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. 2018 Nov 14;23(11):2969. doi: 10.3390/molecules23112969

Chemical Profiles of Incense Smoke Ingredients from Agarwood by Headspace Gas Chromatography-Tandem Mass Spectrometry

Wen-Yi Kao 1,2,†, Chien-Yun Hsiang 3,†, Shih-Ching Ho 2, Tin-Yun Ho 4,*, Kung-Ta Lee 1,*
PMCID: PMC6278519  PMID: 30441810

Abstract

Agarwood, the resinous wood in the heartwood of Aquilaria trees, has been used as incense in traditional Chinese medicine for its sedative, aphrodisiac, carminative, and anti-emetic effects. Grading of agarwood is usually based on its physical properties. Therefore, it is important to develop analytic methods for judgment and grading of agarwood. Here, we created a headspace (HS) preheating system that is combined with gas chromatography-mass spectrometry (HS GC-MS) to analyze the chemical constituents in the incense smoke produced by agarwood. Incense smoke generated in the HS preheating system was injected directly to GC-MS for analysis. A total of 40 compounds were identified in the incense smoke produced by Kynam agarwood, the best agarwood in the world. About half of the compounds are aromatics and sesquiterpenes. By analyzing chemical constituents in the incense smoke produced by Vietnamese, Lao, and Cambodian varieties of agarwood, we found that butyl hexadecanoate, butyl octadecanoate, bis(2-ethylhexyl) 1,2-benzenedicarboxylate, and squalene were common in the aforementioned four varieties of agarwoods. 2-(2-Phenylethyl) chromone derivatives were identified only in the incense smoke produced by Kynam agarwood, and were the major ingredient (27.23%) in the same. In conclusion, this is the first study that analyzes chemical profiles of incense smoke produced by agarwood using HS GC-MS. Our data showed that 2-(2-phenylethyl) chromone derivatives could be used to assess quality of agarwoods. Moreover, HS GC/MS may be a useful tool for grading quality of agarwood.

Keywords: agarwood, headspace preheated system, gas chromatography-mass spectrometry, 2-(2-phenylethyl)chromone

1. Introduction

Agarwood, also called Gaharu, Chén-xīang, Jin-koh, Kyara, or Oud, is the resinous wood formed in the heartwood of Aquilaria trees. Aquilaria species, including A. sinensis (Lour.) Glig., A. agallocha Roxb., and A. malaccensis Lam., taxonomically belong to Thymelaeaceae and are found mainly in Southeast Asia, such as Vietnam, Indonesia, Laos, Cambodia, and Malaysia [1,2]. Agarwood and its oil are important and useful natural substances that have been used to produce valuable products. Agarwood has been used as incense in religious ceremonies for centuries as well as in traditional Chinese medicine for its sedative, aphrodisiac, carminative, and anti-emetic effects [3,4,5,6,7,8,9]. The value of agarwood depends on its quality. However, grading of agarwood tends to be discretionary and subjective, because agarwood is usually graded based on its physical properties, such as color, density, resin formation, by sensory panels [1,2,10]. Kynam, also known as Kanankoh in Japan, is regarded to have the highest quality among different varieties of agarwood [11]. The high resin content, better water sinking quality, and darker color of Kynam enable it to produce a special and pleasant scent [1]. Kynam has been considered to be the noblest spiritual wood, both in ancient societies and in modern times, because of its scarcity. At present, the cost of Kynam agarwood is about 350 Euro per gram [12]. Kynam agarwood has a very complex scent, and even grading professionals cannot easily distinguish it from others. Therefore, it is important and essential to develop analytic methods for judgment and grading of agarwood.

Chemical constituents of agarwood have been studied by several research teams [1,3,13]. Previous studies identified chemical constituents of agarwood essential oils or organic solvent extracts with column chromatography or spectroscopic techniques [14,15]. Other studies prepared agarwood essential oils, hydrodistillates, or solvent extracts for gas chromatography (GC) or multidimensional GC analysis [16,17]. However, analysis of chemical constituents in agarwood solvent extracts is not suitable for differentiation of agarwood because agarwood is usually used to produce incense smoke. A few research studies analyzed chemical ingredients in the incense smoke produced by heated agarwood [11,18,19]. For example, Ishihara et al. [11] analyzed chemical constituents in the incense smoke that was trapped in the Tenax TA adsorbent resin, and extracted in diethyl ether for GC and GC-mass spectrometry (MS) analysis. They found 53 chemical compounds in the incense smoke produced by high quality (Kanankoh) and low quality (Jinkoh) varieties of Vietnamese agarwood, and most of them were sesquiterpenes and aromatics. Hung et al. [18] prepared an agarwood extract by heating a sealed vial that contained agarwood powder in a water bath at 90 °C for 30 min. The extract was absorbed in a solid-phase microextraction (SPME) needle with polydimethylsiloxane (PDMS) fiber for 30 min, and then desorbed for GC-MS analysis. They found six peaks that could be used for evaluating the quality or price of the most expensive agarwood. Zhou et al. [19] collected incense smoke produced by agarwood and incense sticks, absorbed the different kinds of collected incense smoke in glass fiber pads, and extracted substances from the glass fiber pads with dichloromethane for GC-MS analysis. Nevertheless, these studies analyzed chemical constituents in the incense smoke that was either absorbed in matrices or extracted in solvents.

Analysis of chemical constituents in the incense smoke produced by agarwood may be an approach to grading of agarwood. Most of past research studies determine chemical ingredients in agarwood solvent extracts [13,14,15,16,17]. However, analysis of chemical constituents in solvent extracts is not suitable in the case of agarwood because it is usually used to produce incense smoke. In this work, we used a headspace (HS) preheating system that is combined with GC-MS (HS GC-MS) to extract heavier sample matrices for GC analysis. Incense smoke produced by heated agarwood was directly analyzed with GC-MS. We could obtain more authentic chemical profiles of incense smoke produced by agarwood without solid-phase extraction. Moreover, by comparing chemical constituents in the incense smoke produced by different grades of agarwood, we found that HS GC/MS-MS could be used to grade quality of agarwood.

2. Results and Discussion

2.1. Morphological Observation of Various Grades of Agarwoods

Agarwood is traditionally graded based on its physical properties, such as resin content, water sinking quality, color, scent/aroma, as well as agarwood-inducing method, formation time, and place of origin [1,2]. Both Kynam and Vietnamese varieties of agarwood showed first-grade colors and had softer scents (Figure 1A,B). However, the Kynam variety fully sank in water, while the Vietnam variety merely partially submerged in water. Both the Lao and Cambodian varieties of agarwood fully floated up, showed second-grade colors, and had sweet scents (Figure 1C,D). However, the sweet scent of Cambodian agarwood was induced by insect infection.

Figure 1.

Figure 1

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Morphological observation of Kynam (A), Vietnam (B), Liao (C), and Cambodian agarwoods (D).

2.2. HS GC-MS/MS Analysis

Chemical constituents of agarwood have been intensively studied by several research teams [1,3,13]. Different extraction methods have been developed to determine chemical composition of essential oils and related compounds from agarwood chips using GC, GC-MS, solid phase microextraction, GC-flame ionization detector, GC-olfactometry, or comprehensive two-dimensional gas chromatography (GC×GC) [20]. Most studies suggest that hydrodistillation is the method of choice for determining the essential oil content of agarwood [7]. Agarwood has only a slight scent at room temperature, and releases a pleasant aroma when heated. Burning incense is a common traditional practice in many families and most temples in Asia, for religious reasons and for its pleasant smell [1,2]. Incense smoke contains many kinds of fragrant sesquiterpenes and aromatics [11]. Without interphase mass transfer, how to obtain a large amount of incense smoke is an important parameter for efficient analysis. Therefore, we prepared the incense smoke of agarwood using a HS preheating system, and tested the incubation times and temperatures to obtain the maximum amount of analytes. Previous studies extracted agarwood oils through incubation at 40 °C for 10 min, or at 90 °C for 30 min [18]. In this study, we prepared the incense smoke of agarwood through incubation at 150 °C for 30 min, and injected the smoke into GC-MS/MS for chemical profiling analysis (Figure 2).

Figure 2.

Figure 2

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Experimental design and incense smoke detection process. Diagram shows that the incense smoke of agarwoods was generated by headspace preheated system and the smoke was injected directly to GC-MS for analysis.

2.3. Chemical Profiles of Kynam Agarwoods Using HS GC/MS-MS

Figure 3 and Figure S1 shows the GC chemical fingerprint of incense smoke produced by Kynam agarwood. The detection period was setup from 2.5 min to 65 min. Forty peaks were identified in the GC-MS/MS analysis, and the identified compounds are summarized in Table 1. As shown in Figure 4, these compounds belong to different kinds of chemical categories, including aromatics (33%), sesquiterpenes (38%), alkanes (8%), terpene (3%), triterpenes (3%), and others (18%). Since sesquiterpenes and triterpenes were mostly identified in agarwoods, other terpenes, except sesquiterpenes and triterpenes, were classified as “terpene” in this study. Moreover, the major component in the incense smoke of Kynam was 2-(2-phenylethyl) chromone (27.23%).

Figure 3.

Figure 3

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GC-MS chemical fingerprint of incense smoke from Kynam agarwood. Number indicates the identified compound, and the details of compounds are listed in Table 1.

Table 1.

List of compounds identified in the incense smoke from Kynam agarwood by GC-MS/MS.

No. Compound LRI a Retention Time (min) Content b (%) Detected m/z Chemical Formula Type c
1 Benzaldehyde 1581 5.296 0.17 ± 0.05 106 C7H6O Ar
2 exo-7-(2-Propenyl)bicyclo [4.2.0]oct-1(2)-ene 1720 11.137 0.04 ± 0.02 148 C11H16 Other
3 4-Methoxybenzaldehyde 1731 11.617 0.14 ± 0.05 136 C8H8O2 Ar
4 1,8-Nonadiene-3-yne, 2,8-dimethyl-7-methylene- 1734 11.717 0.04 ± 0.03 160 C12H16 Other
5 Cubenene 1818 15.273 0.03 ± 0.02 204 C15H24 S
6 α-Longipinene 1823 15.471 0.26 ± 0.12 204 C15H24 S
7 Tricyclo[5 2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8-trimethyl- 1860 17.017 1.30 ± 0.42 220 C15H24O Ar
8 Naphthalene 1885 18.086 0.18 ± 0.22 202 C15H22 Ar
9 δ-Guaiene 1888 18.196 0.13 ± 0.08 204 C15H24 S
10 Nootkatene 1895 18.502 0.12 ± 0.06 202 C15H22 Ar
11 Kessane 1910 19.119 0.39 ± 0.29 222 C15H26O Ar
12 4a,5-Dimethyl-3-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalen-1-ol 1928 19.863 0.31 ± 0.07 220 C15H24O Ar
13 cis-α-Santalol 1962 21.312 0.28 ± 0.09 220 C15H24O S
14 β-Vetivenene 1969 21.593 0.10 ± 0.02 202 C15H22 T
15 γ-Eudesmol 1998 22.799 0.06 ± 0.06 222 C15H26O S
16 10s,11s-Himachala-3(12),4-diene 2017 23.61 0.27 ± 0.12 204 C15H24 Ar
17 Agarospirol 2022 23.808 0.21 ± 0.04 222 C15H26O S
18 β-Guaiene 2031 24.209 0.53 ± 0.24 204 C15H24 S
19 Cubenol 2041 24.606 1.24 ± 0.43 222 C15H26O S
20 α-epi-7-epi-5-Eudesmol 2057 25.296 0.95 ± 0.28 222 C15H26O S
21 Bulnesol 2074 26.021 0.26 ± 0.18 222 C15H26O S
22 α-Tetralone 2089 26.624 0.53 ± 0.26 218 C15H22O Ar
23 α-Kessyl acetate 2110 27.497 0.91 ± 1.31 280 C17H28O3 Ar
24 (1R,7S,E)-7-Isopropyl-4,10-dimethylenecyclodec-5-enol 2120 27.941 0.33 ± 0.32 220 C15H24O Ar
25 10-epi-Elemol 2141 28.822 0.19 ± 0.15 222 C15H26O S
26 Ledol 2156 29.445 0.53 ± 0.10 222 C15H26O S
27 Longiverbenone 2217 32.017 0.82 ± 0.74 218 C15H22O Other
28 Cryptomeridiol 2230 32.552 1.95 ± 1.10 240 C15H28O2 S
29 2aS,3aR,5aS,9bR)-2a,5a,9-Trimethyl-2a,4,5,5a,6,7,8,9b-octahydro-2H-naphtho[1–b]oxireno[2–c]furan 2243 33.081 0.40 ± 0.45 234 C15H22O2 Ar
30 10-epi-γ-Eudesmol 2324 36.506 2.21 ± 0.19 222 C15H26O S
31 Sandaracopimarinal 2413 40.221 2.19 ± 0.57 286 C20H30O Ar
32 γ-Gurjunene 2470 42.629 0.52 ± 0.10 204 C15H24 S
33 Butyl hexadecanoate 2542 45.628 0.75 ± 0.46 312 C20H40O2 Other
34 2-(2-Phenylethyl)chromone 2613 48.64 26.19 ± 1.0 250 C17H14O2 Other
35 Butyl octadecanoate 2635 49.538 1.13 ± 0.69 340 C22H44O2 Other
36 1-Iodo-octacosane 2640 49.743 0.05 ± 0.03 520 C28H61I Ak
37 1-Iodo-eicosane 2680 51.447 0.09 ± 0.06 408 C20H41I Ak
38 Bis(2-ethylhexyl) 1,2-benzenedicarboxylate 2700 52.277 0.34 ± 0.38 390 C24H38O4 Other
39 Squalene 2806 56.472 1.19 ± 1.30 410 C30H50 Tri
40 Nonacosane 2900 60.669 0.08 ± 0.05 408 C29H60 Ak
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a LRI: Linear retention index as tested on SH-Rxi-5Sil column using the series of C8–40 n-alkanes; b Values are the mean ± standard error (n = 3); c Ak: alkane; Ar: aromatics; S: sesquiterpene; T: terpene; Tri: triterpene.

Figure 4.

Figure 4

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Pie chart showing chemical classification of 40 compounds identified in the incense smoke from Kynam agarwood.

In 1993, Ishihara et al. [11] analyzed two kinds of agarwood incense smoke obtained with solvent extraction. Fifty components were identified in higher quality agarwood, while 36 components were identified in low quality agarwood. The incense smoke of both higher and low-quality agarwood was composed mainly of sesquiterpenes (53% and 61%, respectively) and aromatics (44% and 31%, respectively). The major components in higher quality agarwood were guaia-1(10), 11-dien-15-oic acid (9.92%), and 2-(2-phenylethyl) chromone (5.83%). Hung et al. [18] identified 17 components that could be used as markers for classification and differentiation of agarwood. 2-(2-Phenylethyl) chromone derivatives (flindersiachromone) were also identified in most expensive agarwoods.

A total of 132 constituents have been identified in solvent extracts from different varieties of agarwood in the past 5 decades, and they belong to sesquiterpenes (52%), 2-(2-phenylethyl) chromone derivatives (41%), and aromatics (1%) [3]. In this study, we found that aromatics and sesquiterpenes were the two major chemical categories in the incense smoke of Kynam agarwood as analyzed with HS GC-MS. The difference may result from the extraction procedure or varieties of agarwood.

Sesquiterpenes and 2-(2-phenylethyl) chromone derivatives were the two predominant constituents in agarwood [2]. As chromone derivatives could not be pyrolyzed [11], Espinoza et al. [20] used direct analysis with real time time-of-flight mass spectrometry (DART-TOFMS) to analyze 60 commercial agarwood chips without extraction, and identified the presence of key ions and the characteristics of 2-(2-phenylethyl) chromones. They found that 8–16 target chromone ions were present in each sample. These results showed that the highly oxidized agarwood chromones were specific to Aquilaria spp.

2.4. Chemical Profiles of Different Grades of Agarwoods Using HS GC/MS-MS

To further compare chemical constituents in the incense smoke produced by Vietnamese, Lao, and Cambodian varieties of agarwood with those in the same produced by the Kynam variety, we prepared the incense smoke using a HS preheating system, and the smoke was directly injected to GC for chemical identification. Lao agarwood shares similar physical properties with Cambodian agarwood, and both varieties exhibited similar patterns in GC (Figure 5 and Figure S2). A total of 110 constituents were identified in the incense smoke produced by four varieties of agarwood, including 40 compounds in the Kynam variety, 25 compounds in the Vietnamese variety, 45 compounds in the Lao variety, and 31 compounds in the Cambodian variety. Four compounds were commonly identified in the four varieties of agarwood, while 28, 13, 26, and 19 compounds were specifically identified in the Kynam, Vietnamese, Lao, and Cambodian varieties of agarwood, respectively (Figure 5D and Table 2). Butyl hexadecanoate, butyl octadecanoate, bis(2-ethylhexyl) 1,2-benzenedicarboxylate, and aqualene were commonly found in the incense smoke produced by the four varieties of agarwood. Ishihara et al. [11] analyzed the smoke profiles of two different varieties of Vietnamese agarwood that were absorbed in Tenax TA, and found small amounts of benzaldehyde and 2-(2-phenylethyl) chromone were present in both varieties of Kynam (Kanakoh) agarwood. Ismail et al. [21] analyzed the chemical constituents of agarwood oil that was absorbed in PDMS and divinylbenzene-carboxen-PDMS, and found that 4-phenyl-2-butanone was one of the major compounds that contributed to the scent. 4-Methoxy-benzaldehyde was the first compound identified in the incense smoke of agarwood in this study, whereas it had not been identified in agarwood smoke or oils in other studies. These findings indicate that the identification of constituents in agarwood is affected by adsorbents. Therefore, more authentic chemical profiles of agarwood can be achieved with the use of incense smoke without matrix absorption.

Figure 5.

Figure 5

Figure 5

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GC-MS chemical fingerprint of incense smoke from agarwoods. (A) Vietnam agarwood. (B) Liao agarwood. (C) Cambodian agarwood. Number indicates the compound specifically present in the agarwood. (D) Venn diagram of 110 compounds identified in the incense smoke from Kynam (K), Vietnam (V), Liao (L), and Cambodian (Ca) agarwoods.

Table 2.

Compounds commonly or specifically present in the incense smoke of agarwoods.

Compound Retention Time (min) LRI Detected m/z Chemical Formula Agarwoods a
Butyl hexadecanoate 45.628 2542 312 C17H40O2 K, V, L, C
Butyl octadecanoate 49.538 2635 340 C22H44O2 K, V, L, C
Bis(2-ethylhexyl) 1,2-benzenedicarboxylate 52.277 2700 390 C24H38O4 K, V, L, C
Squalene 56.472 2800 410 C30H50 K, V, L, C
Benzaldehyde 5.296 1581 106 C7H6O K
4-methoxy-Benzaldehyde 11.617 1731 136 C8H8O2 K
1,8-Nonadien-3-yne, 2,8-dimethyl-7-methylene- 11.717 1734 160 C12H16 K
Cubenene 15.275 1818 204 C15H24 K
α-Longipinene 15.471 1823 204 C15H24 K
Tricyclo[5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8-trimethyl- 17.017 1860 220 C15H24O K
Naphthalene 18.086 1885 202 C15H22 K
δ-Guaiene 18.196 1888 204 C15H24 K
Nootkatene 18.502 1895 202 C15H22 K
4a,5-Dimethyl-3-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalen-1-ol 19.863 1928 220 C15H24O K
cis-α-Santalol 21.312 1962 220 C15H24O K
β-Vetivenene 21.593 1969 202 C15H22 K
γ-Eudesmol 22.799 1998 222 C15H26O K
10s,11s-Himachala-3(12),4-diene 23.610 2017 204 C15H24 K
β-Guaiene 24.209 2031 204 C15H24 K
Cubenol 24.606 2041 222 C15H26O K
α-Tetralone 26.624 2089 218 C15H22O K
α-Kessyl acetate 27.497 2110 280 C17H28O3 K
(1R,7S,E)-7-Isopropyl-4,10-dimethylenecyclodec-5-enol 27.941 2120 220 C15H24O K
Ledol 29.445 2156 222 C15H26O K
Longiverbenone 32.017 2217 218 C15H22O K
2aS,3aR,5aS,9bR)-2a,5a,9-Trimethyl-2a,4,5,5a,6,7,8,9b-octahydro-2H-naphtho[1–b]oxireno[2–c]furan 33.081 2243 234 C15H22O2 K
10-epi-γ-Eudesmol 36.506 2324 222 C15H26O K
Sandaracopimarinal 40.221 2413 286 C20H30O K
γ-Gurjunene 42.629 2470 204 C15H24 K
2-(2-Phenylethyl)chromone 48.640 2613 250 C17H14O2 K
1-iodo-Triacontane 49.743 2640 548 C22H44O2 K
Eicosane, 1-iodo- 51.447 2680 408 C20H41I K
2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene 17.354 1868 204 C15H24 L
Dehydrodeoxybaimuxinol 19.844 1927 220 C15H24O L
Rosifoliol 22.267 1985 222 C15H26O L
1H-Cyclopropa[a]naphthalene, 1a,2,3,3a,4,5,6,7b-octahydro-1,1,3a,7-tetramethyl-, [1aR-(1aα,3aα,7bα)]- 23.598 2017 204 C15H24 L
Guaiol acetate 23.692 2019 222 C15H26O L
Isovalencenol 24.782 2045 218 C15H22O L
4-isopropenyl-1-methoxymethoxymethyl-cyclohexene 25.046 2051 196 C12H20O2 L
trans-α-Bisabolene 25.447 2061 204 C15H24 L
β-Patchoulene 25.667 2066 204 C15H24 L
Androstan-17-one, 3-ethyl-3-hydroxy-, (5α)- 25.938 2072 318 C21H34O2 L
Acetate, (2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydro-1-naphthalenyl) ester 26.337 2082 250 C16H26O3 L
Dehydrofukinone 27.707 2115 218 C15H22O L
Neoisolongifolene, 8,9-epoxy- 33.235 2246 218 C15H22O L
6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalen-2-ol 34.576 2278 220 C15H24O L
(S)-cis-Verbenol 36.717 2329 152 C10H16O L
Card-20(22)-enolide, 3,5,14,19-tetrahydroxy-, (3β,5β)- 38.348 2368 406 C23H34O6 L
Espatulenol 39.248 2390 220 C15H24O L
5-Isopropenyl-2-methylcyclopent-1-enecarboxaldehyde 39.504 2396 150 C10H14O L
Levoverbenone 39.650 2399 150 C10H14O L
Cyclobutene, 4,4-dimethyl-1-(2,7-octadienyl)- 39.908 2405 190 C14H22 L
Alloaromadendrene oxide-(1) 40.407 2417 220 C15H24O L
3-Oxatricyclo[20.8.0.0(7,16)]triaconta-1(22),7(16),9,13,23,29-hexaene 41.858 2452 406 C29H42O L
Andrographolide 42.100 2458 350 C20H30O5 L
Docosa-2,6,10,14,18-pentaen-22-al, 2,6,10,15,18-pentamethyl-, all-trans 48.062 2600 384 C27H44O L
2,6,10-trimethyl-Tetradecane 49.731 2639 240 C17H36 L
Heptadecanal 51.965 2693 254 C17H34O L
4,5-di-epi-Aristolochene 17.343 1868 204 C15H24 V
α-Costal 25.156 2054 218 C15H22O V
6,7-Dimethyl-1,2,3,5,8,8a-hexahydronaphthalene 25.964 2073 162 C12H18 V
Dihydrocolumellarin 39.222 2389 234 C15H22O2 V
Columellarin 40.694 2424 232 C15H20O2 V
Isovelleral 44.081 2505 232 C15H20O2 V
Octadecanoic acid 45.149 2530 284 C18H36O2 V
1-Hexacosene 45.755 2545 364 C26H52 V
Pentadecanal- 46.405 2560 226 C15H30O V
n-Pentadecanol 47.783 2593 228 C15H32O V
2-Nonadecanone 48.071 2600 282 C19H38O V
Dotriacontane, 1-iodo- 52.442 2704 576 C32H65I V
Tetracosanal 53.568 2731 352 C24H48O V
α-Ylangene 14.047 1789 204 C15H24 C
5,5-dimethyl-4-(3-oxobutyl)-Spiro[2.5]octane 15.489 1824 208 C14H24O C
Diepicedrene-1-oxide 17.013 1860 220 C15H24O C
7-epi-α-Cadinene 17.220 1865 204 C15H24 C
α-Guaiene 17.385 1869 204 C15H24 C
Alloaromadendrene oxide-(2) 19.874 1928 220 C15H24O C
Diethyl phthalate 21.888 1976 222 C12H14O4 C
Hinesol 23.982 2026 222 C15H26O C
cis-Eudesm-6-en-11-ol 24.456 2037 222 C15H26O C
Longipinane, (E)- 25.371 2059 206 C15H26 C
Pentadecanoic acid 35.990 2312 242 C15H30O2 C
Palmitoleic acid 39.168 2388 254 C16H30O2 C
Hexadecane 45.448 2537 226 C16H34 C
trans-Geranylgeraniol 48.077 2600 290 C20H34O C
Tetradecanal 48.432 2608 212 C14H28O C
Hexadecanal 50.261 2652 240 C16H32O C
Octacosane 51.439 2680 394 C28H58 C
Oxirane, hexadecyl- 51.970 2693 268 C18H36O C
Heptacosane 53.037 2718 380 C27H56 C
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a K: Kynam; V: Vietnam; L: Liao; C: Cambodian.

2.5. Chemical Ingredients of Different Grades of Agarwoods

Sesquiterpenoids and 2-(2-phenylethyl) chromone derivatives are two predominant constituents in agarwood [1,2,3]. Sesquiterpenoids, the essential ingredients in luxury perfumes, have been identified in different varieties of agarwood. In addition to sesquiterpenes, 2-(2-phenylethyl) chromone derivative (flindersiachromone, 2-phenethyl-4H-chromen-4-one) is another key reference constituent in agarwood. 2-(2-Phenylethyl)chromone derivatives can only be extracted in solvents or supercritical CO2, and are never found in hydrodistillates [22,23,24]. Substitute chromones in different grades of agarwood have also been studied. 2-(2-Phenylethyl) chromone derivatives can be detected in 76% of agarwood chips using direct analysis with DART-TOFMS without chemical extraction; however, DART-TOFMS is only used for distinguishing Aquilaria genus from others instead of agarwood grading [20]. One previous study showed that 2-(2-phenylethyl)chromone derivatives were found in incense smoke produced by Vietnamese agarwood, with a GC peak area of 5.83% in Kanankoh (high quality variety of agarwood) [11]. Another study also showed that 2-(2-phenylethyl)chromone derivatives were present only in expensive agarwood [15]. In this study, we found that 2-(2-phenylethyl) chromones were identified in the incense smoke of the Kynam variety (27.23%), but not in the same of Vietnamese, Lao, or Cambodian variety. So far, 39 different 2-(2-phenylethyl) chromone derivatives have been identified in different varieties of agarwood [20,25]. In line with these studies, our findings suggest that analysis of 2-(2-phenylethyl) chromone derivatives can be used to assess quality of agarwood. Moreover, the contents of 2-(2-phenylethyl) chromone derivative in triple duplicates of Kynam smoke were 25.18%, 27.23%, and 26.17% (Supplementary Materials Figure S1). Therefore, our data suggested that the HS GC/MS developed in this study can be a useful tool for grading quality of agarwood through direct analysis of incense smoke.

3. Materials and Methods

3.1. Reagents

All chemicals were purchased from Sigma (St. Louis, MO, USA), unless indicated.

3.2. Agarwood Materials

All natural agarwoods, including Kynam, Vietnam, Liao and Cambodia, were purchased from Chenglin Inc. (Yunlin, Taiwan). Agarwoods used in this study were identified and qualified by The International Tropical Timber Organization. The voucher specimen has been deposited in Development Center for Biotechnology. The agarwood chips were pulverized into a fine powder before use.

3.3. Sample Preparation

The incense smoke of agarwood was prepared using HS preheated system. Agarwood powder (50 mg) was weighted and placed in a 20 mL glass vial. The vial was sealed with Teflon, shaken, and incubated at 150 °C for 30 min in a headspace autosampler oven (Model HS-20, Shimadzu Corporation, Kyoto, Japan).

3.4. GC-MS/MS Analysis

Chemical constituents of incense smoke were analyzed by GS-MS/MS. Incense smoke (1 mL) was injected into GC-MS/MS (Model GC-2010 Plus and GCMS-TQ8040, Shimadzu Corporation, Kyoto, Japan). Temperature of sample line and transfer line was kept at 150 °C Ultra-high purity helium (99.99%) was used as the carrier gas at a flow rate of 5.1 mL/min. The chromatographic separation was conducted with SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm inner diameter, 0.25 μm thickness, Shimadzu Corporation, Kyoto, Japan). The column temperature program was set as follows: 60 °C initial temperature and subsequently ramped to 140 °C at a rate of 6 °C/min, held at 140 °C for 5 min, ramped to 160 °C at a rate of 2 °C/min, held at 160 °C for 5 min, ramped to 280 °C at a rate of 5 °C/min, held at 280 °C for 10 min. The GC-MS interface temperature was maintained at 250 °C. Electron ionization was used as the ionization method, and the ion source temperature was set at 230 °C. The mass spectra obtained with full scan and the mass ranged from m/z 45 to 550 were used for determining the unknown components.

Raw data were processed using GCMS solution Version 4.41 (Shimadzu Corporation, Kyoto, Japan). Mass spectral fragmentation patterns were compared with those stored in the NIST Mass Spectral Library (nist14), which is built up by using the pure substances and the mass spectra from literatures. In order to get the linear retention index values of the volatile compounds, a series of n-alkanes calibration standard (C8–C40) was run in the same condition (Figure S3). A Venn diagram was generated by eulerr program of R package [26].

4. Conclusions

So far, grading of agarwoods is flexible and subjective, because of the absence of standard grading system. Scent is the most important parameter for the quality assessment of agarwoods. In this work, we established a simple, automated, robust, and high-throughput method to analyze the incense smoke generated from a minimum amount of agarwood sample by HS GC-MS/MS. In comparison with the incense smoke constituents in Vietnam, Liao, and Cambodian agarwoods, we found that Kynam agarwood was composed of more complex constituents. Moreover, 2-(2-phenylethyl)chromone derivative was present in Kynam instead of Vietnam, Liao, and Cambodia agarwoods, suggesting that the content of 2-(2-phenylethyl)chromone derivative could be applied to grade the quality of agarwoods.

Supplementary Materials

The Supplementary Materials are available online.

Click here for additional data file. (1.1MB, pdf)

Author Contributions

Methodology, W.-Y.K. and S.-C.H.; Supervision, T.-Y.H. and K.-T.L.; Writing—original draft, W.-Y.K. and C.-Y.H.; Writing—review & editing, C.-Y.H., T.-Y.H. and L.-T.L.

Funding

This research was funded by Ministry of Economic Affairs (Small Business Innovation Research Program 1Z1030845), Ministry of Science and Technology (MOST105-2320-B-039-017-MY3 and MOST107-2320-B-039-029-MY3), and China Medical University (CMU104-H-01 and CMU104-H-02).

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the agarwoods are available from the authors.

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Supplementary Materials

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