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www.rsc.org/methods Page 1 of 23 Analytical Methods

1 2 3 1 Analysis of volatile compounds in spp. by headspace solidphase 4 5 6 2 microextraction and GC×GCTOFMS 7 8 3 Stanislau Bogusz Junior a, d *, Paulo Henrique Março b, Patrícia Valderrama b, Flaviana 9 10 c c c 11 4 Cardoso Damasceno , Maria Silvana Aranda , Cláudia Alcaraz Zini , Arlete Marchi

12 d e 13 5 Tavares Melo , Helena Teixeira Godoy 14 15 6 16 17 18 7 a Federal University of the Jequitinhonha and Mucuri (UFVJM), Institute of Science and 19 20 8 Technology, Diamantina, MG, . 21 Manuscript 22 23 9 b Technological Federal University of Paraná (UTFPR), Campo Mourão, PR, Brazil. 24 25 c 26 10 Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 27 28 d Horticultural Center, Campinas Agronomic Institute (IAC), Campinas, SP. 29 11 30 e 31 12 University of Campinas (UNICAMP), School of Food Engineering, Campinas, SP, Accepted 32 33 13 Brazil. 34 35 36 14 * Corresponding author : +55 (38) 35321200; [email protected] 37 38 39 15

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 1

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1 2 3 16 Abstract 4 5 6 17 A suitable method based on headspace solidphase microextraction (HSSPME), 7 8 18 comprehensive twodimensional gas chromatography with timeofflight mass 9 10 19 spectrometry detector (GC × GCTOFMS) and chemometric approach was used aiming 11 12 20 the investigation of the volatile fraction of Brazilian Capsicum peppers: malagueta ( C. 13 14 21 frutescens), dedodemoça (C. baccatum ) and murupi ( C. chinense). A total of 184 15 16 17 22 volatiles compounds were identified in the three pepper samples and 123 of these 18 19 23 compounds were first described in Brazilian peppers. In addition, during Brazilian chili 20 21 24 peppers maturation, as maturation time increases, it was noted that the majority of Manuscript 22 23 25 volatile compounds responsible for green odor notes disappeared being replaced to 24 25 26 26 others whit fruity notes. The chemometric analysis (PCA) was able to separate samples 27 28 27 according to their constituents, were malagueta was characterized by branched , 29 30 28 murupi by terpenes and dedodemoça by the presence of aldehydes and terpenes.

31 Accepted 32 33 29 Keywords: Comprehensive twodimensional gas chromatography, 34 35 30 L. , C. chinense Jacq. , C. baccatum (Willd) Eshbaugh, volatile compounds, principal 36 37 31 component analysis. 38 39

40 32 Methods 41 42 43 33 1. Introduction 44 45 34 Peppers from the genus Capsicum are very popular in various parts of the 46 47 1, 2 48 35 world, mainly due to their attributes of color, and aroma. They can be 49 50 36 consumed as fresh, dried, preserved or in pepper sauces. Industrially, they are used as 51 Analytical 52 37 coloring and flavor agents in a variety of types of food. 3 As a consequence of the 53 54 38 biochemical processes that occur during maturation, differences in the flavor of 55 56 39 Capsicum can be perceived as a result of the degree of maturation. 4 57 58 59 60 2

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1 2 3 40 Although pungency is one of the most important attributes of Capsicum fruits, previous 4 5 41 studies analyzed the pepper volatile fraction, since the chemical compounds present in 6 7 42 this fraction directly affect the flavor. 5, 6 Moreover, the perception of the complex 8 9 10 43 volatile mixture of the compounds is an important part of the consumers selection 11 12 12 44 criteria for the acceptance of food. Furthermore, the knowledge of volatile 13 14 45 composition is an important tool for differentiating between the Capsicum types and 15 16 46 also to establish measures for authenticity, quality control, guaranty of authenticity, 17 18 21 47 fraud prevention and assurance of origin. In addition, the food industry has an interest 19 20 21 48 in obtaining concentrated aroma of peppers for flavored products without necessarily Manuscript 22 7 23 49 giving pungency to food. 24 25 26 50 In the studies of peppers volatile fractions different extraction methods have 27 28 51 been used, such as simultaneous steam distillationsolvent extraction (SDE), purge and 29 30 52 trap and solid phase microextraction (SPME), but independent of the extraction

31 Accepted 32 53 method, gas chromatography (GC) coupled to mass spectrometry (MS) was the 33 34 standard instrumental methodology used to investigate the volatile fraction. 7, 8, 9 35 54 36 37 55 However, even the most modern GC system is not able to separate all the compounds in 38 39 56 the volatile fraction of complex samples as peppers. Then, it is common to have non

40 Methods 41 57 identified peaks on the chromatograms, those peaks commonly are constituted by two or 42 43 58 three coeluted compounds. An alternative to the analysis of complex samples is the 44 45 46 59 comprehensive twodimensional gas chromatography (GC × GC), a powerful separation 47 48 60 technique, whose resolving power is higher than that of conventional chromatography 49 50 61 because the analytes in GC × GC are separated with the use of two sequential 51 Analytical 52 62 chromatographic columns with different separation characteristics. 22, 23 The entire 53 54 55 63 effluent from the first column is reinjected in the second by the modulator which, by 56 57 58 59 60 3

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1 2 3 64 focusing effect, decreases the peak width and increases the height of each peak, which 4 5 65 increases analytical sensitivity. 10 6 7 8 66 Following the former investigations on the chemistry of the volatiles in 9 10 67 Capsicum 11, 12 , this study aimed the development of a GC × GC methodology suitable 11 12 68 for the characterization of the volatile fraction of malagueta ( C. frutescens ), dedode 13 14 69 moça ( C. bacccatum ) and murupi ( C. chinense ) Brazilian pepper varieties. Static 15 16 17 70 headspace solidphase microextraction (HSSPME) and GC × GCTOFMS were used to 18 19 71 identify the characteristic compounds of each variety and assess the volatile compounds 20 21 72 that could differentiate these samples. In order to extract the maximum information Manuscript 22 23 73 from data, Principal Component Analysis was employed to search for relationship 24 25 26 74 among samples and variables. 27 28 75 29 30

31 76 2. Material and methods Accepted 32 33 34 77 2.1 Materials 35 36 78 The SPME fiber used was a 50/30 µm 37 38 39 79 divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) from Supelco

40 Methods 41 80 (Bellefonte, PA, USA). The gas chromatography columns tested in this study are: DB5 42 43 81 – (5%phenyl)methylpolysiloxane, DB1 – 100% dimethylpolysiloxane, DB17 – (50% 44 45 82 phenyl)methylpolysiloxane, DBWAX – polyethylene glycol purchased from Agilent 46 47 48 83 Technologies (Wilmington, DE, USA) and RTLC50 (dimethyl [50% liquid crystal] 49 50 84 polysiloxane) purchased from Restek Corporation, (Bellefonte, PA, USA). 51 Analytical 52 53 85 2.2 Samples 54 55 86 Approximately 2 kg of samples, namely, malagueta, dedodemoça, and murupi 56 57 87 pepper were botanically identified and supplied by the Campinas Agronomic Institute 58 59 60 4

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1 2 3 88 (IAC, Campinas, SP, Brazil). The genotypes selected for the study were obtained from 4 5 89 the germplasm bank of the Horticultural Center of the IAC. The plants were cultivated 6 7 90 in 2009 from January to May, using similar fertilizer and irrigation treatments. They 8 9 10 91 were harvested at two maturity stages: the physiological stage (maximum size 11 12 92 development, but still immature) and the commercial stage (complete development of 13 14 93 both size and color). The peppers were harvested in the morning and immediately 15 16 94 transported to the laboratory in order to be analyzed. The period between harvest and 17 18 95 analysis was no longer than 48 hours. 19 20 21 96 2.3 Samples Preparation Manuscript 22 23 24 97 The volatiles were extracted by HSSPME according to previously published 25 26 98 procedure. 11, 12 Whole pepper fruits were grounded in a blender in 100 g batches, and 27 28 99 1.00 g aliquots of the ground material were weighed into 15.0 mL SPME flasks with 29 30 100 screw tops and PTFE/silicon septa (Supelco Bellefonte, PA, USA). The fiber was

31 Accepted 32 33 101 prepared before use according to the manufacturer instructions. The volatiles extraction 34 35 102 conditions were: equilibration for 15 min followed by extraction during 80 minutes at 36 37 103 40 ºC. After extraction, the fiber was placed in the gas chromatograph injector and the 38 39 104 analytes were desorbed in the split mode (1:20) at 250 ºC for 1.0 minute. After each

40 Methods 41 42 105 extraction and desorption procedure, the fiber was reconditioned during 15 minutes at 43 44 106 250 ºC, this additional procedure was employed to eliminate analyte carry over between 45 46 107 extractions. All of the samples were analyzed in triplicate and the results are mean 47 48 108 values. 49 50 Analytical 51 109 2.4 GC × GC analysis 52 53 54 110 The twodimensional chromatographic analyses were carried out in the 55 56 111 Comprehensive Twodimensional Gas Chromatography MultiUser Nucleus of the 57 58 112 Institute of Chemistry of the Federal University of Rio Grande do Sul (UFRGS), Brazil. 59 60 5

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1 2 3 113 In the beginning, a comprehensive twodimensional gas chromatography system 4 5 114 coupled to a flameionization detector (GC × GCFID) was used to analyze the pepper 6 7 115 volatiles in order to find out the best chromatographic and injection conditions. The 8 9 10 116 chromatograph used was the GC × GCFID HP6890 (Agilent Technology, USA), 11 12 117 equipped with a LECO cryogenic modulator (LECO Corporation, St. Joseph, MI, 13 14 118 USA). The capillary column sets used to optimize the chromatographic separation are 15 16 119 listed in Table 1. 17 18 19 120 The GC × GCFID operational parameters were: injector at 250 ºC; temperature 20 21 121 programming for the primary and secondary ovens: 40 ºC (0.2 min), with increments of Manuscript 22 23 1 122 3 ºC min to 230 ºC, remaining at this temperature for 4 min; carrier gas (hydrogen) at a 24 25 1 26 123 constant flow rate of 1.0 mL min and detector at 250 ºC. In order to optimize the 27 28 124 chromatographic conditions it was tested: modulation periods of 4, 5, 6 and 8 seconds, 29 30 125 and split ratios of 1:10; 1:20; 1:30 and 1:50.

31 Accepted 32 33 126 The operational parameters used in the GC × GCTOFMS were set as being 34 35 127 injector temperature of 250 ºC and split ratio of 1:20; column set DB5 (30 m x 0.25 36 37 128 mm i.d. x 0.25 µm stationary phase) and DBWAX (2.60 m x 0.1 mm i.d. x 0.1 µm 38 39 129 stationary phase); temperature gradient in the primary oven of 40 ºC (0.2 min), with

40 Methods 41 1 42 130 increments of 3 ºC min to 230 ºC, remaining at this temperature during 4 min; 43 1 44 131 secondary oven 45 ºC (0.2 min), with increments of 3 ºC min to 235 ºC, remaining at 45 46 132 this temperature during 4 min; carrier gas (helium) at a constant flow rate of 1.3 mL 47 48 133 min 1; modulation period of 6 s; interface at 240 ºC; electron ionization source at 200 ºC 49 50 51 134 electronic ionization at +70 eV; detector at 100 Hz, monitoring the mass range from 40 Analytical 52 53 135 400 m/z and a multichannel plate voltage of 1.7 kV. The identification criteria 54 55 136 considered for the volatile compounds present in the Capsicum samples were minimum 56 57 137 similarity search of 80% and LTPRI filtering (± 5 units). 13 58 59 60 6

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1 2 3 138 4 5 6 139 2.5 Statistical analysis 7 8 140 The Principal Components Analysis was carried out by using the Statistica v. 7 9 10 11 141 software (Statsoft Inc., Tulsa, OK, USA). 12 13 142 14 15 16 143 3. Results and discussion 17 18 19 144 3.1 Analysis of the Brazilian Capsicum pepper volatiles by GC × GCTOFMS 20 21 145 After establishing the operational parameters for the GC × GCFID system, the Manuscript 22 23 24 146 analysis was carried out in the GC × GCTOFMS. Figure 1 demonstrates the GC × GC 25 26 147 TOFMS chromatogram for the immature malagueta pepper sample, highlighting the 27 28 148 main chemical classes found in the sample. These classes were found grouped or 29 30 149 structured in specific regions, since chromatographic structure is a result of the

31 Accepted 32 150 organization between compounds belonging to the same chemical class or group found 33 34 35 151 in the same chromatographic separation space. The importance of such structuring is the 36 37 152 possibility of applying the same chromatographic method to other similar samples, 38 39 153 where compounds belonging to a specific chemical class trends to keep its positions in

40 Methods 41 154 the same place at the structure. 10, 13, 14, 15 42 43 44 155 Table 2 illustrates the results obtained in the identification of the volatile 45 46 156 compounds from malagueta, murupi and dedodemoça peppers in ripe and unripe 47 48 49 157 maturation states by HSSPME and GC × GCTOFMS. Considering all the samples 50 51 158 analyzed, an amount of 184 volatiles were identified and grouped according to the Analytical 52 53 159 following chemical classes: alkanes (26), alcohols (20), aldehydes (17), ketones (8), 54 55 160 esters (68), ethers (3), terpenes (40) pyrazine (1) and sulfur compound (1). 56 57 58 59 60 7

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1 2 3 161 A large number of esters and terpenes were detected in the chili peppers, which 4 5 162 was expected since it has been previously reported in literature. 17, 18 Methyl and ethyl 6 7 163 esters provide strong fruity notes in foods whereas terpenes provide wood, floral, fruity 8 9 12, 17, 18 10 164 and spices notes. Aldehydes are also especially important because of its low odor 11 12 165 threshold described in sniffing analyzes of Capsicum as green, cucumber, pungent or 13 14 166 herbaceous odor notes. 17, 18 On the other hand, alcohols have a higher odor threshold 15 16 167 when compared to aldehydes and so their importance for the food aroma is relatively 17 18 12 168 less to aldehydes. Shortchain ketones, especially methyl ketones have powerful 19 20 21 169 aroma, while pyrazines are considered strong odor compounds in Capsicum (odor notes Manuscript 22 17 23 170 of and green). Finally, the presence of aliphatic, aromatic and branched 24 25 171 alkanes in Capsicum peppers has been reported in the literature related to 26 27 172 biosynthesis and degradation processes of carotenoids. 18 28 29 30 173 For all peppers investigated in this research, the ripening process decreases the

31 Accepted 32 174 amount of aldehydes, which may explain the more pleasant and attractive aroma that 33 34 exhibited the ripped Capsicum peppers. Additionally, the biosynthesis of Capsicum 35 175 36 37 176 esters is described in literature as being directly related to the quantity and availability 38 39 177 of the alcohols present in the sample. 18 In the maturation process of Brazilian peppers,

40 Methods 41 178 the number of alcohols decreased in all of the samples and, in some cases, this reduction 42 43 179 occurred by giving place to the appearance of new esters like heptyl hexanoate, (E)3 44 45 46 180 hexenyl butyrate, 3methyl butanoate, (Z)3hexenyl isopentanoate, methyl 47 48 181 methacrylate and , presents only in ripe peppers. Moreover, in this 49 50 182 study pyrazines were found in all of the pepper samples and in all maturation stages. 51 Analytical 52 53 183 The different analyzed peppers displayed characteristic compounds, identified 54 55 184 by upper case letters in Table 2. For malagueta pepper, 104 compounds were identified 56 57 185 in which 43 were only detected in this sample. These characteristic compounds consist 58 59 60 8

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1 2 3 186 mainly of esters (total of 18) and alcohols (9). Whereas a total of 103 compounds were 4 5 187 identified in murupi peppers, in which 40 were only detected in this sample. The 6 7 188 characteristic compounds were referred as being mainly constituted of terpenes (14) and 8 9 10 189 esters (13). Furthermore, in dedodemoça peppers it was detected and identified 68 11 12 190 compounds, in which 28 were exclusive to this sample. The characteristic compounds 13 14 191 consisted mainly of terpenes (9) and aldehydes (9). In this way, it was identified 123 15 16 192 compounds, described for the first time in Brazilian peppers. 17 18 19 193 By comparing these GC × GC results with those obtained by using one 20 21 194 dimensional gas chromatography (GCMS) for Capsicum peppers from Brazil, in the Manuscript 22 23 195 first case, a greater number of volatiles were identified probably due to the high 24 25 26 196 resolving power of GC × GC. As an example, when were used onedimensional 27 28 197 chromatography only 83 volatiles were identified in malagueta, 77 in murupi and 49 in 29 30 198 dedodemoça pepper. 13 To better understand, the similarities among the different

31 Accepted 32 199 pepper species, the standard recognizing method of Principal Component Analysis was 33 34 employed. 35 200 36 37 201 38 39

40 202 3.2 Principal components analysis Methods 41 42 43 203 Principal Component Analysis is a method mainly used to describe samples 44 45 204 present in an ndimensional space order for pattern recognition and is able to extract the 46 47 205 relevant information from a given data set of a multivariate nature to aid in 48 49 206 understanding the model. PCA has been reported in the literature to describe various 50 Analytical 51 24 52 207 problems involving food and agricultural matrices. This is an unsupervised 53 54 208 exploratory technique which reduces the dimensions of an initial multivariate dataset to 55 56 209 a smaller number of uncorrelated variables with maximized variance, i.e., that permits 57 58 210 the analysis of a dataset using the most important variables. Multivariate data analysis 59 60 9

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1 2 3 211 methods, either supervised or unsupervised, used to reduce the dimensionality of 4 5 212 multivariate dataset and provide aid to identify differences or similarities among the 6 7 213 samples. PCA is a primary tool among the various multivariate data analysis methods. It 8 9 10 214 is an unsupervised method and samples are clustered or separated due to similarities 11 25 12 215 among profiles. 13 14 216 In this research, thirty volatile compounds which were common to more than 15 16 17 217 one pepper species and provides important odors notes to the peppers were evaluated by 18 19 218 Principal Components Analysis. To this, peak area was used and the PCA method was 20 21 219 made with mean center preprocess. The objective was determining the similarities and Manuscript 22 23 220 differences of these volatiles in those peppers. Figures 2 and 3 shows the sample scores 24 25 26 221 and loadings plots, respectively, regarding on the two first principal components which 27 28 222 captured 69.7% of the total variance explained. 29 30 223 The malagueta pepper samples were separated by the negative part of the Factor

31 Accepted 32 33 224 2 in the scores plot. In accordance with the loadings plot, malagueta pepper were mainly 34 35 225 characterized by compounds arising from the degradation of amino acids, such as the 36 37 226 branched esters methyl hexanoate, isoamyl isoisobutyrate and hexyl isovalerate (fruit, 38 39 7, 17 227 aromas) and by products formed from the degradation of fatty acids such as the

40 Methods 41 17, 18 42 228 aliphatic hexyl butyrate and hexyl hexanoate (with fruity notes). Compounds 43 44 229 as alcohols, aldehydes, ketones and furan were also responsible for the separation of 45 46 230 these samples e.g. 2methylbutanal, 3methylbutanal (green, almond, burnt, malty 47 48 231 notes), 1penten3one (pungent) and 2pentylfuran (buttery, green beanlike). 49 50 Analytical 51 232 The scores plot shows that murupi pepper was separated trough the positive part 52 53 233 of Factors 1 and 2 and it was characterized, according to Figure 3, mainly by products 54 55 234 from the terpenoid pathway, such sesquiterpenes copaene (woody) and δcadinene 56 57 19 58 235 (green, sweet) , and also by products from amino acid degradation such as the 59 60 10

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1 2 3 236 aromatic ester methyl salicylate (sweet) and the methyl esters as isopentyl isovalerate 4 5 237 (with fruity aromas) 17 . 6 7 8 238 On the other hand, the dedodemoça pepper was located at the positive part of 9 10 239 Factor 2 and negative part of Factor 1 in the scores plot. The loadings plot shows that 11 12 240 dedodemoça pepper was characterized by the presence of products derived from fatty 13 14 241 acids, such as the aldehydes (E)2hexanal (applelike, fruity, green), hexanal (grass, 15 16 17 242 tallow, fat) and the aliphatic ester (fruity) and compounds derived from 18 19 243 amino acid degradation such as 2isobutyl3methoxypyrazine (green peppers). 17, 19, 20 20 21 Manuscript 22 244 23 24 245 4. CONCLUSIONS 25 26 27 246 The methodology developed for comprehensive twodimensional gas 28 29 247 chromatography and PCA was successfully applied to the analysis of the volatile 30

31 Accepted 32 248 fractions from malagueta, murupi and dedodemoça peppers in two stages of 33 34 249 maturation. An amount of 184 volatile compounds was identified, being 123 of it 35 36 250 described for the first time in Brazilian peppers. The Principal Components Analysis 37 38 251 indicated that malagueta pepper is mainly described by compounds from branched ester 39

40 Methods 252 group, while murupi peppers are considered sesquiterpenes abundant and the dedode 41 42 43 253 moça peppers characterized mainly by the presence of aldehydes and methoxypyrazine. 44 45 254 46 47 48 255 5. REFERENCES 49 50 51 256 1. J. S. Pruthi. Adv Food Res Suppl ., 1980, 1449. Analytical 52 53 257 2. E. T. Sousa, F.M. Rodrigues, C.C. Martins, F.S. Oliveira, P.A.P. Pereira, J.B. 54 55 258 Andrade. Microchem. J. , 2006, 82,142–149. 56 57 58 59 60 11

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1 2 3 259 3. J. Pino, M. González, L. Ceballos, A.R. CenturiónYah, J.TrujilloAguirred, L. 4 5 260 LatournerieMoreno, E. SauriDuch. Food Chem ., 2007, 104, 16821686. 6 7 261 4. M. M. Mazida, M.M. Salleh, H. Osman. J. Food Comp. Anal. , 2005, 18, 427 437. 8 9 10 262 5. J. Pino, E. SauriDuch, R. Marbot. Food Chem. , 2006, 94, 394–398. 11 12 263 6. S. van Ruth, E. Boscaini, D. Mayr, J. Pugh, M. Posthumus. Int. J. Mass Spectrom., 13 14 264 2003, 223–224, 55–65. 15 16 265 7. J. Pino, V. Fuentes, O. Barrios. Food Chem. , 2011, 125, 860–864. 17 18 266 8. C.M. Wu, S.E. Liou. J. Agric. Food Chem. , 1986, 34, 770772. 19 20 21 267 9. D.R. Cremer, K. Eichner. J. Agric. Food Chem. , 2000, 48, 24542460. Manuscript 22 23 268 10. M.P. Pedroso, E.C. Ferreira, L.W. Hantao, S. Bogusz Jr., F. Augusto. J. Sep. 24 25 269 Sci., 2011, 34, 1547–1554. 26 27 270 11. S. Bogusz Junior, A.M.T. Melo, C.A. Zini, H.T. Godoy. J. Chromatogr. A. , 2011, 28 29 30 271 1218, 33453350.

31 Accepted 32 272 12. S. Bogusz Junior, A. M. Tavares, J. Teixeira Filho, C. A. Zini, H. T. Godoy. Food 33 34 273 Res. Int. , 2012, 48, 98–107. 35 36 274 13. C. von Mühlen, C.A. Zini, E.B. Caramão, P.J. Marriott. Quím. Nova , 2007, 30, 682 37 38 275 687. 39

40 Methods 41 276 14. J. Beens, J. Blomberg, P. J. Schoenmakers. J. High Resol. Chromatogr. , 2000, 23, 42 43 277 182–188. 44 45 278 15. Z. L. Cardeal, M.D.R. Gomes da Silva, P.J. Marriott. Rapid Commun. Mass 46 47 279 Spectrom. , 2006, 20, 2823–2836. 48 49 50 280 16. C. von Mühlen, C.A. Zini, E.B. Caramão, P.J. Marriott. J.Chromatogr.A. , 2008, 51 Analytical 52 281 1200, 3442. 53 54 282 17. H. Kollmannsberger, A. RodrıguezBurruezo, S. Nitza, F. Nuez. F. J. Sci. Food 55 56 283 Agric. , 2011, 91, 1598–1611. 57 58 59 60 12

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1 2 3 284 18. A. RodríguezBurruezo, H. Kollmannsberger, M.C. GonzálezMas, S. Nitz, F. 4 5 285 Nuez. J. Agric. Food Chem. , 2010, 58, 4388–4400. 6 7 286 19. W. Schwab, R. Roscher. Recent Res. Dev. Phytochem. , 1997, 1, 643–673. 8 9 10 287 20. W. Schwab, R. DavidovichRikanati, E. Lewinsohn. The Plant J. , 2008, 54, 712– 11 12 288 732. 13 14 289 21. J. Pino, V. Fuentes, O. Barrios. Food Chem ., 2011, 125, 860–864. 15 16 290 22. Z. Lui, J. B. Phillips. J. Chromatogr. Sci. , 1991, 29, 227–231. 17 18 291 23. J. B. Phillips, J. Beens, J. Chromatogr., A 1999, 856,331–347. 19 20 21 292 24. R. H. S. Andrade, L. S. Nascimento, G. E. Pereira, F. Hallwass, A. P. S. Paim. Manuscript 22 23 293 Microchem. J,. 2013, 110, 256262. 24 25 294 25. S. Wold. Chemom. Intell. Lab. Syst. , 1987, 2, 3752. 26 27 295 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 13

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1 2 3 296 Figure captions 4 5 6 297 Figure 1 : Chromatogram of the volatile compounds from immature malagueta peppers 7 8 298 obtained by HSSPME and GC × GCTOFMS, with the main chemical classes 9 10 11 299 respective structuring. 12 13 300 Figure 2: Scores plot regarding on the first two principal components obtained from 14 15 301 PCA of the volatile compounds of malagueta, murupi and dedodemoça peppers. 16 17 18 302 Figure 3: Loadings plot regarding on the first two principal components obtained from 19 20 303 PCA of the volatile compounds of malagueta, murupi and dedodemoça peppers. 21 Manuscript 22 23 304 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 14

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1 2 3 305 Table 1: Column sets used in the GC x GCFID system. 4 5 Set Phase 1D – dimension Phase 2D – dimension 6 7 1 DB5 30m x 0.25mm i.d. x 0.25µm DBWAX 2.60m x 0.1mm i.d. x 0.1µm 8 9 2 DB5 30m x 0.25mm i.d. x 0.25µm DB17 2.60m x 0.1mm i.d. x 0.1µm 10 11 3 DB5 30m x 0.25mm i.d. x 0.25µm RTLC50 2.60m x 0.1mm i.d. x 0.1µm 12 13 4 DBWAX 30m x 0.25mm i.d. x 0.25µm DB17 2.60m x 0.1mm i.d. x 0.1µm 14 15 5 DBWAX 30m x 0.25mm i.d. x 0.25µm DB1 1.1m x 0.15mm i.d. x 0.1µm 16 17 18 306 DB5 – (5%phenyl)methylpolysiloxane 19 307 DB1 – 100% dimethylpolysiloxane 20 308 DB17 – (50%phenyl)methylpolysiloxane 21 309 DBWAX – polyethylene glycol Manuscript 22 310 RTLC50 (dimethyl [50% liquid crystal] polysiloxane) 23 311 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 15

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1 2 3 312 Table 2: Volatile compounds from Capsicum peppers in two ripening stages and obtained by 4 5 313 HSSPME and GC × GCTOFMS. Analytical conditions in the text. A: unripe malagueta pepper 6 7 314 (C. frutescens ); B: ripe malagueta pepper ( C. frutescens ); C: unripe murupi pepper (C. chinense) ; 8 9 10 315 D: ripe murupi pepper (C. chinense) ; E: unripe dedodemoça pepper ( C. baccatum var. 11 12 316 pendulum ); F: ripe dedodemoça pepper ( C. baccatum var. pendulum ). 13 14 317 15 Name SI LTPRI cal. LTPRI lit. A B C D E F 16 Alkanes 17 heptane c 909 701 700 1 x 18 19 (E)2methyl3octene b 906 851 x x

20 ethylbenzene a 926 863 864 1 x

21 b 4ethyl2,3dimethylhex2ene 801 995 x Manuscript 22 2,7dimethyl1,7octadiene a 807 1021 x 23 3hexyl1,1dimethylcyclopentane b 880 1247 x x 24 25 2methyldodecane 870 1265 1266 1 x x x

26 tridecane 905 1301 1300 1 x x x x x 27 (E)3methyldec4ene b 813 1345 x x 28 3,7dimethylocta1,6diene b 833 1354 x x 29 30 2methyl1tridecene a 885 1354 x

b 31 (E)6tetradecene 900 1354 x x Accepted

32 2methyltridecane 923 1365 1365 0 x x x x x x 33 3methyltridecane 876 1373 1371 2 x x x x 34 (E)5tetradecene a 923 1386 1387 1 x x 35 36 (E)4tetradecene 904 1393 x x

37 tetradecane 952 1401 1400 1 x x x x x x 38 2methyl1tetradecene 906 1447 1445 2 x x x x x x 39 (E)5tridecene 892 1483 x x x

40 Methods 41 pentadecane 928 1501 1500 1 x x x x x x 42 2methylpentadecane 923 1565 1564 1 x x x x

43 3methylpentadecane 871 1571 1570 1 x x x x x x 44 hexadecane 941 1601 1600 1 x x x x 45 2methylhexadecane a 895 1665 1666 1 x x 46 8heptadecene a 901 1681 1677 4 x 47 48 heptadecane a 929 1701 1700 1 x x

49 Alcohols 50 3methyl2butanol 908 693 692 1 x x x Analytical 51 1penten3ol 858 694 694 0 x x x x 52 1pentanol c 926 767 768 1 x 53 54 4methyl1pentanol 951 837 838 1 x x x x

55 (Z)3hexen1ol a 947 858 860 2 x x 56 1hexanol 877 870 865 5 x x x x 57 3methyl1pentanol b 884 870 872 2 x 58 59 60 16

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1 2 3 2heptanol a 895 903 900 3 x x

4 6methyl2heptanol a 855 968 x

5 1octen3ol c 844 981 983 2 x 6 (R)2octanol a 916 1003 998 5 x x 7 5methyl5octen2ol a 816 1050 x x 8 9 3ethyl2heptanol a 844 1081 x

10 5,5dimethylcyclohex3en1ol b 801 1195 x

11 2nonanol a 886 1103 1100 3 x x 12 2decanol b 828 1182 1186 4 x x 13 3cyclohexene1ethanol a 813 1281 x 14 15 3cyclopentyl1propanol b 831 1343 x x

16 3,3dimethylcyclohexanol b 845 1381 x x 17 (E)2hexadecacen1ol a 890 1483 x x 18 Aldehydes 19 3methylbutanal 871 675 671 4 x x x 20 21 2methylbutanal 867 680 677 3 x x x x Manuscript 22 pentanal c 883 702 698 4 x 23 hexanal 890 802 802 0 x x x x x 24 2hexenal c 877 846 841 5 x x 25 26 (E)2hexenal 939 854 853 1 x x x x

27 heptanal c 922 903 901 2 x

28 (Z)2heptenal c 923 959 956 3 x 29 (E,E)2,4heptadienal 811 1001 1000 1 x x 30 2ethyl hexanal a 860 1041 x 31 Accepted 32 2phenylacetaldehyde a 835 1053 1052 1 x

33 (E)2octenal c 923 1061 1060 1 x

34 (Z)dec7enal b 822 1156 x x 35 (E,Z)2,6nonadienal c 875 1156 1155 1 x 36 (E)2nonenal c 934 1163 1161 2 x 37 38 2,4decadienal c 898 1299 1297 2 x

39 pentadecanal c 922 1718 1717 1 x

40 Methods Ketones 41 3methyl2butanone a 801 677 673 4 x 42 43 1penten3one 896 696 691 5 x x x x x x 44 2,3pentanedione b 871 702 696 6 x

45 2heptanone c 837 891 887 4 x 46 1octen3one c 846 979 975 4 x 47 2nonanone 916 1092 1089 3 x x x 48 2decanone a 914 1193 1191 2 x x 49 50 2undecanone a 843 1259 1257 2 x x Analytical 51 Esters 52 ethyl acetate 941 614 610 4 x x x x x x 53 methyl methacrylate c 884 712 710 2 x 54 55 ethyl propionate c 905 714 711 3 x

56 methyl isovalerate a 929 775 770 5 x x

57 methyl 2methylbutanoate 884 775 771 4 x x 58 59 60 17

Analytical Methods Page 18 of 23

1 2 3 a 940 824 821 3 x x

4 methyl 3methyl2butenoate c 951 844 842 2 x

5 ethyl 2methylbutanoate a 914 848 846 2 x 6 a 908 901 898 3 x 7 2pentyl propionate a 820 918 916 2 x 8 9 methyl hexanoate 911 924 922 2 x x x

10 αmethylbutyl isobutyrate a 837 962 958 4 x

11 ethyl 4methylpentanoate a 875 964 963 1 x x 12 817 977 972 5 x x x 13 a 892 998 996 2 x 14 15 isobutyl 2methylbutyrate b 915 1003 1002 1 x

16 (Z)3hexenyl acetate b 898 1005 1001 4 x 17 isobutyl isovalerate b 940 1006 1003 3 x x 18 isoamyl isobutyrate 950 1011 1007 4 x x x x x 19 butyl 2methylbutanoate a 894 1041 1039 2 x 20 21 pentyl isobutyrate 942 1049 1047 2 x x x x Manuscript 22 4methylhexyl acetate b 821 1082 x x 23 methyl 6methyl heptanoate a 838 1087 x 24 944 1093 1092 1 x x x 25 26 isopentyl 2methylbutanoate 895 1100 1099 1 x x x x

27 methyl benzoate c 891 1101 1096 5 x

28 isopentyl isovalerate 920 1106 1105 1 x x x x x 29 heptyl acetate b 879 1112 1110 2 x x 30 3methyl butanoate b 856 1117 1116 1 x 31 Accepted 32 methyl octanoate a 884 1123 1120 3 x

33 hexyl 2methyl2propenoate a 839 1135 x

34 (E)3hexenyl butyrate 883 1139 x x x 35 pentyl 2methylbutyrate 905 1139 x x x x 36 hexyl isobutyrate 926 1142 1139 3 x x x x 37 38 (Z)3hexenyl butyrate 899 1147 1142 5 x x x x

39 3methyl3butenyl 3methylbutanoate 877 1147 x x x

40 b Methods 3methyl2butenyl pentanoate 825 1149 x x 41 isopentyl pentanoate a 917 1154 1152 2 x 42 hexyl butyrate 870 1191 1188 3 x x x x x 43 44 octanoic acid, ethyl ester a 853 1196 1194 2 x

45 methyl salicylate 926 1202 1201 1 x x x x x 46 (Z)3hexenyl isopentanoate 879 1235 1235 0 x x 47 hexyl 2methylbutanoate 939 1237 1234 3 x x x x 48 hexyl isovalerate 871 1241 1240 1 x x x x x 49 50 (E)2hexenyl pentanoate b 838 1246 1243 3 x x Analytical 51 isopentyl hexanoate 921 1251 1250 1 x x 52 hexyl pentanoate 819 1252 1247 5 x x x 53 hexyl 3methyl2butenoate 858 1287 x x x x 54 55 heptyl pivalate 846 1298 x x x x

56 benzyl isobutyrate 820 1301 x x

57 7ethoxy3heptene b 822 1318 x x 58 59 60 18

Page 19 of 23 Analytical Methods

1 2 3 heptyl 2methylbutanoate 869 1335 1332 3 x x x x

4 heptyl pentanoate a 806 1341 x x

5 (Z)3hexenylpyruvate 851 1343 x x x x 6 (E)4hexenyl hexanoate c 839 1347 x 7 8 hexyl hexanoate 827 1352 1348 4 x x x x x x 9 (Z)3hexenyl hexanoate 890 1381 1379 2 x x x

10 benzyl 3methylbutanoate 892 1391 1387 4 x x x x

11 octyl pivalate b 814 1398 x 12 octyl pentanoate b 819 1403 x x 13 octyl 2methylbutanoate b 879 1433 1430 3 x x 14 15 octyl isopentanoate b 876 1440 1434 6 x x

16 nheptyl hexanoate a 813 1450 1448 2 x 17 (Z)3decenyl acetate a 826 1474 x 18 phenylethyl pivalate b 835 1497 x x 19 benzyl hexanoate 810 1516 x x 20 21 benzoic acid nhexyl ester b 873 1549 1545 4 x x Manuscript 22 hexyl benzoate a 847 1550 1549 1 x x 23 Ether 24 2ethylfuran 940 702 702 x x x 25 26 2pentylfuran 880 991 990 1 x x x x x x 27 (Z)1ethoxy4methylpent2ene 803 1244 x x x

28 Pyrazine 29 2isobutyl3methoxypyrazine 874 1180 1180 0 x x x x x x 30 Sulfur 31 Accepted 32 2pentylthiophene 905 1166 1164 2 x x x

33 Terpenes

34 αpinene 879 926 925 1 x x 35 αtricyclene 894 926 923 3 x x x 36 (R)αpinene c 921 937 933 4 x 37 38 camphene c 937 955 952 3 x x

39 sabinene c 875 977 973 4 x

40 a Methods βmyrcene 843 990 988 2 x x 41 βpinene c 878 990 985 5 x x 42 αphellanderene c 854 1009 1004 5 x 43 44 ocymene c 931 1029 1028 1 x

45 limonene 911 1032 1031 1 x x x x 46 eucalyptol c 920 1037 1035 2 x x 47 (Z)βocimene a 822 1047 1045 2 x x 48 γterpinolene c 882 1090 1089 1 x 49 50 terpinen4ol c 804 1189 1184 5 x Analytical 51 βcyclocitral a 847 1227 1224 3 x x 52 αcubenene b 908 1356 1352 4 x x 53 αlongipinene 866 1364 1360 4 x x x x 54 55 ylangene 869 1379 1375 4 x x x x

56 copaene 889 1386 1382 4 x x x x x

57 βcubebene b 904 1398 1394 4 x x 58 59 60 19

Analytical Methods Page 20 of 23

1 2 3 αionone a 851 1428 1427 1 x x

4 (E)αionone b 860 1430 1428 2 x x

5 caryophyllene 911 1434 1432 2 x x x 6 germacrene D b 845 1443 1442 1 x 7 aromadendrene b 800 1452 1451 1 x 8 9 (E)βfarnesene 871 1456 1454 2 x x x x

10 αcaryophyllene 922 1470 1467 3 x x x

11 ()germacrene D 866 1474 1470 4 x x x 12 9epiβcaryophyllene 879 1482 1477 5 x x x x 13 γmuurolene b 914 1486 1481 5 x x 14 15 (E)βionone 893 1486 1485 1 x x x x

16 longifolene(v4) b 888 1492 x 17 2epi(E)βcaryophyllene b 862 1498 x x 18 αselinene b 892 1502 1500 2 x x 19 6epiβcubebene b 879 1506 x x 20 21 alloaromadedrene b 859 1515 1511 4 x Manuscript 22 δcadinene 897 1527 1524 3 x x x x x 23 Lcalamenene b 840 1532 1530 2 x x 24 cadinadiene1,4 b 883 1544 1539 5 x x 25 26 8,9dehydroneoisolongifolene b 819 1547 x x 27 318 28 319 R.T 1 = retention time in seconds for the first dimension. 29 320 R.T = retention time in seconds for the second dimension. 30 2 321 SI = Similarity of the spectra obtained with those of the libraries.

31 Accepted 32 322 LTPRI Cal. = Experimental linear temperature programmed retention index. 33 323 LTPRI Lit. = Literature linear temperature programmed retention index. 34 324 = LTPRI Cal. LTPRI Lit. 35 325 a = compounds only characteristic of malagueta peppers. 36 326 b = compounds only characteristic of murupi peppers. 37 327 c = compounds only characteristic of dedodemoça peppers. 38 328 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 20

Page 21 of 23 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 Figure 1: Chromatogram of the volatile compounds from immature malagueta peppers obtained by HS-SPME 25 and GC × GC-TOFMS, with the main chemical classes respective structuring. 896x438mm (87 x 87 DPI) 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Analytical Methods Page 22 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 Figure 2: Scores plot regarding on the first two principal components obtained from PCA of the volatile 33 compounds of malagueta, murupi and dedo-de-moça peppers. 34 254x190mm (96 x 96 DPI) 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 Page 23 of 23 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 Figure 3: Loadings plot regarding on the first two principal components obtained from PCA of the volatile 33 compounds of malagueta, murupi and dedo-de-moça peppers. 34 254x190mm (96 x 96 DPI) 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60