Accepted Manuscript

A comprehensive study on the phenolic profile of widely used culinary herbs

and spices: rosemary, thyme, oregano, cinnamon, cumin and bay

Anna Vallverdú-Queralt, Jorge Regueiro, Miriam Martínez-Huélamo, José

Fernando Rinaldi Alvarenga, Leonel Neto Leal, Rosa M. Lamuela-Raventos

PII: S0308-8146(14)00004-1
DOI: http://dx.doi.org/10.1016/j.foodchem.2013.12.106
Reference: FOCH 15223

To appear in: Food Chemistry

Received Date: 1 October 2013

Revised Date: 16 December 2013
Accepted Date: 31 December 2013

Please cite this article as: Vallverdú-Queralt, A., Regueiro, J., Martínez-Huélamo, M., Alvarenga, J.F., Leal, L.N.,
Lamuela-Raventos, R.M., A comprehensive study on the phenolic profile of widely used culinary herbs and spices:
rosemary, thyme, oregano, cinnamon, cumin and bay, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/
j.foodchem.2013.12.106

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 1 A comprehensive study on the phenolic profile of widely used culinary herbs and

2 spices: rosemary, thyme, oregano, cinnamon, cumin and bay.

4 Anna Vallverdú-Queralta,b, Jorge Regueiroc, Miriam Martínez-Huélamoa,b, José

5 Fernando Rinaldi Alvarengad, Leonel Neto Leale and Rosa M. Lamuela-Raventosa,b*

a
6 Nutrition and Food Science Department, XaRTA, INSA. Pharmacy School, University

7 of Barcelona, Spain.
b
8 CIBER Fisiopatología de la Obesidad y la Nutrición (CIBERobn), Instituto de Salud

9 Carlos III, Spain.

c
10 Nutrition and Food Science Group, Department of Analytical and Food Chemistry,

11 Faculty of Food Science and Technology, University of Vigo, Spain.

d
12 Department of Food and Nutrition. School of Pharmaceutical Science. São

13 Paulo State University, Brazil.

e
14 Nutreco Research and Development, The Netherlands.

15

16

*
17 Corresponding author: Nutrition and Food Science Department, XaRTA, INSA

18 Pharmacy School, University of Barcelona, Spain. Telephone +34-934034843. Fax +34-

19 934035931; e-mail lamuela@ub.edu

20
21 ABSTRACT

22 Herbs and spices have long been used to improve the flavour of food without being

23 considered as nutritionally significant ingredients. However, the bioactive phenolic

24 content of these plant-based products is currently attracting interest.

25 In the present work, liquid chromatography coupled to high-resolution/accurate mass

26 measurement LTQ-Orbitrap mass spectrometry was applied for the comprehensive

27 identification of phenolic constituents of six of the most widely used culinary herbs

28 (rosemary, thyme, oregano and bay) and spices (cinnamon and cumin). In this way, up

29 to 52 compounds were identified in these culinary ingredients, some of them, as far as

30 we know, for the first time. In order to establish the phenolic profiles of the different

31 herbs and spices, accurate quantification of the major phenolics was performed by

32 multiple reaction monitoring in a triple quadrupole mass spectrometer. Multivariate

33 statistical treatment of the results allowed the assessment of distinctive features among

34 the studied herbs and spices.

35

36

37 Key words: Culinary herbs, spices, polyphenols, Rosemary, Thyme, Oregano,

38 Cinnamon, Bay, Cumin, LC–ESI-LTQ-Orbitrap,

39

40

41

42

43

44
45 1. Introduction

46 Since ancient times, herbs and spices have been used all over the world to enhance or

47 improve the flavour of food due to their sensory properties, and also as preservative

48 agents (Kivilompolo & Hyotylainen 2007; Park 2011; Shan, Cai, Sun, & Corke 2005).

49 However, most of their potential health-promoting properties have received little

50 attention. Recent research has shown culinary herbs and spices to be a dietary source of

51 bioactive polyphenols (Hinneburg, Damien Dorman, & Hiltunen, 2006; Wojdyło,

52 Oszmiański, & Czemerys, 2007), which has stimulated the study of their phenolic

53 composition and antioxidant properties. Several culinary herbs and spices are now

54 known to have beneficial effects for human health, including digestive stimulant, anti-

55 inflammatory, antimicrobial, antioxidant and anticarcinogenic activities (Shobana &

56 Akhilender Naidu 2000; Velioglu, Mazza, Gao, & Oomah 1998; Zheng & Wang 2001),

57 which are attributed to the predominant polyphenol compounds in these plant materials.

58 Moreover, the volatile constituents (essential oils) that are the main cause for use of

59 these plants can significantly contribute to biological activity (Inouye, Takizawa, &

60 Yamaguchi, 2001).

61 Recently, there has been growing awareness of the importance of a high dietary content

62 of phenolic compounds, such as flavonoids and hydroxycinnamic acids, because of their

63 apparent multiple biological effects, including metal chelation, free-radical scavenging,

64 inhibition of cellular proliferation, modulation of enzymatic activity and signal

65 transduction pathways (Del Rio, Rodriguez-Mateos, Spencer, Tognolini, Borges, &

66 Crozier, 2013).

67 Although the contribution of several widely-used culinary herbs and spices to the total

68 intake of dietary polyphenols has been previously investigated (Halvorsen et al. 2002;

69 (Halvorsen et al. 2002; Hinneburg et al., 2006; Wojdyło et al., 2007), a comprehensive
70 identification of their phenolic profile is still lacking, mainly due to the wide variety of

71 structures of these natural compounds and unavailability of commercial standards. In

72 this context, high-resolution/accurate mass measurement (HR/AM) mass spectrometry

73 techniques have been demonstrated to be a reliable tool for the structural elucidation of

74 unknown compounds in complex samples (Vallverdú-Queralt, Jáuregui, Medina-

75 Remón, Andrés-Lacueva & Lamuela-Raventós, 2010). Among the HR/AM systems,

76 linear ion trap quadrupole-Orbitrap-mass spectrometry (LTQ-Orbitrap-MS) delivers

77 single-stage mass analysis providing molecular mass information, two-stage mass

78 analysis (MS/MS) and multi-stage mass analysis (MSn) with useful structural

79 information. Zhou et al. have recently identified the phenolics of Sarcandra glabra by

80 non-targeted high-performance liquid chromatography fingerprinting and targeted

81 electrospray ionisation tandem quadrupole mass spectrometry/time-of-flight mass

82 spectrometry analyses (Zhou, Liang, Lv, Hu, Zhu, Si , & Wu, 2013).

83 The objective of this work was therefore to extensively study the phenolic profile of

84 several widely-used culinary herbs (rosemary, thyme, oregano and bay) and spices

85 (cinnamon and cumin) by liquid chromatography coupled to electrospray ionisation

86 LC–ESI-LTQ-Orbitrap mass spectrometry. The high-resolution MS analyses revealed

87 the presence of 51 phenolic compounds, some of them hitherto unreported in culinary

88 herbs and spices. Quantification of major compounds was also carried out by LC

89 coupled to triple quadrupole mass spectrometry (LC–ESI-QqQ) using multiple reaction

90 monitoring (MRM) mode. The quantification levels of phenolic compounds allowed the

91 identification of distinguishing features among Lamiaceae, Lauraceae and Apiaceae

92 botanical families.

93

94
 95 2. Materials and methods

96 2.1. Standards and reagents

97 All samples and standards were handled without exposure to light. Caffeic, ferulic, p-

98 coumaric, protocatechuic, syringic, rosmarinic, p-hydroxybenzoic and chlorogenic acid

99 (5-O-caffeoylquinic acid), quercetin, catechin, epicatechin, ABTS: 2,2’azino-bis(3-

100 ethylbenzothiazoline-6-sulfonic acid), Trolox: (±)-6-hydroxy-2,5,7,8-

101 tetramethylchromane-2-carboxylic acid 97% and manganese dioxide were purchased

102 from Sigma-Aldrich (Madrid, Spain); DPPH: 2,2-diphenyl-1-picrylhydrazyl from

103 Extrasynthèse (Genay, France). Ethanol, methanol and HPLC-grade formic acid were

104 obtained from Scharlau (Barcelona, Spain) and ultrapure water (Milli-Q) from Millipore

105 (Billerica, MA). Samples were stored at 4 ºC and protected from light until analysis.

106

107 2.2. Extraction and analysis of polyphenols

108 2.2.1. Samples

109 Dried and ground rosemary (Rosmarinus officinalis), oregano (Origanum vulgare),

110 thyme (Thymus vulgaris), bay leaf (Laurus nobilis), cumin (Cuminum cyminum) and

111 cinnamon (Cinnamomum zeylanicum) were sourced by Nutreco B.V., Amersfoort, the

112 Netherlands. According to product specifications, the countries of origin were China

113 (oregano, cumin, cinnamon and bay) and Spain (rosemary and thyme). Spices were

114 extracted with a hydroalcoholic solvent, centrifuged, concentrated and dried. The dried

115 spices were ground (particle size range: 500 to 600 µm) and stored at ‒20 °C in

116 darkness.

117

118

119 2.2.2. Extraction of polyphenols

120 Each dried spice was divided into 3 portions, each extracted, and each extract analysed

121 twice in a darkened room with a red safety light to avoid photodegradation of the

122 analytes following a previously reported procedure (Vallverdú-Queralt et al., 2010) with

123 minor modifications. Briefly, samples (1 g) were extracted with 5 mL of 50 % ethanol

124 in ultrapure water with 0.1 % formic acid, sonicated for 5 min and centrifuged at 3000 g

125 for 10 min at 4 ºC. The extraction procedure was repeated twice with the plant material

126 residue. Both supernatants were combined and the organic solvent was evaporated

127 under a nitrogen flow. Finally, extracts were reconstituted up to 5 mL with 0.1 % of

128 formic acid in water.

129 A solid-phase extraction (SPE) procedure was carried out to eliminate potential

130 interferences from plant extracts. Oasis mixed-mode anion-exchange cartridges and

131 Oasis MAX 96-well plates (30 mg, 30 µm) from Waters (Milford, USA) were used

132 following a previously reported procedure (Vallverdú-Queralt et al., 2010). Firstly, 1

133 mL of methanol and subsequently 1 mL of sodium acetate (50 mmol/L, pH 7) were

134 loaded into Oasis® MAX cartridges from Waters to equilibrate it; then, 1mL of each

135 extract was diluted with 1 mL of Milli-Q water and acidified with 34 µL of hydrochloric

136 acid (35%) before being loaded into the cartridges separately. These were rinsed with

137 sodium acetate (50 mmol/L, pH 7; 5% methanol). The polyphenols were eluted with

138 1800 µL of methanol (2% formic acid). The eluted fractions were evaporated under

139 nitrogen flow, and the residue was reconstituted with water (0.1 % formic acid) up to

140 250 µL and filtered through a 13 mm, 0.45 µm PTFE filter (Waters) into an insert-

141 amber vial for HPLC analysis. Samples were stored at ‒20 ºC until analysis.

142

143

144 2.2.3. LC-LTQ-Orbitrap-MS and LC-MS/MS analyses

145 For accurate mass measurements, a LTQ Orbitrap Velos mass spectrometer (Thermo

146 Scientific, Waltham, MA) equipped with an ESI source was used operating in negative

147 ion mode. Specific parameters were as follows: spray voltage, 4 kV; sheath gas, 20

148 (arbitrary units); auxiliary gas, 10 (arbitrary units); sweep gas, 2 (arbitrary units); and

149 capillary temperature, 275 °C. Default values were used for most other acquisition

150 parameters (FT Automatic gain control (AGC) target 5·× 10 5 for MS mode and 5·× 104

151 for MSn mode). Plant extracts were first analysed in full MS mode at a resolution of

152 60000 (at m/z 400). Successive analyses were done in MSn mode with the Orbitrap

153 resolution set at 30000 (at m/z 400). The most intense ions detected in full scan

154 spectrum were selected for the data-dependent scan. Parent ions were fragmented by

155 high-energy C-trap dissociation (HCD) with normalised collision energy of 45% and an

156 activation time of 100 ms. The maximum injection time was set to 100 ms with two

157 micro scans for MS mode and to 1000 ms with one micro scan for MSn mode. The mass

158 range was from m/z 100 to 1000.

159 Instrument control and data acquisition were performed with Xcalibur 2.0.7 software

160 (Thermo Fisher Scientific). An external calibration for mass accuracy was carried out

161 the day before the analysis according to the manufacturer’s guidelines.

162 The liquid chromatograph was an Accela system (Thermo Scientific, Hemel

163 Hempstead, UK) equipped with a quaternary pump, a photodiode array detector (PDA)

164 and a thermostated autosampler. A reversed-phase column Atlantis T3 C18 (100 × 2.1

165 mm, 3 µm) from Waters (Milford, MA) maintained at 25 ºC was used. Gradient elution

166 was performed with water/0.1% formic acid (v/v), acetonitrile/0.1% formic acid (v/v) at

167 a constant flow rate of 0.350 mL/min, and injection volume was 5 µL. An increasing

168 linear gradient of solvent B was used. Separation was carried out in 36 min under the

169 following conditions: 0 min, 10 % B; 1 min, 10% B; 15 min, 30% B; 22 min, 50% B; 28
170 min, 100% B; 34 min, 100% B, 36 min, 10% B. The column was equilibrated for 6 min

171 prior to each analysis. These conditions were adapted from a previous study with some

172 modifications (Vallverdú-Queralt, Rinaldi de Alvarenga, Estruch, & Lamuela-Raventos,

173 2013).

174 The elemental composition of the detected compounds was based on their accurate mass

175 measurements and isotopic patterns, and then searched for identification in the

176 Dictionary of Natural Products (Chapman & Hall/CRC), the MOTO database

177 (http://appliedbioinformatics.wur.nl/moto) and the Plant Metabolic Network. The

178 interpretation of the observed MS/MS spectra in comparison with those found in the

179 literature was the main tool for tentative identification of polyphenols.

180 Quantification of the previously identified compounds was performed by LC–ESI-

181 MS/MS using an Agilent series 1100 HPLC instrument (Agilent, Waldbronn, Germany)

182 coupled to an API 3000 triple quadrupole mass spectrometer (PE Sciex, Concord,

183 Ontario, Canada) equipped with a Turbo Ionspray source, which was operated in

184 negative-ion mode. Separation was carried out under the same chromatographic

185 conditions used during the identification step. Specific mass spectrometer parameters

186 were as follows: spray voltage, ‒3.5kV; nebuliser gas (N2), 10 (arbitrary units); curtain

187 gas (N2), 12 (arbitrary units); collision gas (N2), 4 (arbitrary units); focusing potential,

188 ‒200 V; entrance potential, ‒10 V; drying gas (N2), heated to 400 ºC and introduced to a

189 flow rate of 6000 cm3/min. The declustering potential and collision energy were

190 optimised for each compound in infusion experiments: individual standard solutions (10

191 µg/mL) dissolved in 1:1 (v/v) mobile phase were infused at a constant flow rate of 5

192 µL/min using a model syringe pump (Harvard Apparatus, Holliston, MA). Collision

193 energy and declustering potential are shown in Table 1.

194 For quantification purposes, data was acquired in multiple reaction monitoring (MRM)

195 mode, tracking the transition of parent and product ions specific for each compound.

196 Quantification of polyphenols was performed by the internal standard method. The

197 method of internal standards is used to improve the precision of quantitative analysis.

198 The internal standard was ethyl gallate (400ng/g) and results were expressed as µg/g dry

199 weight (DW).

200

201 2.2.4. Analysis of total polyphenols

202 For the total polyphenols (TP) assay, each sample was analysed three times; 20 µL of

203 the eluted fractions from SPE were mixed with 188 µL of Milli-Q water in a thermo

204 microtitre 96-well plate (nuncTM, Roskilde, Denmark), and 12 µL of Folin-Ciocalteau

205 (F–C) 2N reagent and 30 µL of sodium carbonate (200 g/L) were added, following the

206 procedure described by Vallverdú-Queralt, Medina-Remon, Martinez-Huelamo,

207 Jauregui, Andres-Lacueva and Lamuela-Raventos (2011a). The mixtures were

208 incubated for 1 h at room temperature in the dark. After the reaction period, 50 µL of

209 Milli-Q water were added and the absorbance was measured at 765 nm in a UV/Vis

210 Thermo Multiskan Spectrum spectrophotometer (Vantaa, Finland). Results were

211 expressed as mg of gallic acid equivalents (GAE)/g DW.

212

213 2.2.5. Antioxidant capacity

214 The culinary herb extracts prepared for polyphenol analysis were also analysed for their

215 antioxidant capacity (AC). The AC was measured using an ABTS+ radical

216 decolorisation assay and DPPH assay (Vallverdu-Queralt, Medina-Remon, Casals-Ribes,

217 Amat, & Lamuela-Raventos, 2011b).

218 ABTS+ assay

219 1 mM Trolox (antioxidant standard) was prepared in methanol. Working standards were

220 obtained by diluting 1 mM Trolox with methanol. Solutions of known Trolox

221 concentration were used for calibration. An ABTS+ radical cation was prepared by

222 passing a 5 mM aqueous stock solution of ABTS (in methanol) through manganese

223 dioxide powder. Excess manganese dioxide was filtered through a 13 mm 0.45 µm filter

224 PTFE (Waters). Then, 245 µL of ABTS+ solution were added to 5 µL of Trolox or to

225 herb extracts (0.1% formic acid in water) and the solutions were stirred for 30 s. The

226 homogenate was shaken vigorously and kept in darkness for 1 h. Absorption of the

227 samples was measured on a UV/Vis Thermo Multiskan Spectrum spectrophotometer at

228 734 nm and methanol blanks were run in each assay. Results were expressed as mmol

229 Trolox equivalents (TE)/g DW.

230 DPPH assay

231 The antioxidant capacity was also studied through the evaluation of the free radical-

232 scavenging effect on the DPPH radical. Solutions of known Trolox concentration were

233 used for calibration. Five microlitres of herb extracts (0.1% formic acid in water) or

234 Trolox were mixed with 250 µL of methanolic DPPH (0.025 g L‒1). The homogenate

235 was shaken vigorously and kept in darkness for 30 min. Absorption of the samples was

236 measured on the spectrophotometer at 515 nm. Results were expressed as mmol TE

237 100/g DW.

238

239 2.3. Statistical analysis

240 The significance of the results and statistical differences were analysed using

241 Statgraphics plus v. 5.1 software (StatPoint, Inc., Herndon, VA). Data were analysed by

242 multifactor analysis of variance and a Duncan multiple range test was applied to

243 determine differences between means, with a significance level of p = 0.05.

244 Additionally, correlations among variables were evaluated using principal component

245 analysis (PCA) to cluster culinary herbs according to their polyphenol profile. PCA is a

246 multivariate statistical technique that allows us to visualise the original arrangement of

247 plant herbs in an n-dimensional space, by identifying the directions in which most of the

248 information is retained.

249

250 3. Results and discussion

251 3.1. Phenolic profile of culinary herbs and spices

252 Culinary herbs and spices are interesting for their content of bioactive compounds that

253 may exert beneficial effects on human health. Table 2 shows a list of 51 phenolic

254 compounds identified by LC–ESI-LTQ-Orbitrap along with their retention times (RT),

255 accurate mass measurements (acc. mass), molecular formula (MF), mDa of error

256 between the mass found and the accurate mass of each polyphenol and the MS/MS

257 fragment ions used for identification. Phenolic compounds were identified by

258 comparing retention times and their masses with those of 24 authentic standards.

259 Identification of the remaining 27 compounds without available standards was based on

260 accurate mass measurements of the [M ‒ H]‒ ion and fragment ions. The fragmentation

261 patterns of the majority of these compounds have been previously identified in other

262 works (Vallverdú-Queralt, Jáuregui, Di Lecce, Andrés-Lacueva, & Lamuela-Raventós, 2011c;

263 Vallverdú-Queralt et al., 2010).

264

265 Caffeic (m/z 179) and caffeic-O-hexoside (m/z 341), protocatechuic (m/z 153),

266 rosmarinic (m/z 359), 3-, 4- and 5-O-caffeoylquinics (m/z 353), coumaroylquinic (m/z

267 337), ferulic-O-hexoside (m/z 355), ferulic (m/z 193), p-coumaric (m/z 163),

268 homovanillic-O-hexoside (m/z 343), gallic (m/z 169), syringic (m/z 197), p- and m-
269 hydroxybenzoic (m/z 137) acids and kaempferol-3-O-glucoside (m/z 447), kaempferol

270 (m/z 285) and quercetin (m/z 301) were detected in all the culinary herbs and spices.

271 Several trimeric proanthocyanidins (m/z 863) and one hexamer (m/z 1727) were also

272 detected in cinnamon and cumin. The proanthocyanidin hexamer identified through its

273 [M ‒ 2H]2‒ ion was confirmed by the 0.5 Da mass differences between the isotopic

274 peaks. The selected resolution of 60000 (at m/z 400) allowed determination of the

275 charge state with high accuracy. The proanthocyanidin hexamer showed doubly-charged

276 ions at m/z 863 corresponding to monoisotopic masses of 1727.3730. The most common

277 classes of proanthocyanidins consist of subunits of catechin, epicatechin, and their gallic

278 acid esters (B-type oligomers). However, the hexamers and trimers found in this study

279 were A-type oligomers (Figure 1), which are structural variations of proanthocyanidin

280 oligomers with the formation of a second interflavanoid bond by C‒O oxidative

281 coupling. Due to the complexity of this conversion, A-type proanthocyanidins are not

282 encountered in nature as frequently as the B-type oligomers (Lazarus, Adamson,

283 Hammerstone, & Schmitz 1999).

284 To our knowledge, three of the polyphenols identified in this work are reported for the

285 first time in these plant extracts. Thus, while apigenin-C-hexoside-C-hexoside (m/z 593)

286 has been previously found in marjoram (Kaiser, Carle, & Kammerer 2013), it has been

287 hitherto unidentified in rosemary and oregano. Sinapic acid-C-hexoside (m/z 385) was

288 also identified for the first time in rosemary and thyme. Lastly, dicaffeoylquinic acid

289 (m/z 515) was detected in rosemary, thyme, oregano, cinnamon and cumin. Hossian et

290 al. (2010) identified dicaffeoylquinic acids in rosemary, thyme, oregano, sage, and basil

291 but, as far as we know, this is the first time they have been reported in cumin and

292 cinnamon. Mass spectra of those compounds identified for the first time are shown in.

293 Figure 2a-c.

294 Consistent differences (p < 0.05) in TP content were observed among the different herbs

295 and spices (Table 3), ranging from 1.12 mg GAE/g DW in bay to 5.82 mg GAE/g DW

296 in cinnamon. A similar pattern was observed in their antioxidant capacities. The ABTS +

297 assay gave results between 0.72 mmol TE/g DW and 4.13 mmol TE/g DW for bay and

298 cinnamon, respectively. The DPPH assay presented 0.30 mmol TE/g DW for bay and

299 2.16 mmol TE/g DW for cumin. The radical-scavenging capacities of oregano,

300 rosemary and thyme extracts have been previously observed in different model systems

301 (Erkan, Ayranci, & Ayranci 2008; Miura, Kikuzaki, & Nakatani 2002; Vichi, Zitterl-

302 Eglseer, Jugl, & Franz 2001).

303

304 3.2. Pattern of similarities among herbs and spices according to the phenolic

305 composition

306 The HPLC-MS/MS performance parameters are reported in Table 4. Recoveries ranged

307 from 85% and 111% and repeatability was less than 8% for all the analytes. Limits of

308 detection (LODs) were between 1.7·× 10‒4 for chlorogenic acid and 8.9 ×·10‒3 for

309 quercetin. The quantification levels of the main polyphenols observed revealed

310 distinctive features among culinary herbs and spices. The results of the quantitative

311 determination of the target polyphenols are summarised in Table 5. The statistically

312 significant differences (p < 0.05) found between the herbs and spices for each

313 polyphenol are highlighted with different superscripts. An HPLC chromatogram of bay,

314 including identification of each peak, is shown in Figure 3.

315 The main phenolic acid in the studied culinary herbs was found to be rosmarinic acid,

316 which varied from 0.39 µg/g DW in bay to 157 µg/g DW in rosemary, being the

317 dominant phenolic compound in oregano, thyme and rosemary. It should be noted that

318 the three species showing similarities - oregano, thyme and rosemary - all belong to the
319 Lamiaceae family. These results are in accordance with another study analysing 26

320 spice extracts (Shan et al., 2005). Rosmarinic acid, along with other compounds also

321 present in rosemary (i.e., carnosol, rosmanol, epi-rosmanol, among others), has shown

322 potent antioxidant activities, and is well correlated with total antioxidant activity

323 (Herrero, Plaza, Cifuentes, & Ibáñez 2010). A similar pattern was observed for p-

324 hydroxybenzoic acid, with the highest levels found in rosemary (15.2 µg/g DW) and the

325 lowest in bay (1.14 µg/g DW).

326 The highest levels of caffeic acid (6.56‒12.6 µg/g DW) were observed in oregano,

327 thyme and rosemary, with lower amounts found in cinnamon, cumin and bay (0.44 to

328 3.06 µg/g DW). As mentioned above, it should be noted that species showing

329 similarities - oregano, thyme and rosemary - belong to the Lamiaceae family. Caffeic

330 acid has been previously identified in oregano and rosemary (Agiomyrgianaki & Dais

331 2012; Herrero et al., 2010). The results for caffeic acid reported by Kivilompolo et al.

332 are in line with our study, with less than 50 µg/g DW found in rosemary and oregano,

333 although they describe higher levels in thyme (129 µg/g DW) (Kivilompolo &

334 Hyotylainen 2007; Park 2011). Papageorgiou et al. reported higher levels of caffeic

335 acid in rosemary (300 to 1500 µg/g DW) than in our study, but with undetectable

336 amounts in bay (Papageorgiou, Mallouchos, & Komaitis 2008). Differences in phenolic

337 acid levels from those in the literature can be attributed to genotypic and environmental

338 differences within species, choice of plant parts tested, when samples were taken and

339 determination methods.

340 Syringic acid showed the same pattern as the aforementioned phenolic acids, with

341 rosemary containing the highest amount (3.46 µg/g DW), followed by oregano (1.26

342 µg/g DW), while bay and cumin showed the lowest levels (0.40‒0.47 µg/g DW).

343 Kivilompolo et al. found syringic acid below 50 µg/g DW in thyme, with undetectable
344 levels in the other herb extracts analysed (Kivilompolo & Hyotylainen 2007; Park

345 2011). In contrast, Hossain et al. reported the presence of syringic acid in thyme,

346 rosemary, oregano and bay (Hossain, Rai, Brunton, Martin-Diana & Barry-Ryan, 2010).

347 Levels of chlorogenic acid were similar in all culinary herbs and spices, with the

348 exception of cumin, which contained 4.18 µg/g DW. Results in line with our study are

349 reported in the literature (Hossain et al., 2010).

350 Levels of protocatechuic acid were highest in cinnamon (10.2 µg/g DW) followed by

351 oregano (9.94 µg/g DW) and rosemary (8.42 µg/g DW), with the lowest in bay and

352 thyme (2.05‒2.55 µg/g DW). Our results for protocatechuic acid are higher than those

353 reported by Papageorgiou et al., who found levels between 3.20 and 4.50 µg/g DW for

354 rosemary, 0.10 and 2 µg/g DW for oregano, and undetectable amounts in bay

355 (Papageorgiou et al., 2008). In another study investigating the phenolic content of 26

356 common spice extracts from 12 botanical families, protocatechuic acid was not detected

357 in any of the species studied in our work, instead being found in sweet basil, dill, star

358 anise and coriander (Shan et al., 2005).

359 In contrast with the aforementioned phenolic acids, levels of p-coumaric acid were

360 highest in bay (9.64 µg/g DW), followed by rosemary (5.57 µg/g DW) and oregano

361 (4.90 µg/g DW), with the lowest levels observed in cumin (0.74 µg/g DW). Coumaric

362 acid is one of the main compounds found in all herbs and spices, together with

363 chlorogenic and p-hydroxybenzoic acids (Lv et al. 2012; Miron, Plaza, Bahrim, Ibáñez,

364 & Herrero 2011; Shan et al., 2005). Lastly, ferulic acid levels were highest in bay and

365 oregano (2.12‒2.15 µg/g DW) and lowest in cinnamon (0.33 µg/g DW), in accordance

366 with other studies detecting these compounds (Baatour et al. 2012; Shan et al., 2005).

367 Shan et al. reported between 0.85 and 7.55 µg/g DW ferulic acid in rosemary, and

368 between 1.30 and 4.90 µg/g DW in oregano, with none detected in bay.
369 Catechin and epicatechin were quantified in cumin and cinnamon, being under the

370 detection limits in the other studied herbs. Catechin levels ranged from 14.1 µg/g DW to

371 16.1 µg/g DW in cumin and cinnamon, respectively. Similarly, epicatechin levels were

372 6.43 µg/g DW for cumin and 7.25 µg/g DW for cinnamon. Catechin and epicatechin

373 have been previously identified by other authors (Shan et al., 2005).

374 Quercetin has been previously detected in rosemary, oregano, sage, bay and thyme

375 (Hossain et al., 2010). In our study, quercetin levels ranged between 0.32 µg/g DW in

376 oregano and 7.50 µg/g DW in cumin, in accordance with another study that reported

377 between 0.20 and 6 µg/g DW quercetin in rosemary, 0.20 and 2.30 µg/g DW in

378 oregano, and none in bay (Papageorgiou et al., 2008).

379 A PCA was carried out to discriminate among culinary herbs and spices (Figure 4)

380 according to their phenolic profile. The two principal components (PC1 and PC2)

381 obtained for each herb or spice accounted for 79.89 % of the variability of the original

382 data. The closer the location of variable Y (= loading) to the axis origin, the lower its

383 contribution to the class distinction among herbs and spices. Thus, plant metabolites

384 such as protocatechuic and chlorogenic acid showed a low discriminating power (Figure

385 4), while large loadings for variables such as catechin, epicatechin, rosmarinic and

386 caffeic acids were highly discriminatory. It can be clearly observed that catechin,

387 epicatechin and quercetin are highly correlated with cumin and cinnamon. In contrast,

388 bay, thyme and oregano, which are situated in the middle and bottom of the plot, are

389 related to low levels of these metabolites and higher levels of p-coumaric and ferulic

390 acids. On the other hand, rosemary, which is situated in the upper-right hand side of the

391 plot, is highly correlated with rosmarinic, caffeic, syringic and p-hydroxybenzoic acids.

392
393 In summary, high-resolution mass spectrometry provided a powerful tool for the

394 identification of polyphenolic diversity in culinary herbs and spices of the families

395 Lamiaceae (rosemary, thyme and oregano), Apiaceae (cumin) and Lauraceae (cinnamon

396 and bay), even in the absence of standards. Quantification levels of phenolic compounds

397 revealed distinguishing features among these plant families. Our results show that these

398 culinary ingredients are rich in phenolic constituents and demonstrate good antioxidant

399 capacities, and the use of them in cooking and food processing may have beneficial

400 effects for human health.

401
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510
511
512 Table 1: Optimized parameters for
DP CE513 MRM conditions
Protocatechuic acid -40 -20
p-Hydroxybenzoic acid -40 -20514
Chlorogenic acid -40 -20
Catechin -50 -25515
Caffeic acid -40 -20
516
Syringic acid -40 -20
Epicatechin -50 -25517
p-Coumaric acid -40 -20
Ferulic acid -50 -20518
Rosmarinic acid -40 -20
Quercetin -50 -30519

520

521 DP: declustering potential; CE: collision energy

522
Table 2: List of compounds identified in culinary herbs and spices

RT
Compound (min) [M ‒ H]‒ MS/MS ions Acc Mass Mda MF detected in

1 Gallic acid* 1.43 169 125 (100) 169.0142 0.8 C7H6O5 R,T,O,Ci,Cu,B

2 Vanillic acid-O-hexoside 1.50 329 329 (10), 167 (100) 329.0877 0.7 C14H18O9 R,T,O,B

3 Syringic acid* 1.70 197 182 (40), 167 (40), 197.0455 0.5 C9H10O5 R,T,O,Ci,Cu,B

4 Caffeic acid-O-hexoside 1 2.10 341 179(100), 135 (10) 341.0877 0.7 C15H18O9 R,T,O,Ci,Cu,B

Neochlorogenic acid (3-O-

5 caffeoylquinic acid ) 2.13 353 191 (100), 179 (40), 135 (20) 353.0877 0.8 C16H18O9 R,T,O,Ci,Cu,B

6 Protocatechuic acid* 2.36 153 153 (40), 109 (90) 153.0193 0.4 C7H6O4 R,T,O,Ci,Cu,B

7 Caffeic acid-O-hexoside 2 2.82 341 179(100), 135 (10) 341.0877 0.9 C15H18O9 R,T,O,Ci,Cu,B

8 Homovanillic acid-O-hexoside 1 3.14 343 181 (100), 137 (10) 343.1034 0.7 C15H20O9 R,T,Ci,B

9 3-O-p-Coumaroylqunic acid 3.29 337 191 (10), 163 (100) 337.0930 1.5 C16H18O8 Cu

10 Caffeic acid-O-hexoside 3 3.30 341 179(100) 341.0877 0.7 C15H18O9 R,T,O,Ci,Cu,B

11 p-Hydroxybenzoic acid* 3.52 137 93 (100) 137.0244 0.4 C7H6O3 R,T,O,Ci,Cu,B

Chlorogenic acid (5-O-

12 caffeoylquinic acid )* 3.58 353 191 (100) 353.0877 0.9 C16H18O9 R,T,O,Ci,Cu,B
13 Catechin* 3.69 289 245 (100) 289.0718 1.4 C15H14O6 Ci,Cu

14 Coumaric acid-O-hexoside 1 3.70 325 163 (100), 119 (20) 325.0928 0.8 C15H18O8 R,T,O,Ci,B

15 m-Hydroxybenzoic acid* 3.89 137 93 (100) 137.0244 0.5 C7H6O3 R,T,O,Ci,Cu,B

Cryptochlorogenic acid (4-O-

16 caffeoylquinic acid) 3.91 353 191(50), 173 (100), 135 (20) 353.0877 0.4 C16H18O9 R,T,O,Ci,Cu,B

17 Homovanillic acid 4.07 181 137 (100) 181.0506 0.2 C9H10O4 T,O,B

18 Proanthocyanidin trimer 1 4.65 863 711 (60), 575 (100), 287 (10) 863.1829 1.5 C45H36O18 Ci,Cu

19 Caffeic acid* 4.84 179 135 (100) 179.0349 0.3 C9H8O4 R,T,O,Ci,Cu,B

20 Proanthocyanidin trimer 2 5.02 863 711 (60), 575 (100), 287 (10) 863.1829 1.3 C45H36O18 Ci,Cu

21 Epicatechin* 5.32 289 245 (100) 289.0718 1.3 C15H14O6 Ci,Cu

22 Apigenin-C-hexoside-C-hexoside 5.36 593 503 (30), 473 (100), 383 (20), 353 (40), 593.1511 0.7 C27H30O15 R,O

23 4-O-p-Coumaroylqunic acid 5.67 337 191 (20), 173 (100), 163 (30) 337.0930 0.1 C16H18O8 R,T,O,Ci,Cu,B

24 Ferulic acid-O-hexoside 5.78 355 193 (100) 355.1034 0.9 C16H20O9 R,T,Ci

25 Coumaric acid-O-hexoside 6.03 325 163 (100), 119 (20) 325.0928 1.2 C15H18O8 B

26 Sinapic acid-C-hexoside 6.95 385 325 (50), 295 (100), 265 (70), 223 (25) 385.1139 0.6 C17H22O10 R,T

27 Vanillic acid 7.03 167 167 (50), 152 (20), 108 (50) 167.0350 0.4 C8H8O4 T,B
28 Proanthocyanidin trimer 3 7.48 863 711 (60), 575 (100), 287 (10) 863.1829 1.8 C45H36O18 Ci,Cu

29 Kaempferol-O-dihexoside 7.70 609 447 (60), 285 (100) 609.1460 1.2 C27H30O16 O

30 Coumaric acid* 7.90 163 119 (100) 163.0400 0.3 C9H8O3 R,T,O,Ci,Cu,B

31 Rosmarinic acid-O-hexoside 8.11 521 359 (100) 521.1300 0.2 C24H26O13 R,O

32 Ferulic acid* 8.22 193 193 (5), 178 (40), 149 (10), 134 (80) 193.0506 0.3 C10H10O4 R,T,O,Ci,Cu,B

1006 (20), 863 (100), 755 (50), 575 (40), 1727.3730

33 Proanthocyanidin hexamer 8.30 863 287 (10) [M-2H]2- 2.6 C90H72O36 Ci,Cu

34 Rutin* 8.68 609 301 (100) 609.1460 1.4 C27H30O16 R,T,Ci,Cu,B

35 Kaempferol-3-O-rutinoside* 8.79 593 285 (100) 593.1511 0.5 C27H30O15 R,T,O,Cu,B

36 Quercetin-3-O-glucoside* 9.08 463 301 (100) 463.0881 1.3 C21H20O12 R,T,O,Ci,Cu

37 Kaempferol-3-O-glucoside* 9.15 447 285 (100) 447.0932 0.5 C21H20O11 R,T,O,Ci,Cu,B

38 Dicaffeoylquinic acid 1 10.03 515 353 (100), 173 (10), 179 (8) 515.1194 0.5 C25H24O12 R,T,O,Ci,Cu

39 Naringenin-C-hexoside* 10.81 433 373(50), 343(50), 303 (20) 433.1140 0.4 C21H22O10 R,T,Ci

40 Hesperidin* 10.91 609 301 (100) 609.1825 0.9 C28H34015 R,T,O

41 Apigenin-7-O-glucoside* 10.93 431 269 (100) 431.0983 1.1 C21H20O10 R,T,O

42 Rosmarinic acid* 12.05 359 197 (30), 161 (100) 359.0772 1.1 C18H16O8 R,T,O,Ci,Cu,B
43 Naringenin-O-hexuronide 13.99 447 271 (100),175 (10) 447.0932 1.2 C21H20O11 Cu

44 Kaempferol* 15.24 285 285 (40), 151 (100) 285.0405 0.9 C15H10O6 R,T,O,Ci,Cu,B

45 Quercetin* 15.33 301 301 (10), 151 (100) 301.0353 0.5 C15H10O7 R,T,O,Ci,Cu,B

46 Naringenin* 17.40 271 271 (15), 151 (100) 271.0611 1.1 C15H12O5 B

47 Apigenin* 17.55 269 269 (10), 151 (100) 269.0455 0.6 C15H10O5 R,T,O,Ci

48 Hesperetin* 17.91 301 286 (30), 151 (100) 301.0718 1.2 C16H14O6 R,T,O,Cu,B

49 Rosmanol 19.00 345 301 (100) 345.1707 0.8 C20H26O5 R,O

50 Carnosol 21.90 329 329 (10), 285 (100) 329.1758 0.6 C20H26O4 R,O

51 Carnosic acid 23.09 331 331 (70), 287 (100) 331.1915 0.8 C20H28O4 R,T,O,Ci

R: Rosemary; T:Thyme; O: Oregano; Cu: Cumin; Ci: cinnamon; B: bay

*Comparison with standard

Table 3. Total polyphenols (mg GAE/g DW) and antioxidant capacity of culinary herbs and spices through ABTS+ and DPPH assays (mmol TE/g DW)

expressed as mean ± SD. Different letters in the columns represent statistically significant differences (p < 0.05).

Herbs/
TP ABTS+ DPPH
Spices
Rosemary 5.02 ± 0.43a 2.39 ± 0.17a 1.98 ± 0.17a

Thyme 3.36 ± 0.14b 1.38 ± 0.13b 1.15 ± 0.06b

Oregano 2.23 ± 0.18c 1.34 ± 0.13b 0.78 ± 0.07c

Cumin 4.98 ± 0.31a 3.26 ± 0.29c 2.16 ± 0.06d

Cinnamon 5.82 ± 0.44d 4.13 ± 0.43d 1.88 ± 0.10e

Bay 1.12 ± 0.08e 0.72 ± 0.07e 0.30 ± 0.02f

GAE: gallic acid equivalents; TE: Trolox equivalents; SD: standard deviation.
Table 4. Performance parameters of the HPLC-MS/MS methodology

LODa Recoveries Repeatibility

(µg/g DW) (%) (RSD%)b
Protocatechuic acid 2.4·× 10-4 95.30 ± 2.22 5.69
-4
p-Hydroxybenzoic acid 6.8·× 10 90.12 ± 1.56 4.21
Chlorogenic acid 1.7·× 10-4 91.23 ± 2.44 7.66
Catechin 1.2·× 10-3 89.20 ± 1.98 6.37
-3
Caffeic acid 2.6·× 10 98.50 ± 1.33 4.43
Syringic acid 2.7·× 10-3 92.43± 1.42 5.15
-3
Epicatechin 1.7·× 10 89.44 ± 2.09 7.76
p-Coumaric acid 1.3·× 10-3 94.56 ± 2.33 4.94
-4
Ferulic acid 2.8·× 10 94.20 ± 2.59 4.85
Rosmarinic acid 2.0·× 10-3 111.30 ± 3.22 5.33
-3
Quercetin 8.9·× 10 85.81 ± 3.03 3.29

a
LOD: limit of detection
b
RSD: relative standard deviation
 Table 5: Quantification of individual polyphenols (mean ± SD) of culinary herbs expressed as µg/g DW. Different letters in the columns represent statistically significant

differences (p < 0.05).

Chlorogenic p-Coumaric p-Hydroxybenzoic Protocatechuic Rosmarinic

Caffeic acid Catechin Epicatechin Ferulic acid Syringic acid Quercetin
acid acid acid acid acid
Rosemary 12.58 ± 0.44a <LOD 1.07 ± 0.07a <LOD 1.99 ± 0.08a 5.57 ± 0.30a 15.16 ± 1.00a 8.42 ± 0.30a 156.90 ± 3.20a 3.46 ± 0.12a 1.36 ± 0.04a

Thyme 6.56 ± 0.41b <LOD 0.70 ± 0.08b <LOD 1.03 ± 0.04b 2.74 ± 0.21b 4.38 ± 0.34b 2.55 ± 0.37b 84.04 ± 2.75b 0.92 ± 0.02b 0.79 ± 0.03b

Oregano 10.60 ± 0.46c <LOD 0.71 ± 0.10b <LOD 2.15 ± 0.11c 4.90 ± 0.23c 2.49 ± 0.10c 9.94 ± 0.55c 52.02 ± 1.74c 1.26 ± 0.05c 0.32 ± 0.01c

Cinnamon 0.45 ± 0.02d 16.14 ± 1.38a 0.12 ± 0.01c 7.25 ± 0.64a 0.33 ± 0.02d 2.24 ± 0.09d 1.19 ± 0.04d 10.16 ± 0.53d 0.73 ± 0.09d 0.77 ± 0.05d 7.45 ± 0.22d

Cumin 3.06 ± 0.16e 14.08 ± 0.92b 4.18 ± 0.27d 6.43 ± 0.45b <LOQ 0.74 ± 0.02e 1.61 ± 0.06e 3.44 ± 0.15e 3.29 ± 0.12e 0.47 ± 0.02e 7.50 ± 0.32d

Bay 0.44 ± 0.01d <LOD 0.13 ± 0.01c <LOD 2.12 ± 0.16c 9.64 ± 0.46f 1.14 ± 0.03d 2.05 ± 0.10f 0.39 ± 0.01f 0.40 ± 0.02e <LOQ
 1 FIGURE CAPTIONS

2 Figure 1. Mass spectra of a Proanthocyanidin trimer (m/z 863)

3 Figure 2a: Mass spectra of sinapic acid-C-hexoside (m/z 385); figure 2b: mass spectra

4 of dicaffeoylquinic acid (m/z 515); figure 2c: mass specrtra of apigenin-C-hexoside-C-

5 hexoside (m/z 593)

6 Figure 3. HPLC chromatogram of Bay. 1. Protocatechuic acid; 2. p-Hydroxybenzoic

7 acid; 3. Chlorogenic acid; 4. Catechin; 5. Caffeic acid; 6. Syringic acid; 7. Epicatechin;

8 8. p-Coumaric acid; 9. Ferulic acid; 10. Rosmarinic acid; 11. Quercetin.

9 Figure 4. Biplot of samples representing the polyphenol profile of culinary herbs.

10
Figure 1
Figure 2a
Figure 2b
Figure 2c
Figure 3
Figure 4
523 LTQ-Orbitrap-MS was applied for the identification of polyphenols of herbs

524 52 phenolic compounds were identified in culinary herbs and spices

525 Multivariate analysis allowed the assessment of distinctive features among herbs

526 Some polyphenols were reported for the first time in culinary herbs and spices

527

528