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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 23 Analytical Methods 1 2 3 1 Analysis of volatile compounds in Capsicum spp. by headspace solid-phase 4 5 6 2 microextraction and GC×GC-TOFMS 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, Brazil. 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) 3532-1200; [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 Analytical Methods Page 2 of 23 1 2 3 16 Abstract 4 5 6 17 A suitable method based on headspace solid-phase microextraction (HS-SPME), 7 8 18 comprehensive two-dimensional gas chromatography with time-of-flight mass 9 10 19 spectrometry detector (GC × GC-TOFMS) 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), dedo-de-moça (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 esters, 29 30 28 murupi by terpenes and dedo-de-moça by the presence of aldehydes and terpenes. 31 Accepted 32 33 29 Keywords: Comprehensive two-dimensional gas chromatography, Capsicum frutescens 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 spices in various parts of the 46 47 1, 2 48 35 world, mainly due to their attributes of color, pungency 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 chili pepper can be perceived as a result of the degree of maturation. 4 57 58 59 60 2 Page 3 of 23 Analytical Methods 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 distillation-solvent extraction (SDE), purge and 29 30 52 trap and solid phase micro-extraction (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 two-dimensional 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 Analytical Methods Page 4 of 23 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 ), dedo-de- 13 14 69 moça ( C. bacccatum ) and murupi ( C. chinense ) Brazilian pepper varieties. Static 15 16 17 70 headspace solid-phase microextraction (HS-SPME) and GC × GC-TOFMS 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, DB-17 – (50%- 44 45 82 phenyl)-methylpolysiloxane, DB-WAX – polyethylene glycol purchased from Agilent 46 47 48 83 Technologies (Wilmington, DE, USA) and RT-LC50 (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, dedo-de-moça, and murupi 56 57 87 pepper were botanically identified and supplied by the Campinas Agronomic Institute 58 59 60 4 Page 5 of 23 Analytical Methods 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 HS-SPME 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).