0]+ and [Y0]− respectively, and no characteristic minor product ions of luteolin 614 31 support. Gabriela Elena Viacava thanks CONICET and Asociación Universitaria Iberoamericana de 770 Postgrado (AUIP) for her Ph.D. grants. 771 References 772 Abad-García, B., Berrueta, L. A., Garmón-Lobato, S., Gallo, B., & Vicente, F. (2009). A general 773 analytical strategy for the characterization of phenolic compounds in fruit juices by high-774 performance liquid chromatography with diode array detection coupled to electrospray 775 ionization and triple quadrupole mass spectrometry. Journal of Chromatography A, 1216, 776 5398-5415. 777 Abu-Reidah, I. M., Contreras, M. M., Arráez-Román, D., Segura-Carretero, A., & Fernández-778 Gutiérrez, A. (2013). 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Phenolic profile evolution of 882 different ready-to-eat baby-leaf vegetables during storage. Journal of Chromatography A, 883 1327, 118-131. 884 Schütz, K., Carle, R., & Schieber, A. (2006). TaraxacumA review on its phytochemical and 885 pharmacological profile. Journal of Ethnopharmacology, 107, 313-323. 886 Sobolev, A. P., Brosio, E., Gianferri, R., & Segre, A. L. (2005). Metabolic profile of lettuce leaves 887 by high-field NMR spectra. Magnetic Resonance in Chemistry, 43, 625-638. 888 Sofo, A., Lundegårdh, B., Mårtensson, A., Manfra, M., Pepe, G., Sommella, E., De Nisco, M., 889 Tenore, G. C., Campiglia, P., & Scopa, A. (2016). Different agronomic and fertilization 890 systems affect polyphenolic profile, antioxidant capacity and mineral composition of lettuce. 891 Scientia Horticulturae, 204, 106-115. 892 Watson, R. R., Preedy, V. R., & Zibadi, S. (2014). Polyphenols in human health and disease (1st 893 ed.). San Diego: Academic Press. 894 895 36 Figure captions 896 Fig. 1. Chemical structures of phenolic compounds found in butterhead lettuce cultivar. 897 Abbreviations for the phenolic moieties: C, caffeoyl; pCo, p-coumaroyl; F, feruloyl; 898 dhC, dihydrocaffeoyl; Sp, sinapoyl; 4-OH-Bz, 4-hydroxybenzoyl; 3,4-diOH-Bz, 3,4-899 dihydroxybenzoyl; Gal, galloyl; Syr, syringoyl; 4-OH-PhAc, 4-hydroxyphenylacetoyl; 900 Que, quercetin (Z1=OH, Z2=OH); Kaemp, kaempferol (Z1=H, Z2=OH); Lut, luteolin 901 (Z1=OH, Z2=H); Api, apigenin (Z1=H, Z2=H); 6,7-diOH-Cou, 6,7-dihidroxycoumarin. 902 Abbreviations for the non-phenolic moieties: Q, quinic acid; Tar, tartaric acid, Mal, 903 malic acid; Mln, malonic acid; Glcr, glucuronic acid; Glcn, gluconic acid; Hex, hexose; 904 Rha, rhamnose; Rut, rutinose (rhamnosylglucose). R, R1, R2, R3, R4 and R5 in non-905 phenolic moieties can be esterified in position X of phenolic acids or etherified with 906 phenolic OH groups. 907 Fig. 2. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 908 mode of quercetin-3-O-glucuronide. ESI, electrospray ionization. 909 Fig. 3. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 910 mode of eriodictyol-O-glucuronide. ESI, electrospray ionization. 911 912 37 Supplementary material 913 Fig. 1S. Low energy function (F1) base peak chromatograms in positive and negative ion modes 914 and DAD chromatograms at 280 and 370 nm of the butterhead lettuce cultivar. 915 Fig. 2S. Butterhead lettuce cultivar chromatograms extracted from the TIC-MS scan 916 chromatogram in negative ion mode at m/z 353, 341, 385 and 153 of the low energy 917 function (F1). Chromatographic peaks are numbered as in Tables 1. 918 Fig. 3S. Butterhead lettuce cultivar chromatograms extracted from the TIC-MS scan 919 chromatogram in negative ion mode at m/z 337, 311, 331, 451 and 435 of the low energy 920 function (F1). Chromatographic peaks are numbered as in Tables 1. 921 Fig. 4S. Butterhead lettuce cultivar chromatograms extracted from the TIC-MS scan 922 chromatogram in negative ion mode at m/z 151 of the low energy function (F1), and at 923 m/z 301, 285 and 269 of the high energy function (F2). Chromatographic peaks are 924 numbered as in Tables 1. 925 Fig. 5S. Butterhead lettuce cultivar chromatograms extracted from the TIC-MS scan 926 chromatogram in negative ion mode at m/z 287, 177 and 581 of the low energy function 927 (F1), and at m/z 417 of the high energy function (F2). Chromatographic peaks are 928 numbered as in Tables 1. 929 Fig. 6S. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 930 mode of 5-trans-O-caffoylquinic acid. ESI, electrospray ionization. 931 Fig. 7S. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 932 mode of luteolin-7-O-glucuronide. ESI, electrospray ionization. 933 Fig. 8S. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 934 mode of esculetin-6-O-glucoside. ESI, electrospray ionization. 935 Fig. 9S. Low (F1) and high (F2) energy function MS spectra in the negative and positive ion 936 mode of syringaresinol-acetylhexose. ESI, electrospray ionization. 937 1 Table 1 1 Retention times, UV-visible maxima and MSE data of polyphenols identified by UHPLC-DAD-ESI-Q-ToF/MS in the butterhead lettuce cultivar.a, b, c 2 LC DAD ESI(+)QToF/MS ESI()QToF/MS Assignment Nº Rt (min) UV bands (nm) Exp. Acc. Mass [M+H]+ Error (mDa) Formula [M+H]+ Adducts & fragment ions of [M+H]+ m/z Exp. Acc. Mass [MH] Error (mDa) Formula [MH] Adducts & fragment ions of [MH] m/z Tentative identification Phenolic acids Hydroxycinnamic derivatives Caffeoylquinic 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transOCaffeoylquinic acid 323 163.0398 [Caffeoyl+H]+ 179.0348 [CaffeicH] (32) 145.0279 [Caffeoyl+HH2O]+ 173.0437 [QuinHH2O] (4) 135.0448 [Caffeoyl+HCO]+ 135.0446 [CaffeicHCO2] (71) 117.0343 [Caffeoyl+HCOH2O]+ 89.0397 [Caffeoyl+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transOCaffeoylquinic acid 551.1234 [2M+Nacaffeic]+ 191.0561 [QuinH] (100) 377.0846 [M+Na]+ 163.0421 [Caffeoyl+H]+ 145.0279 [Caffeoyl+HH2O]+ 135.0433 [Caffeoyl+HCO]+ 117.0342 [Caffeoyl+HCOH2O]+ 89.0396 [Caffeoyl+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transOCaffeoylquinic acid 324 551.1234 [2M+Nacaffeic]+ 191.0556 [QuinH] (100) 377.0846 [M+Na]+ 179.0343 [CaffeicH] (1) 163.0421 [Caffeoyl+H]+ 173.0449 [QuinHH2O] (3) 145.0279 [Caffeoyl+HH2O]+ 135.0443 [CaffeicHCO2](2) 135.0433 [Caffeoyl+HCO]+ 117.0342 [Caffeoyl+HCOH2O]+ 89.0396 [Caffeoyl+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Caffeoylquinic acid 709.1981 [2M+H]+ 191.0557 [QuinH] (100) 163.0397 [Caffeoyl+H]+ 179.0344 [CaffeicH] (12) 145.0128 [Caffeoyl+HH2O]+ 135.0441 [CaffeicHCO2] (21) 135.0463 [Caffeoyl+HCO]+ 117.0333 [Caffeoyl+HCOH2O]+ 89.0383 [Caffeoyl+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transOCaffeoylquinic acid 163.0445 [Caffeoyl+H]+ 173.0458 [QuinHH2O] (13) 145.0325 [Caffeoyl+HH2O]+ 135.0408 [Caffeoyl+HCO]+ 117.0364 [Caffeoyl+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Caffeoylquinic acid 316 551.1199 [2M+Nacaffeic]+ 191.0557 [QuinH] (100) 377.0841 [M+Na]+ 173.0449 [QuinHH2O] (3) 163.0400 [Caffeoyl+H]+ 145.0284 [Caffeoyl+HH2O]+ 135.0443 [Caffeoyl+HCO]+ 117.0346 [Caffeoyl+HCOH2O]+ 89.0396 [Caffeoyl+HH2O2CO]+ 7 15.06  C16H19O9 163.0399 [Caffeoyl+H]+ 353.0876 0.3 C16H17O9 191.0578 [QuinH] (100) 4cisOCaffeoylquinic acid 145.0287 [Caffeoyl+HH2O]+ 179.0314 [CaffeicH] (5) 135.0446 [Caffeoyl+HCO]+ 173.0455 [QuinHH2O] (2) 117.0278 [Caffeoyl+HCOH2O]+ Table1 2 LC DAD ESI(+)QToF/MS ESI()QToF/MS Assignment Nº Rt (min) UV bands (nm) Exp. Acc. Mass [M+H]+ Error (mDa) Formula [M+H]+ Adducts & fragment ions of [M+H]+ m/z Exp. Acc. Mass [MH] Error (mDa) Formula [MH] Adducts & fragment ions of [MH] m/z Tentative identification p-Coumaroylquinic 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Coumaroylquinic acid 361.0892 [M+Na]+ 191.0467 [QuinH] 147.0451 [pCoumaroyl+H]+ 163.0393 [pCoumaricH] 119.0500 [pCoumaroyl+HCO]+ 119.0496 [pCoumaricHCO2] 91.0556 [pCoumaroyl+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Coumaroylquinic acid 361.0907 [M+Na]+ 173.0449 [QuinHH2O] 147.0453 [pCoumaroyl+HH2O]+ 163.0390 [pCoumaricH] 119.0500 [pCoumaroyl+HH2OCO]+ 119.0491 [pCoumaricHCO2] 91.0561 [pCoumaroyl+HH2O2CO]+ Caffeoyltartaric acid 10 9.06 301 sh, C13H13O9 311.0526 12.3 C13H11O9 293.0287 [CaftarHH2O] Caffeoyltartaric acid 323 179.0349 [CaffeicH] 149.0227 [TartaricH] 135.0432 [CaffeicHCO2] p-Coumaroyltartaric acid 11 15.63 310 C13H13O8 295.0457 0.3 C13H11O8 163.0393 [pCoumaricH] pCoumaroyltartaric acid 149.0104 [TartaricH] 119.0481 [pCoumaricHCO2] Caffeoylmalic acid 12 9.05 301 sh, 297.0585 2.5 C13H13O8 319.0429 [M+Na]+ 295.0448 0.6 C13H11O8 591.0983 [2MH] Caffeoylmalic acid 323 163.0404 [Caffeoyl+H]+ 179.0345 [CaffeicH] 145.0297 [Caffeoyl+HH2O]+ 135.0446 [CaffeicHCO2] 135.0447 [Caffeoyl+HCO]+ 133.0275 [MalicH] 117.0348 [Caffeoyl+HCOH2O]+ 115.0032 [MalicHH2O] 89.0397 [Caffeoyl+HH2O2CO]+ 105.0342 [MalicHCO] Dicaffeoylquinic acids and caffeoylquinic acid glycosides 13 5.86  517.1548 0.9 C22H29O14 539.1364 [M+Na]+ 515.1402 0.1 C22H27O14 353.0869 [CafquinH] Caffeoylquinic acidhexoside 355.1038 [Mhexosyl]+ 191.0548 [QuinH] 163.0415 [Caffeoyl+H]+ 145.0310 [Caffeoyl+HH2O]+ 135.0449 [Caffeoyl+HCO]+ 117.0385 [Caffeoyl+HCOH2O]+ 89.0399 [Caffeoyl+HH2O2CO]+ 14 7.56  C22H29O14 539.1367 [M+Na]+ 515.1402 0.1 C22H27O14 Caffeoylquinic acidhexoside 15 20.20 321 517.1423 7.7 C25H25O12 539.1155 [M+Na]+ 515.1194 0.4 C25H23O12 353.0871 [CafquinH] 1,5diOCaffeoylquinic acid 499.1237 [M+HH2O]+ 335.0771 [CafquinHH2O] 355.0985 [Cafquin+H]+ 191.0558 [QuinH] 163.0403 [Caffeoyl+H]+ 179.0349 [CaffeicH] 145.0159 [Caffeoyl+HH2O]+ 135.0448 [CaffeicHCO2] 135.0451 [Caffeoyl+HCO]+ 117.0350 [Caffeoyl+HCOH2O]+ 89.0404 [Caffeoyl+HH2O2CO]+ 16 20.63 326 517.1332 1.4 C25H25O12 539.1155 [M+Na]+ 515.1186 0.4 C25H23O12 353.0866 [CafquinH] 3,5diOCaffeoylquinic acid 499.1230 [M+HH2O]+ 335.0761 [CafquinHH2O] 355.1016 [Cafquin+H]+ 191.0556 [QuinH] 163.0401 [Caffeoyl+H]+ 179.0347 [CaffeicH] 145.0291 [Caffeoyl+HH2O]+ 135.0446 [CaffeicHCO2] 135.0450 [Caffeoyl+HCO]+ 117.0346 [Caffeoyl+HCOH2O]+ 89.0401 [Caffeoyl+HH2O2CO]+ 3 LC DAD ESI(+)QToF/MS ESI()QToF/MS Assignment Nº Rt (min) UV bands (nm) Exp. Acc. Mass [M+H]+ Error (mDa) Formula [M+H]+ Adducts & fragment ions of [M+H]+ m/z Exp. Acc. Mass [MH] Error (mDa) Formula [MH] Adducts & fragment ions of [MH] m/z Tentative identification 17 24.17 331 517.1423 7.7 C25H25O12 539.1165 [M+Na]+ 515.1190 0.0 C25H23O12 353.0860 [CafquinH] 4,5diOCaffeoylquinic acid 499.1228 [M+HH2O]+ 335.0802 [CafquinHH2O] 473.2006 [M+HCO2]+ 179.0347 [CaffeicH] 355.0161 [Cafquin+H]+ 173.0449 [QuinHH2O] 163.0395 [Caffeoyl+H]+ 135.0441 [CaffeicHCO2] 135.0447 [Caffeoyl+HCO]+ 117.0347 [Caffeoyl+HCOH2O]+ 89.0400 [Caffeoyl+HH2O2CO]+ p-Coumaroylcaffeoylquinic acids 18 23.58 312 501.1384 1.3 C25H25O11 523.1219 [M+Na]+ 499.1233 0.7 C25H23O11 353.0868 [MHcoumaroyl] 3pCoumaroyl4caffeoylquinic acid 483.1295 [M+HH2O]+ 337.0916 [MHcaffeoyl] 163.0399 [Caffeoyl+HH2O]+ 191.0560 [QuinH] 147.0446 [pCoumaroyl+H]+ 179.0353 [CaffeicH] 145.0279 [Caffeoyl+H2H2O]+ 163.0398 [pCoumaricH] 135.0455 [Caffeoyl+HH2OCO]+ 135.0452 [CaffeicHCO2] 119.0497 [pCoumaroyl+HH2OCO]+ 119.0503 [pCoumaricHCO2] 117.0335 [Caffeoyl+H2H2OCO]+ 91.0550 [pCoumaroyl+HH2O2CO]+ 89.0398 [Caffeoyl+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coumaroyl] 4Caffeoyl5pcoumaroylquinic acid 483.1281 [M+HH2O]+ 337.0928 [MHcaffeoyl] 147.0445 [pCoumaroyl+H]+ 191.0553 [QuinH] 119.0493 [pCoumaroyl+HCO]+ 179.0342 [CaffeicH] 91.0550 [pCoumaroyl+H2CO]+ 163.0390 [pCoumaricH] 135.0448 [CaffeicHCO2] 119.0490 [pCoumaricHCO2] Dicaffeoyltartaric acids 20 10.53 301 sh, C22H19O12 497.0677 [M+Na]+ 473.0719 0.1 C22H17O12 947.1354 [2MH] diOCaffeoyltartaric acid 324 457.0698 [M+HH2O]+ 311.0402 [CaftarH] 295.0577 [CaftarHH2O]+ 293.0296 [CaftarHH2O] 163.0397 [Caffeoyl+H]+ 179.0345 [CaffeicH] 145.0292 [Caffeoyl+HH2O]+ 149.0091 [TartaricH] 135.0448 [Caffeoyl+HCO]+ 135.0443 [CaffeicHCO2] 117.0343 [Caffeoyl+HCOH2O]+ 105.0339 [TartaricHCO2] 89.0396 [Caffeoyl+HH2O2CO]+ 21 12.54 301 sh, C22H19O12 295.0563 [CaftarHH2O]+ 473.0713 0.7 C22H17O12 311.0387 [CaftarH] mesodiOCaffeoyltartaric acid 323 163.0398 [Caffeoyl+H]+ 293.0297 [CaftarHH2O] 145.0288 [Caffeoyl+HH2O]+ 179.0346 [CaffeicH] 135.0446 [Caffeoyl+HCO]+ 149.0126 [TartaricH] 117.0341 [Caffeoyl+HCOH2O]+ 135.0448 [CaffeicHCO2] 89.0398 [Caffeoyl+HH2O2CO]+ 105.0343 [TartaricHCO2] Other hydroxycinnamic acid derivatives 22 5.39  343.1098 6.9 C15H19O9 365.0878 [M+Na]+ 341.0905 3.2 C15H17O9 Caffeic acidhexoside 163.0394 [Caffeoyl+H]+ 145.0104 [Caffeoyl+HH2O]+ 135.0497 [Caffeoyl+HCO]+ 89.0401 [Caffeoyl+HH2O2CO]+ 23 5.64  C15H19O9 365.0833 [M+Na]+ 341.0854 1.9 C15H17O9 179.0330 [CaffeicH] Caffeic acidhexoside 163.0389 [Caffeoyl+H]+ 135.0435 [CaffeicHCO2] 145.0289 [Caffeoyl+HH2O]+ 135.0473 [Caffeoyl+HCO]+ 117.0309 [Caffeoyl+HCOH2O]+ 24 6.08 301 sh, C15H19O9 365.0844 [M+Na]+ 341.0873 0.0 C15H17O9 179.0348 [CaffeicH] Caffeic acidhexoside 325 135.0452 [CaffeicHCO2] 10 LC DAD ESI(+)QToF/MS ESI()QToF/MS Assignment Nº Rt (min) UV bands (nm) Exp. Acc. Mass [M+H]+ Error (mDa) Formula [M+H]+ Adducts & fragment ions of [M+H]+ m/z Exp. Acc. Mass [MH] Error (mDa) Formula [MH] Adducts & fragment ions of [MH] m/z Tentative identification 106 24.50  C30H39O14 621.2183 0.0 C30H37O14 417.1548 [MHacetylhexosyl] Syringaresinolacetylhexose 402.1313 [MHacetylhexosylCH3] 387.1078 [MHacetylhexosyl2CH3] 359.1111 [MHacetylhexosyl2CH3CO] 181.0503 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)] 166.0268 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)CH3] 151.0044 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)2CH3] 123.0065 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)2CH3CO] 107 24.63  C30H39O14 621.2181 0.2 C30H37O14 417.1546 [MHacetylhexosyl] Syringaresinolacetylhexose 402.1313 [MHacetylhexosylCH3] 387.1074 [MHacetylhexosyl2CH3] 359.1084 [MHacetylhexosyl2CH3CO] 181.0503 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)] 166.0269 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)CH3] 151.0041 [MHacetylhexosyl2CH3OOHC6 H2CHO2(CH2CH)2CH3] 108 19.22  C28H39O13 581.2239 0.5 C28H37O13 341.1392 [MHhexosylCH3COOHH2O] Dimethoxyhexosyllariciresinol 329.1390 [MHhexosylCH3COOH2CH3] 109 19.39  C28H39O13 581.2238 0.4 C28H37O13 359.1494 [MHhexosylCH3COOH] Dimethoxyhexosyllariciresinol 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] Dimethoxyhexosyllariciresinol 329.1392 [MHhexosylCH3COOH2CH3] 111 16.37  C34H49O18 743.2742 2.0 C34H47O18 581.2249 [MHhexosyl] Dimethoxydihexosyllariciresinol 359.1494 [MH2hexosylCH3COOH] 341.1383 [MH2hexosylCH3COOHH2O] 329.1392 [MH2hexosylCH3COOH2CH3] 3 a Fragment ions produced in MS were named according to Ma et al. (1997)(Ma, Li, Van den Heuvel, & Claeys, 1997). 4 b Abbreviations: Caffeic, caffeic acid; Cafquin, caffeoylquinic acid; Caftar, caffeoyltartaric acid; DiHBZ, dihydroxybenzoic acid; DiHBZhex, 5 dihydroxybenzoic acid-hexoside; DihydroCaf, dihydrocaffeic acid; Gallic, gallic acid; HBZ, hydroxybenzoic acid; hex, hexose; 4-hydroxyphenylacetic, 4-6 hydroxyphenylacetic acid; 4-hydroxyphenylacetichex, 4-hydroxyphenylacetic acid-hexoside ; Malic, malic acid; pCoumaric, p-coumaric acid; Quin, quinic 7 acid; Tartaric, tartaric acid; sh, shoulder. 8 c Abundances of the fragment ions of caffeoylquinic acids in the negative mode are given in parenthesis. 9 OR1 HOOC OR5 OR3 OR4 Q OR2 OR1 HO OH O O Tar 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 Glcr O HO HO OH OR O HO H3C HO O OH Rut 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 Syr H3CO OCH3 OH XO OO H3CO OCH3 OCH3 H3CO HO OH Syringaresinol O HO OCH3 H3CO OH OH Lariciresinol Lignans O HO HO O 6,7-diOH-Cou Coumarins Que / Kaemp / Lut / Api O Z2 OH O HO Z1 OH O O OH HO OH OH Eriodictyol Flavonoids 1 2 3 4 5 6 7 8 2‘ 3‘ 4‘ 5‘ 6‘ AC B Phenolic Figure 1 revised Click here to download Figure(s): FoodChem_Fig1_Alonso-Salces_revised.eps m/z 50 100 150 200 250 300 350 400 450 500 % 0 100 477.0675 299.0200 301.0347 Scan ESIF1: 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 ESIF2: 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 Figure 2 Click here to download Figure(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 ESIF2: 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 ESIF1: 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 Figure 3 Click here to download Figure(s): FoodChem_Fig3_Alonso-Salces.eps