Original article revista.iq.unesp.br | Vol. 48 | n. 4 | 2023 | 27 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 Variation in the chemical composition of essential oils from Mangifera indica L. leaves by comprehensive two-dimensional gas chromatography Claudia Andrea Lima Cardoso1+ , Elina Bastos Caramão2 , Thiago Luis Aguayo de Castro1 , Euclesio Simionatto3 1. Mato Grosso do Sul State University , Postgraduate in Natural Resources, Dourados, Brazil. 2. Tiradentes University , Department of Industrial Biochemistry, Aracaju, Brazil. 3. Mato Grosso do Sul State University , Chemistry Course, Naviraí, Brazil. +Corresponding author: Claudia Andrea Lima Cardoso, Phone: +55 67 3902-2651, Email address: claudia@uems.br ARTICLE INFO Article history: Received: August 06, 2022 Accepted: July 24, 2023 Published: October 03, 2023 Keywords: 1. GC×GC/TOFMS 2. Mango 3. Mass spectrometry 4. Variety ‘coração de boi’ 5. Variety ‘espada’ Section Editors: Assis Vicente Benedetti ABSTRACT: The leaves of Mangifera indica L. have been used in the medical system of India to treat diseases such as asthma, dysentery, cough, leucorrhea, jaundice, pain, and malaria. The analysis of different varieties of the same species is intended to determine if the compounds have a differential distribution. The present study investigates the volatile compounds from the leaves of two M. indica varieties extracted by hydrodistillation and analyzed by comprehensive two- dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOFMS). The number of compounds identified by GC×GC/TOFMS was superior to that obtained by gas chromatography/mass spectrometry (GC/MS) for the same variety of M. indica. This study demonstrates the applicability of the GC×GC/TOFMS for the comprehensive profiling of essential oils from M. indica, in which 125 and 95 compounds were identified in the varieties ‘espada’ and ‘coração de boi’, respectively. These results show that the compositions of the two analyzed essential oils present differences concerning the GC×GC/TOFMS and conventional chromatography technique, the GC/MS. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 mailto:claudia@uems.br https://orcid.org/0000-0002-4907-0056 https://orcid.org/0000-0002-7045-5874 mailto:elinaufrgs@gmail.com https://orcid.org/0000-0002-8127-1990 mailto:thiagoaguayo@gmail.com https://orcid.org/0000-0003-2029-8003 mailto:euclesio@uems.br https://ror.org/02ggt9460 https://ror.org/015xjsg96 https://ror.org/02ggt9460 Original article revista.iq.unesp.br 28 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 1. Introduction Plant-derived essential oils are known and primarily used for their biological properties (Mesquita et al., 2015). Combined with this, the major interest of the pharmaceutical, food and cosmetics industries in the use of new oils as well as the consumer receptivity to new products of natural origin, transformed the evaluation methods of these plants into widely used tools in the search for new products (Aćimović et al., 2022). The proportion of individual compounds in the oil composition differs from trace levels to over 90% (Bassolé and Juliani, 2012). Then, the complete separation and the correct identification of the essential oil compounds appear to be very important to a better understanding of the mechanisms involved in those biological activities and the prospection of new active compounds (Cagliero et al., 2022). Mangifera indica L., belonging to the Anarcadiaceae family, is one of the 40 species of the Mangifera genus that can be found in tropical and subtropical regions of Southeastern Asia, Africa, and Latin America (Nikhal and Mahajan, 2010). Its fruits are considered multifunctional foods. However, other parts of this plant, such as bark, flowers, branches, and leaves, have also bioactive compounds (Gupta et al., 2022). The leaves of M. indica have been used in the medical system of India to treat diseases such as asthma, dysentery, cough, leucorrhea, jaundice, pain, and malaria (Basha et al., 2011). In Brazil, the leaves are used as analgesic, anti-inflammatory and the treat hepatitis (Oliveira et al., 2022). The study of the aqueous extracts of the bark of a selected variety of M. indica resulted in a pharmaceutical formula, commercially named Vimang. The volatile constituents of M. indica fruits present a considerable variation in their chemical composition, which has been extensively investigated (Dzamić et al., 2010). The variability of the volatile constituents can be influenced by factors such as the stage of development, variety, and extraction method (Pino et al., 2005). The essential oils of M. indica leaves from Egyptian varieties have antimicrobial activity (Ouf et al., 2021). The latex essential oil of M. indica from the ‘rosa’ and ‘espada’ varieties showed cytotoxic activity against HL-60 human tumor cells (Ramos et al., 2014). The volatile compounds in M. indica were usually obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry (GC/MS) (Ansari et al., 2000; Berenbaum et al., 1985; Dzamić et al., 2010; Moreno et al., 2010; Oliveira et al., 2017; Pino et al., 2005). Due to the volatility and polarity of essential oils components, capillary gas chromatography is the preferable technique for their analysis because essential oils are generally complex mixtures of components with similar physicochemical characteristics (Aspromonte et al., 2019; Rubiolo et al., 2010). However, the satisfactory separation of a complex sample requires a higher peak capacity. In this case, comprehensive two- dimensional gas chromatography (GC×GC), a relatively new technique, can be the best alternative (Keppler et al., 2018). Comprehensive GC×GC, idealized by Liu and Phillips (1991), has since emerged as the most powerful separation technique for analyzing volatile compounds. The satisfactory separation in complex samples, such as some essential oil, requires a higher peak capacity, achieved using GC×GC. In this technique, two independent separation mechanisms are used to resolve the compounds of complex samples within a single analysis, based on applying two GC columns with different stationary phases connected in series, with a transfer device defined as a modulator. The modulator’s function is continuously isolating, reconcentrating, and introducing small portions of the first (1D) effluent onto a second column (2D). The time required to complete this process is defined as the modulation period. Each 1D peak is modulated several times, preserving the 1D separation (Adahchour et al., 2008; Stefanuto et al., 2021). The GC×GC has the advantage of increasing the resolution and sensitivity of the analysis due to the concentration of the sample fraction through the modulation process allowing the detection of compounds in trace levels as well as the separation of related compounds in the second dimension (Baharum et al., 2010). GC×GC is the most powerful separation system now available when combined with mass spectrometry (MS). The time-of-flight mass spectrometer (TOFMS) can obtain high spectra acquisition rates for the correct peak assignment and quantification in GC×GC. However, its high cost limits its laboratory utilization. Some studies used GC×GC with a time-of-flight mass spectrometry detector (GC×GC/TOFMS) to analyze essential oils (Eyres et al., 2007; Ieri et al., 2019; Jalali et al., 2012; Rubiolo et al., 2010; Wang et al., 2012). The results obtained by these studies showed an important improvement in the characterization of these samples by GC×GC. In the present study, the volatile compounds of two M. indica were analyzed by GC×GC/TOFMS to evaluate the difference between the compounds in the varieties, allowing an adequate selection for medicinal and industrial purposes. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 Original article revista.iq.unesp.br 29 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 2. Experimental 2.1 Samples The leaves of the M. indica variety were collected in Campo Grande/MS, Brazil. The ‘espada’ variety (collected at 20°30’7” S and 54°37’17” W) and the ‘coração de boi’ variety (20°30’13” S and 54°37’14” W) were identified by Dr. Ronaldo Posella Zaccaro (Centro Universitário Moura Lacerda, Ribeirão Preto/SP, Brazil) and deposited with voucher specimens’ numbers CM105 and CM 107, respectively. All the used solvents and reference standards (linear alkanes) were HPLC grade (JT Baker and Sigma Aldrich). The collection was recorded in the SisGen, number AF9B3C3. 2.2 Essential oil Each essential oil was isolated from a 400 g sample of fresh leaves of M. indica by hydrodistillation using a Clevenger-type apparatus. The essential oils were recovered, dried with anhydrous sodium sulfate, transferred to dark vials, and finally stored at –4 °C for further analysis. Before gas chromatographic analysis, the essential oils (1 mg) were diluted in 1 mL n-hexane. The essential oil yield calculated based on fresh leaves was 0.2% for ‘coração de boi’ and 0.3% for ‘espada’. 2.3 Chromatographic analysis A GC×GC/TOFMS Pegasus-IV system (LECO, St. Joseph, USA) was equipped with a liquid nitrogen quad- jet modulator and CTC Combi Pal autosampler (CTC Analytics, Carrboro, NC, USA). Electron ionization was 70 eV, the mass acquisition was performed in the range of 50 to 550 amu at 100 Hz, and the detector voltage was –1,706 V. The injector, transfer line and detector temperature were maintained at 250 °C. A conventional column set was employed: DB-5 (5% phenyl–95% dimethylpolysiloxane) with 60 m length, an internal diameter of 0.25 mm, and 0.10 μm of film thickness in the first dimension and a DB-17ms (50% phenyl–50% dimethylpolysiloxane) with 2.15 m length, the internal diameter of 0.18 mm and 0.18 μm of film thickness. Both columns were acquired from Agilent Technologies – J&W Scientific (Palo Alto, CA, USA). The temperature program of the first column started at 50 °C for 5 min, heating at 3 °C min–1 till 250 °C. The second column temperature was maintained 10 °C above the temperature of the first column. The modulation period was 10 s, and the Hot pulse was 40% of the modulation period. ChromaTOF software version 3.32 was employed for data processing the total ion current chromatogram, including tools such as peak finder and mass spectra deconvolution. Data processing was performed using a signal-to-noise ratio equal to three. The criterium for accepting a detected compound was a minimum of 80% similarity with the library. Temperature-programmed retention indices (Mota et al., 2013) were calculated using a mixture of linear alkane (C6-C30), which was analyzed under the same conditions as the chromatographic analysis of the samples. The volatile components’ identification was based on comparing their mass spectra with those of the database NIST 2.0, the comparison of their retention index and mass spectrum (Adams, 2007) and the interpretation of the mass spectrum. The results obtained in this study were compared with the data obtained using GC/MS by Oliveira et al. (2017). 3. Results and discussion Some studies show that the main compounds of the essential oils obtained from mango leaves are sesquiterpenes and monoterpenes (Dzamić et al., 2010; Gerbara et al., 2011; Moreno et al., 2010; Pino et al., 2005). These results of this study corroborate the data on the composition of M. indica leaves in the ‘espada’ and ‘coração de boi’ varieties. The essential oil from mango contains constituents such as α-gurjunene, trans-caryophyllene, α-humulene, α-selinene, and camphor (Kumar et al., 2021). Ramos et al. (2014) identified 25 compounds in the essential oil of M. indica leaves using gas chromatography with flame ionization detection (GC-FID) and GC/MS. Fontenelle et al. (2017) studied the essential oil from different M. indica leaves by GC/MS, obtaining 20 compounds for the ‘Tommy Atkins’ variety, 13 for the ‘rosa’ variety, 6 for the ‘muscat’ variety and 15 for the ‘jasmine’ variety. Ouf et al. (2021) identified 31 compounds in the ‘Alphonso’ variety, 33 compounds in the ‘Sidik’ variety, 29 compounds in the ‘waste’ variety, 26 compounds in the ‘zebda’ variety and 31 compounds in the ‘fagri- kalan’ variety and trans-caryophyllene (8.06–18.88%), α-selinene (4.33–16.92%), and α-humulene (8.48– 25.98%) were found in the higher concentrations. For the ‘coração de boi’ variety, cyperene, (E)-caryophyllene and α-humulene are the predominant compounds. Those compounds were also reported to be the most important in the leaves of the ‘coquinho’ variety by GC/MS analysis (Gerbara et al., 2011). The study performed by Oliveira et al. (2017) identified 23 volatile compounds such as monoterpenes https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 https://www.sciencedirect.com/topics/chemistry/gas-chromatography-with-flame-ionization-detection#:~:text=GC with flame-ionization detection,the carbon number (CN). Original article revista.iq.unesp.br 30 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 and sesquiterpenes from the leaves of M. indica ‘espada’ and ‘coração de boi’ varieties extracted by hydrodistillation and analyzed by GC/MS. In the essential oil of leaves obtained from the ‘espada’ variety, the major compounds were β-selinene (34.90%), cyperene (22.40%), (E)-caryophyllene (16.39%), α-humulene (10.84%), terpinolene (2.31%), and α-selinene (2.31%), while in ‘coração de boi’ variety, the major compounds were cyperene (32.62%), (E)-caryophyllene (26.91%), α-humulene (17.12%), β-selinene (5.70%), myrcene (2.80%), and - phellandrene (2.70%) Oliveira et al. (2017). The ‘espada’ and ‘coração de boi’ varieties generated essential oils of leaves with 125 and 95 tentatively identified compounds using the GC×GC/TOFMS technique. Due to its superior performance over the GC/MS, the GC×GC/TOFMS increased the number of identified peaks in M. indica essential oils. In the column setup used, nonpolar in the 1D and medium polar in the 2D, the compounds are separated in the first dimension based on their different volatilities. In the second dimension, the separation is governed by polarity. Consequently, compounds with similar volatility had similar or even exact retention times in the 1D and will be resolved in the 2D. The GC×GC/TOFMS analyses revealed a complex organic compound mixture (Figs. 1 and 2). Combining a low polar 5% phenyl phase in the first dimension with a medium polar 50% phenyl phase in the second dimension allowed efficient use of the available chromatographic space. Figures 1 and 2 highlight the complexity of the M. indica essential oil and the efficiency of GC×GC to reduce the peak coelution, obtaining pure MS spectra and increasing peak detectability. Figure 1. Color diagram of the essential oils of M. indica ‘espada’ variety obtained by comprehensive two- dimensional gas chromatography with time-of-flight mass spectrometric detector. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 Original article revista.iq.unesp.br 31 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 Figure 2. Color diagram of the essential oils of M. indica ‘coração de boi’ variety obtained by comprehensive two- dimensional gas chromatography with time-of-flight mass spectrometric detector. The experimental linear retention indices show a good agreement between the identified compounds and the linear retention indices reported by literature for 1D- GC (Bogusz Junior et al., 2011). The list of identified compounds is shown in Table 1. Table 1. Percentage composition of essential oils of leaves of M. indica by GC×GC/TOFMS. KIa KIb 1tR 2tR Compound ‘Espada’ (%) ‘Coração de boi’ (%) 769 771 7.83 3.29 2-pentenol 0.05 ± 0.01 - 800 800 8.50 2.02 octane 0.03 ± 0.01 0.05 ± 0.01 800 802 8.50 3.54 caproaldehyde 0.16 ± 0.01 0.16 ± 0.01 848 854 10.17 4.3 2-hexenal 2.10 ± 0.10 1.00 ± 0.10 852 851 10.33 3.97 3-hexen-1-ol 0.44 ± 0.02 0.40 ± 0.03 867 867 10.83 3.85 hexen-1-ol 0.86 ± 0.03 0.81 ± 0.09 867 869 10.83 3.79 santene 1.75 ± 0.09 1.50 ± 0.20 904 902 12.17 4.11 heptanal 0.10 ± 0.01 - 929 930 13.33 3.93 α-thujene 0.06 ± 0.01 0.10 ± 0.01 932 939 13.50 3.5 α-pinene 1.20 ± 0.10 1.01 ± 0.07 946 944 14.17 2.71 valeric acid 3-methyl 0.07 ± 0.01 - 975 975 15.50 3.91 sabinene 0.30 ± 0.01 0.34 ± 0.01 989 991 16.17 3.79 myrcene 0.21 ± 0.01 1.60 ± 0.10 1000 1000 16.67 3.89 m- mentha-1(7).8-diene 0.21 ± 0.01 0.22 ± 0.01 1006 1005 17.00 4.51 α- phellandrene 0.33 ± 0.01 0.23 ± 0.01 1010 1011 17.17 4.01 γ-carene 4.20 ± 0.10 2.70 ± 0.20 1027 1031 18.00 4.14 limonene 0.99 ± 0.05 1.04 ± 0.08 1033 1031 18.33 5.01 β-phellandrene 0.02 ± 0.01 2.80 ± 0.20 1087 1087 21.00 5.22 terpinolene 0.02 ± 0.01 - 1093 1089 21.30 3.21 p-cimenene 0.05 ± 0.01 0.04 ± 0.01 1110 1108 22.17 5.03 maltrol 0.09 ± 0.01 - 1128 1128 23.00 8.03 allo-ocimene 0.09 ± 0.01 - 1134 1134 23.33 5.22 1-terpineol 0.03 ± 0.01 - 1141 1141 23.67 5.73 cis-verbeneol 0.12 ± 0.01 0.22 ± 0.01 1159 1159 24.50 5.61 karahanaenone 0.41 ± 0.03 0.38 ± 0.02 1176 1177 25.33 5.45 terpinen-4-ol 0.35 ± 0.02 0.37 ± 0.01 Continue… https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 Original article revista.iq.unesp.br 32 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 1186 1183 25.83 6.15 p-cimen-8-ol 2.00 ± 0.20 2.90 ± 0.20 1190 1190 26.00 3.36 neo isoverbenol 0.25 ± 0.01 - 1211 1212 27.00 3.21 2.4-nonadienal 0.25 ± 0.01 - 1278 1278 30.00 3.27 isopulegyl acetate 0.30 ± 0.01 - 1300 1300 31.00 3.42 tridecane 0.33 ± 0.02 - 1331 1330 32.50 4.85 acetate cis-piperitol 0.02 ± 0.01 - 1334 1333 32.67 7.38 hexyl tiglate 0.08 ± 0.01 0.40 ± 0.02 1338 1339 32.83 6.59 γ-elemene 0.04 ± 0.01 - 1345 1344 33.17 5.77 verbenol acetate 0.08 ± 0.01 0.17 ± 0.01 1348 1348 33.33 6.12 7-epi -silphiperfol-5-one 0.05 ± 0.01 - 1369 1368 34.33 4.51 cyclosativene 0.42± 0.02 0.42± 0.02 1372 1373 34.50 3.36 isoledene 0.39± 0.01 0.58± 0.02 1376 1376 34.67 4.73 α-copaene 0.02 ± 0.01 0.04 ± 0.01 1379 1379 34.83 6.02 silpheperfol-6-one 0.02 ± 0.01 - 1379 1379 34.83 6.85 methyl cinnamate 0.02 ± 0.01 - 1386 1391 35.17 4.67 β-elemene 2.10 ± 0.20 3.10 ± 0.20 1393 1393 35.50 3.55 jasmone 0.35 ± 0.01 0.47 ± 0.02 1397 1398 35.67 4.53 cyperene 5.00 ± 0.30 7.00 ± 0.30 1405 1404 36.01 4.75 methyl eugenol 0.84 ± 0.04 0.84 ± 0.02 1405 1406 36.01 4.85 italicene 3.50 ± 0.30 2.50 ± 0.10 1409 1409 36.17 5.89 α-gurjunene 0.02 ± 0.01 - 1414 1417 36.33 6.61 4.8-alpha-epoxy caryophyllane 0.02 ± 0.01 - 1414 1417 36.33 5.22 sesquitujene 0.02 ± 0.01 - 1414 1418 36.33 5.10 (e)-caryophyllene 5.20 ± 0.20 6.50 ± 0.30 1423 1423 36.67 5.69 β-duprezianene 0.06 ± 0.01 0.06 ± 0.01 1427 1429 36.83 4.81 cis thujopsene 0.06 ± 0.01 0.05 ± 0.01 1436 1432 37.17 4.95 β-gurjunene 0.47 ± 0.01 0.52 ± 0.02 1445 1444 37.50 5.30 cedrene 1.04 ± 0.07 1.30 ± 0.10 1450 1450 37.67 4.98 epicedrene 0.13 ± 0.01 0.19 ± 0.01 1450 1450 37.67 5.36 cis-muurola-3.5-diene 0.16 ± 0.01 0.15 ± 0.01 1455 1454 37.83 5.23 α –humulene 6.90 ± 0.30 7.20 ± 0.30 1459 1460 38.00 3.46 β-santalene 0.03 ± 0.01 0.02 ± 0.01 1459 1460 38.00 5.23 allo-aromadendrene 2.10 ± 0.20 2.00 ± 0.10 1473 1473 38.50 5.40 α -terpinyl isobutanoate 0.57 ± 0.02 0.50 ± 0.02 1473 1473 38.50 5.22 drima-7.9(11)-diene 4.80 ± 0.20 4.90 ± 0.20 1477 1477 38.67 5.06 β-gurjunene 0.48 ± 0.02 0.45 ± 0.01 1482 1480 38.83 5.25 γ-murolene 0.30 ± 0.01 0.32 ± 0.01 1486 1480 39.00 5.48 germacrene d 0.72 ± 0.02 0.64 ± 0.02 1486 1485 39.00 5.40 β-selinene 10.20 ± 0.20 10.40 ± 0.20 1495 1496 39.33 3.71 asaricinae 0.28 ± 0.01 0.25 ± 0.01 1495 1498 39.33 5.38 α-selinene 6.70 ± 0.10 5.00 ± 0.20 1500 1500 39.50 5.42 biciclogermacrene 0.87 ± 0.01 0.87 ± 0.03 1509 1509 39.83 5.39 farenol 0.22 ± 0.01 0.21 ± 0.01 1509 1509 39.83 5.24 germacrene a 0.60 ± 0.02 0.62 ± 0.01 1517 1518 40.17 5.34 menthyl isovalerate 0.24 ± 0.01 0.24 ± 0.01 1522 1523 40.33 5.51 eugenol acetate 0.85 ± 0.03 0.83 ± 0.02 1526 1526 40.50 5.80 1-phenyl heptan-3-one 0.18 ± 0.01 0.19 ± 0.01 1526 1527 40.50 5.26 vanillin acetate 0.21 ± 0.01 0.21 ± 0.01 1535 1535 40.83 5.58 10-epi-cubenol 0.33 ± 0.01 0.36 ± 0.02 1539 1539 41.00 5.40 α-cadinene 0.13 ± 0.01 0.17 ± 0.01 1543 1543 41.17 5.75 8.14-cedranoxide 0.43 ± 0.02 0.41 ± 0.01 1548 1548 41.33 6.14 silphiperfolan-6-beta-ol 0.02 ± 0.01 - 1557 1557 41.67 6.93 elemicine 0.03 ± 0.01 0.01 ± 0.01 1561 1561 41.83 5.91 germacrene b 1.31 ± 0.08 2.01 ± 0.12 Continue… https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 Original article revista.iq.unesp.br 33 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 1565 1566 42.00 6.32 β-calacorene 0.03 ± 0.01 0.03 ± 0.01 1565 1566 42.00 6.42 davanone b 0.23 ± 0.01 0.32 ± 0.01 1574 1575 42.33 5.67 α-cedrene epoxy 0.28 ± 0.01 0.25 ± 0.01 1574 1575 42.33 6.01 silphiperfol-5-em-3-one a 0.38 ± 0.01 0.35 ± 0.01 1583 1583 42.67 6.26 turmerol 0.75 ± 0.06 0.34 ± 0.01 1583 1583 42.67 6.43 caryophyllene oxide 3.95 ± 0.08 2.15 ± 0.06 1591 1591 43.00 6.36 β-copaen-4-α-ol 0.14 ± 0.01 0.19 ± 0.01 1596 1596 43.17 6.04 turmerone-ar-dihydro 0.60 ± 0.01 0.64 ± 0.02 1600 1600 43.33 3.69 hexadecane 0.01 ± 0.01 - 1600 1601 43.33 5.15 cedrol 0.02 ± 0.01 - 1600 1601 43.33 5.92 guaiacol 0.04 ± 0.01 0.04 ± 0.01 1600 1601 43.33 6.25 β-elemenone 0.05 ± 0.01 0.05 ± 0.01 1605 1605 43.50 6.40 sesquilavandulol 0.88 ± 0.03 0.94 ± 0.03 1609 1608 43.67 6.21 platyphyllol 0.17 ± 0.01 0.15 ± 0.01 1609 1608 43.67 6.06 β-atlantol 0.92 ± 0.03 1.12 ± 0.05 1614 1614 43.83 6.42 ethyl chromone 2 0.44 ± 0.02 0.40 ± 0.01 1614 1614 43.83 6.59 β- biotol 3.40 ± 0.10 4.00 ± 0.20 1618 1618 44.00 6.16 butyl anthranilate 0.12 ± 0.01 - 1632 1631 44.50 6.31 eremoligenol 0.23 ± 0.01 0.27 ± 0.01 1632 1632 44.50 6.55 γ-eudesmol 0.82 ± 0.04 0.89 ± 0.02 1641 1641 44.83 7.52 epoxy allo alloaromadendrene 0.34 ± 0.01 0.31 ± 0.01 1645 1646 45.00 6.15 α-murolol 0.16 ± 0.01 0.13 ± 0.01 1655 1655 45.33 6.55 dihydromyrcene. 1.6 –diol. e 0.14 ± 0.01 0.15 ± 0.01 1655 1655 45.33 6.61 3- tujopsanone 1.64 ± 0.08 1.4 ± 0.1 1659 1659 45.50 6.62 atractilone 0.61 ± 0.02 0.63 ± 0.03 1668 1668 45.83 6.46 citronellyl tiglate e 0.15 ± 0.01 0.12 ± 0.01 1677 1677 46.17 6.6 cadelene 1.04 ± 0.06 0.90 ± 0.10 1686 1686 46.50 6.65 α-bisabolol 0.15 ± 0.01 0.13 ± 0.01 1695 1694 46.83 3.7 germacrone 0.20 ± 0.01 0.22 ± 0.01 1700 1700 47.00 3.79 heptadecane 0.41 ± 0.01 0.43 ± 0.02 1705 1705 47.17 6.68 δ-dodecalactone 0.11 ± 0.01 0.14 ± 0.01 1714 1714 47.50 4.91 cedroxide 0.03 ± 0.01 - 1714 1714 47.50 6.76 α -humulene. 14hydrixy 0.43 ± 0.01 0.52 ± 0.03 1724 1723 47.83 7.03 crisolide 0.44 ± 0.02 0.48 ± 0.03 1738 1740 48.33 7.53 oplopanone 0.29 ± 0.01 0.34 ± 0.01 1748 1748 48.67 7.29 α- oxobisabolone 0.20 ± 0.01 - 1800 1800 50.50 3.85 octadecane 0.23 ± 0.01 0.31 ± 0.01 1897 1898 53.83 4.70 seseline 0.26 ± 0.01 - 1901 1902 54.00 3.91 laurencene 0.26 ± 0.01 - 1963 1962 55.83 5.25 tetrahydro rimuene 1.40 ± 0.10 0.75 ± 0.03 2000 2000 57.00 3.94 eicosane 0.34 ± 0.01 - 2088 2088 59.33 6.16 abietadiene 0.56 ± 0.02 - 2100 2100 60.00 4.03 heneicosane 0.35 ± 0.01 - 2115 2116 60.67 5.14 laurensen-2-one 0.64 ± 0.02 0.73 ± 0.03 2176 2175 62.17 6.63 grandiflorene 0.12 ± 0.01 - aKI: retention index calculated; bKI: retention index literature from Adams (2007); (-): not identified; 1tR: retention time in the first dimension; 2tR: retention time in the second dimension. Furthermore, the GC×GC increased the detectability of the compounds due to using the modulator (Baharum et al., 2010), as can be observed in the increase in the number of compounds. These peaks were present at low concentrations, but the improvement of their signal by GC×GC achieved better mass spectra and separation than in the 1D-GC. In several cases, it was found that, despite using two chromatographic separation columns, some compounds were still coeluting. The essential oils have many isomers with similar retention times and mass spectra, especially sesquiterpenes and oxygenated sesquiterpenes. However, peak deconvolution algorithms allowed for resolving chromatographic solutions and extracting the mass spectrum of each https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v48.4.2023.p27-36 Original article revista.iq.unesp.br 34 Eclética Química, vol. 48, n. 4, 2023, 27-36 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v48.4.2023.p27-36 compound, even in such situations. GC×GC promoted the identification of fivefold more compounds in the two essential oils than GC/MS. The major constituents of ‘espada’ variety essential oil identified by GC×GC/TOFMS were β-selinene (10.2%), α-humulene (6.9%), α-selinene (6.7%), (E)- caryophyllene (5.2%), ciperene (5.0%), Drima-7.9(11)- diene (4.8%), γ-carene (4.2%), caryophyllene oxide (3.95%), italicene (3.5%) and β-biotol (3.4%). Ninety- five compounds were identified in essential oil of the ‘coração de boi’ variety by GC×GC/TOFMS and the major constituents were β-selinene (10.4%), α-humulene (7.2%), ciperene (7.0%), (E)-caryophyllene (6.5%), α- selinene (5.0%), Drima-7.9(11)-diene (4.9%, β-biotol (4.0%) and β-elemene (3.1%). A total of 31 compounds were identified exclusively in the ‘espada’ variety. The two essential oils were characterized by the predominance of β-selinene, (E)-caryophyllene and α- humulene. Also, monoterpenes were found in small concentrations in the two oils. The use of GC×GC provided enhanced efficiency, mainly for minor compounds. The results showed a considerable increase in the number of separated compounds. In addition, the analysis of mass spectra data together with the retention index allowed the identification of three times more compounds which reflected a differentiation between the essential oils studied. 4. Conclusions This study demonstrates the applicability of the GC×GC/TOFMS for the comprehensive profiling of M. indica essential oils. It also indicated that two- dimensional gas chromatography had a superior resolution, making it possible to identify more compounds. One hundred and twenty-five and ninety- five compounds were tentatively identified in the two studied essential oils of the ‘espada’ and ‘coração de boi’ varieties, respectively. These results showed that the compositions of the two analyzed essential oils showed differences in relation to the GC×GC/TOFMS and conventional chromatography technique, the GC/MS. Authors’ contribution Conceptualization: Cardoso, C. A. L.; Data curation: Cardoso, C. A. L.; Formal Analysis: Cardoso, C. A. L.; Castro, T. L. A.; Funding acquisition: Cardoso, C. A. L.; Investigation: Cardoso, C. A. L.; Simionatto, E.; Methodology: Cardoso, C. A. L.; Caramão, E.; Project administration: Cardoso, C. A. L.; Resources: Cardoso, C. A. L.; Software: Cardoso, C. A. L.; Supervision: Cardoso, C. A. L.; Caramão, E.; Validation: Cardoso, C. A. L., Simionatto, E.; Visualization: Cardoso, C. A. L.; Writing – original draft: Cardoso, C. A. L.; Simionatto, E.; Caramão, E.; Writing – review & editing: Cardoso, C. A. L.; Castro, T. L. A. Data availability statement All data sets were generated or analyzed in the current study. Funding Not applicable. Acknowledgments Not applicable. References Aćimović, M.; Rat, M.; Pezo, L.; Lončar, B.; Pezo, M.; Miljković, A.; Lazarević, J. Biological and chemical diversity of Angelica archangelica L.—Case study of essential oil and its biological activity. Agronomy. 2022, 12 (7), 1570. https://doi.org/10.3390/agronomy12071570 Adahchour, M.; Beens, J.; Brinkman, U. A. Recent developments in the application of comprehensive two- dimensional gas chromatography. J. Chromatogr. A. 2008, 1186 (1–2), 67–108. https://doi.org/10.1016/j.chroma.2008.01.002 Adams, R. P. 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