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

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.


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

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).

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). . 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.

Chemical Profiles of Kynam Agarwoods Using HS GC/MS-MS
Figures 3 and 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%).
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. 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. 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%).

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 (Figures 5 and 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) 1,2benzenedicarboxylate, 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 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.

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.   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.   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].

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.