Article

# Distinguishing wild from cultivated agarwood (Aquilaria spp.) using direct analysis in real time and time of-flight mass spectrometry

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## Abstract

It is important for the enforcement of the CITES treaty to determine whether agarwood (a resinous wood produced in Aquilaria and Gyrinops species) seen in trade is from a plantation that was cultivated for sustainable production or was harvested from natural forests which is usually done illegally. We analyzed wood directly using Direct Analysis in Real Time (DART™) ionization coupled with Time-of-Flight Mass Spectrometry (TOFMS). Agarwood was obtained from five countries, and the collection contained over 150 samples. The spectra contained ions from agarwood-specific 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones as well as many other ions. The data was analyzed using either kernel discriminant analysis or kernel principal component analysis. Probability estimates of origin (wild vs cultivated) were assigned to unknown agarwood samples. Analysis of the DART-TOFMS data shows that many of the chromones found in cultivated and wild agarwood samples are similar; however, there is a significant difference in particular chromones that can be used for differentiation. In certain instances, the analysis of these chromones also allows inferences to be made as to the country of origin. Mass Mountaineer™ software provides an estimate of the accuracy of the discriminate model, and an unknown sample can be classified as cultivated or wild. Eleven of the thirteen validation samples (85%) were correctly assigned to either cultivated or wild harvested for their respective geographic provenance. The accuracy of each classification can be estimated by probabilities based on Z scores. The direct analysis of wood for the diagnostic chromones using DART-TOFMS followed by discriminant analysis is sufficiently robust to differentiate wild from cultivated agarwood and provides strong inference for the origin of the agarwood. Copyright © 2013 John Wiley & Sons, Ltd.

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... Quercus rubra L., Cody et al., 2012), between four species of agarwood (Aquilaria spp., Espinoza et al., 2014;Lancaster and Espinoza, 2012), ...
... This leads to the ionization and emissions of compounds from the wood, which then enter the mass analyser. Spectra were acquired in positive ion mode with DART ion source parameters settings as indicated in Lancaster and Espinoza (2012), Espinoza et al. (2014) and McClure et al. (2015). ...
... DART TOFMS for geographical provenancing is currently not an established forensic method. Although the technique in question can and has been tested for provenancing (see Espinoza et al., 2014;Finch et al., 2017 and Chapter 3), this requires finding small chemotype differences within a species based on a region and more exploratory research is needed. Currently there are no standards available for geographical provenancing (except for a few very specific cases), as it is still a very experimental approach. ...
... Novel methods for accurate wood identification have been intensively developed during the last two decades, such as DNA genotyping (Lemes et al. 2010;Tnah et al. 2010;Jolivet and Degen, 2012;Höltken et al. 2012, Degen et al. 2013Hartvig 2015), machine vision using morphological features (Hermanson and Wiedenhoeft 2011;Rosa da Silva et al. 2017;Hermanson 2017;Ravindran et al. 2018;Kobayashi et al. 2019), near-infrared (NIR) spectroscopy , Tsuchikawa et al. 2003Braga et al. 2011;Bergo et al. 2016), and DART-TOFMS and chemometry (Espinoza et al. 2014Deklerck et al. 2017Deklerck et al. , 2019Deklerck et al. , 2020Paredes-Villanueva et al. 2018). These modern methods to date have only been evaluated and applied on a limited number of species that do not include Afzelia spp. ...
... Nevertheless, extracting sufficient amount of DNA from wood products is currently challenging, time-consuming, and expensive. On the other hand, DART-TOFMS requires small (a few mm 3 ) slivers of heartwood specimens and gives an accurate profile of the metabolites of the sample (Espinoza et al. 2014. Moreover, the collection of DART-TOFMS data of wood is quick, cost effective, and relatively non-destructive. ...
... Once a statistically sufficient number of samples is analyzed, the resulting fingerprints show a common pattern that can be used to identify unknown samples (Musah et al. 2015). Metabolite profiles obtained by DART-TOFMS for wood identification are routinely used by the U.S. Fish and Wildlife Service to differentiate between CITES-listed and some look-alike timber species (Lancaster and Espinoza 2012;Espinoza et al. 2014Espinoza et al. , 2015McClure et al. 2015, Evans et al. 2017. ...
Article
• Key message Distinct chemical fingerprints of the wood of Afzelia pachyloba and A. bipindensis demonstrated an effective method for identifying these two commercially important species. Direct analysis in real-time (DART) time-of-flight mass spectrometry (TOFMS) allowed high-throughput examination of chemotypes with vast potential in taxonomic, ecological, and forensic research of wood. • Context Afzelia is a genus of valuable tropical timber trees. Accurate identification of wood is required for the prevention of illicit timber trade as well as for certification purposes in the forest and wood products industry. For many years, particular interest has been focused on attempts to distinguish the wood of A. bipindensis Harms from A. pachyloba Harms due to substantial differences in the commercial values of these two species.• Aims We investigated if wood chemical signatures and microscopy could identify the wood of A. bipindensis and A. pachyloba.• Methods We used two approaches, namely metabolome profiling by direct analysis in real-time (DART) time-of-flight mass spectrometry (TOFMS) and wood microstructure by light microscopy and SEM. In all, we analyzed samples from 89 trees of A. bipindensis, and A. pachyloba.• Results The two species could not be separated by the IAWA standard microscopic wood features. SEM analysis showed considerable variation in the morphology of vestured pits; however, this variation was not species-specific. In contrast, DART-TOFMS followed by unsupervised statistics (Discriminant Analysis of Principal Components) showed distinct metabolome signatures of the two species.• Conclusion DART-TOFMS provides a rapid method for wood identification that can be easily applied to small heartwood samples. Time- and cost-effective classification of wood chemotypes by DART-TOFMS can have potential applications in various research questions in forestry, wood science, tree-ecophysiology, and forensics.
... DART-TOFMS provides an instantaneous small molecule profile for solid samples in an open-air environment, removing the labor-intensive requirement of material preparation in chemical solvent and the potential for sample preparation biases (Cody et al., 2005;Cody, 2013). Differentiation provided by DART-TOFMS metabolite profiles has been used to discriminate wood from many closely related tree species (Cody et al., 2012;Espinoza, 2012a, 2012b;Espinoza et al., 2014Espinoza et al., , 2015. Due to rapid sample preparation (i.e., less than one minute per sample) and the classification accuracy of this method, DART-TOFMS is now used by the U.S. Fish and Wildlife Service to identify CITES-listed species in wood forensics cases, especially when anatomical identification is not possible (Lancaster and Espinoza, 2012a;Espinoza et al., 2014;McClure et al., 2015). ...
... Differentiation provided by DART-TOFMS metabolite profiles has been used to discriminate wood from many closely related tree species (Cody et al., 2012;Espinoza, 2012a, 2012b;Espinoza et al., 2014Espinoza et al., , 2015. Due to rapid sample preparation (i.e., less than one minute per sample) and the classification accuracy of this method, DART-TOFMS is now used by the U.S. Fish and Wildlife Service to identify CITES-listed species in wood forensics cases, especially when anatomical identification is not possible (Lancaster and Espinoza, 2012a;Espinoza et al., 2014;McClure et al., 2015). ...
... spp. wood specimens (Espinoza et al., 2014). These studies tested the ability of DART-TOFMS to discriminate differences at a large spatial scale (e.g., >500 km; Kim et al., 2015), but they did not directly address the ability of DART-TOFMS data to resolve fine-scale intraspecific provenances. ...
Article
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Premise of the study: We investigated whether wood metabolite profiles from direct analysis in real time (time-of-flight) mass spectrometry (DART-TOFMS) could be used to determine the geographic origin of Douglas-fir wood cores originating from two regions in western Oregon, USA. Methods: Three annual ring mass spectra were obtained from 188 adult Douglas-fir trees, and these were analyzed using random forest models to determine whether samples could be classified to geographic origin, growth year, or growth year and geographic origin. Specific wood molecules that contributed to geographic discrimination were identified. Results: Douglas-fir mass spectra could be differentiated into two geographic classes with an accuracy between 70% and 76%. Classification models could not accurately classify sample mass spectra based on growth year. Thirty-two molecules were identified as key for classifying western Oregon Douglas-fir wood cores to geographic origin. Discussion: DART-TOFMS is capable of detecting minute but regionally informative differences in wood molecules over a small geographic scale, and these differences made it possible to predict the geographic origin of Douglas-fir wood with moderate accuracy. Studies involving DART-TOFMS, alone and in combination with other technologies, will be relevant for identifying the geographic origin of illegally harvested wood.
... First such report was published by Hidayat et al. (2010) followed by series of other publications by Ismail et al. (Hidayat et al. 2010;Ismail et al. 2013a;Nor Azah et al. 2013). The methods of analysis includes Z-Score (Ismail et al. 2013a;Nor Azah et al. 2013), Artificial Neural Network ANN (Ismail et al. 2013b) using techniques such as electronic nose (E-nose) (Hidayat et al. 2010), gas chromatography (GC), gas chromatography/mass spectrometric (GC/MS), solid phase micro extraction (SPME) (Ismail et al. 2013c), gas chromatography-flame ionization detector (GC-FID), gas chromatography-olfactometry (GC-O) (Pripdeevech et al. 2011) and comprehensive two dimensional gas chromatography (GC 9 GC) and DART-TOF-MS (Espinoza et al. 2014). Different studies used different methods to identify the compounds which are responsible for the grading, quality and aroma of agarwood (Table 2). ...
... Several other researchers supported the presence of agarospirol as a chief constituent in the A. malaccensis (Meier et al. 2003;Tajuddin et al. 2013). Espinoza et al. (2014) used direct DART-TOFMS method to identify various chromones present in three different Aquilaria species. The reference collection for this study consisted of cultivated and wild samples from three different Aquilaria species obtained from four different geographic landscapes (Borneo, Hainan Island, Thailand, and Vietnam). ...
... The reference collection for this study consisted of cultivated and wild samples from three different Aquilaria species obtained from four different geographic landscapes (Borneo, Hainan Island, Thailand, and Vietnam). Difference in number and type of chromones present in samples and also kDa (Kernal Discriminant analysis) helps in distinguish wild and cultivated samples from the same geographical region, in addition to separating samples based on geographic landscape of origin (Espinoza et al. 2014). ...
Article
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Agarwood is a valuable oleoresin obtained from wounded tree of various genus of Thymelaeaceae family especially from Aquilaria species. Agarwood is regarded as most treasured non-timber forest product used in fragrances as well as medicines. Quality of agarwood plays an important role to define its commercial value. Different countries use different grading pattern to define the quality of agarwood. More than 250 compounds have been identified so far, mostly sesquiterpenoids, chromones and volatile aromatic compounds. GC–MS is the considered as a best method for the identification of chemical profile of the agarwood by various authors. Present review discusses the various methods used to determine quality of agarwood and chemical constituents of infected agarwood tree.
... The characteristic aroma of agarwood is attributable to a complex mixture of numerous volatile sesquiterpene compounds, including agarofurans, cadinanes, eudesmanes, valencanes, eremophilanes, guaianes, prezizanes, vetispiranes and agrospirol. In addition, the resin contains various 2-(2-phenylethyl) chromone derivates Espinoza et al. 2014;Ismail et al. 2014;Subasinghe and Hettiarachchi 2015;Sulaiman et al. 2015;Wong et al. 2015). ...
... The basic skeleton of all agarwood chromones is 2-(2-phenylethyl) chromone, and the base peak of m/z 91 is the phenyl cleavage of 2-(2-phenylethyl) chromone with no further substitution. Various instruments have been used to study 2-(2-phenylethyl) chromone and its derivatives as a major components group of agarwood, including LC-Hires-MS , HPLC-ESI-QTOF-MS (Li et al. 2016), GC-MS (Mei et al. 2013), and direct analysis in real time with time-of-flight mass spectrometry (DART-TOFMS; Lancaster and Espinoza 2012; Espinoza et al. 2014). Among 89 2-(2-phenylethyl) chromones identified, 33 highly oxidized chromones appeared to be unique to agarwood and not present in healthy Aquilaria specimens (Lancaster and Espinoza 2012). ...
... Among 89 2-(2-phenylethyl) chromones identified, 33 highly oxidized chromones appeared to be unique to agarwood and not present in healthy Aquilaria specimens (Lancaster and Espinoza 2012). Espinoza et al. (2014) further distinguished wild agarwood from cultivated agarwood (Aquilaria spp.) through DART-TOFMS. Mei et al. (2013) identified 41 2-(2-phenylethyl) chromones through GC-MS, an identification not previously reported by researchers who used other SPME-GC-MS methods (Nurlaila et al. 2016). ...
Article
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Agarwood is the most valuable resinous wood used in the production of products such as incenses, perfumes, and traditional medicines. However, such production has led to the destruction of wild Aquilaria forests. In the current study, an in vitro culture system was established by combining Aquilaria malaccensis with a quasi-symbiotic bacteria strain, A329, cultured on a medium designed for agarwood production. The quasi-symbiotic bacteria A329 derived from A. malaccensis agarwood has Bacillus-like characteristics, is low in phytopathogens, and can coculture with A. malaccensis with the current designed culture medium. The prolonged culture induced agarwood production at the base of A. malaccensis in vitro. Using high solid-phase microextraction gas chromatography-mass spectrometry, 23 compounds were tentatively identified, including several fingerprinting compounds, agarospirol, eudesmanes, guaianes, valencanes, selinenes, aristolone and 2-(2-phenylethyl) chromone. Using the in vitro system established in this study, the duration of agarwood resin formation can be shortened to 3 months in a well-controlled environment with 100% yield, constituting a promising platform with high commercial value for the selection of Aquilaria spp., the quasi-symbiotic microbes, and the combination of the two.
... Thus, wood metabolome profiling could potentially be used for timber species identification, wood fluorescence analysis and evaluation of the biological degradation of waterlogged or archeological wood samples. However, most previous studies on wood metabolome fingerprinting and profiling have focused on medicinal plants, especially agarwood (Espinoza et al. 2014). Due to their bioactive chemical components and potential as sources of important new drugs, studies on the metabolomes of medicinal plants have become common for the identification and quantification of bioactive metabolites from natural sources (Newman and Cragg 2012;De Combarieu et al. 2015), the quality control of medicinal plants and herbal medicines and combating counterfeit medicines (Wang et al. 2004;Li et al. 2015) and the discovery of metabolic biomarkers (Frédérich et al. 2010). ...
... Both polar and nonpolar small molecule metabolites in wood tissues can be detected by DART-MS. This technique is considered promising for the rapid classification of white oak and northern red oak (Cody et al. 2012), the analysis of selected Dalbergia and trade timber (Lancaster and Espinoza 2012a,b), evaluating and distinguishing agarwood products (Lancaster and Espinoza 2012a,b;Espinoza et al. 2014), the discrimination of selected Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)-protected Araucariaceae (Evans et al. 2017) and source identification of western Oregon Douglas-fir wood cores (Finch et al. 2017). ...
Article
Timber genus identification based on the anatomical features of wood is well established in botany. However, species-level wood identification is not always possible based on traditional wood morphology techniques alone. To compensate for the deficiencies of traditional methods, direct analysis in real time coupled to Fourier transform ion cyclotron resonance mass spectrometry (DART-FTICR-MS) was used to obtain the mass spectral fingerprints of different timber species. Using heartwood samples of two morphologically similar species, Pterocarpus santalinus and Pterocarpus tinctorius , subjected to different treatments, i.e. solvent extractions and powdered samples as well as air-dried samples and samples dried at low and high temperatures, we observed distinct chemical signatures for the wood samples from the two species, enabling rapid species-level identification when multivariate statistical analysis was adopted. The supervised orthogonal partial least squares discriminant analysis (OPLS-DA) models for samples subjected to different treatments all exhibited accurate differentiation performance of the explained fraction of variance of classes (R ² Y = 0.936–0.987) and the cross-validated fraction of variance of classes (Q ² = 0.857–0.949). Compared with solvent types and the physical form of the sample, the drying treatment method had a greater impact on the chemical fingerprint from DART-FTICR-MS. Air-dried wood chips were the optimal samples for the DART-FTICR-MS method coupled with statistical analysis.
... Chromones in agarwood have recently been found to be important for identification purposes and for their pharmacological effects. Analysis of 2-(2-phenylethyl) chromones is promising to distinguish cultivated from wild harvested agarwood, especially the highly oxidized 5,6,7,8-tetrahydro-2-(2phenylethyl) chromones (Espinoza et al. 2014). Some new chromones in agarwood show antitumor immunity (Suzuki et al. 2017), inhibition of innate and adaptive immunity (Guo et al. 2017) and anti-inflammatory properties (Zhu et al. 2016;Huo et al. 2017). ...
... Chromones in agarwood have recently received considerable attention for their importance in identification of quality (Espinoza et al. 2014), pharmacological effects (Zhu et al. 2016;Huo et al. 2017;Suzuki et al. 2017), chemical isolation (Liao et al. 2016(Liao et al. , 2017Xiang et al. 2017;Yang et al. 2017), and their biosynthesis pathways . The determination of chromones in agarwood is a reasonable option for quality control (Lancaster and Espinoza 2012). ...
Article
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Agarwood is the resinous heartwood of Aquilaria species. However, low yields and high costs of existing stimulation methods have led to the need for new techniques to produce agarwood rapidly and effectively. We developed a biological agarwood-inducing technique (Agar-Bit) that produces high yields and quality within 6 months. To better understand agarwood formation by Agar-Bit, dynamic gene expressions of key synthetases pathways of sesquiterpenes and chalcone- related enzymes at different times were determined after both Agar-Bit and the traditional burning chisel drilling (BCD) stimulation on Aquilaria sinensis trees. The qRT-PCR results show that some characteristic synthase genes were expressed at greatly different levels and times compared with the controls. For the Agar-Bit technology, main changes were after the 3rd or 5th month, while BCD expression clearly changed at the 5th month. Essential oils and total chromone contents were simultaneously determined. In the Agar-Bit group, both were higher and similar to natural levels. The Agar-Bit methodology is a new option for producing agarwood as demonstrated by genetic and chemical aspects. The differences in gene expression within 6 months for both groups indicates that the mechanisms of the two methods are different. These findings provide information on genetic variation during the process of agarwood formation.
... To improve species identification of ebony woods, we are currently applying a complementary chemical profiling technique based on DART TOFMS. This chemical profiling method uses species-specific wood chemical composition in order to identify species (Cody et al. 2005) and has been successfully applied to several tree genera, such as Aquilaria (Lancaster & Espinoza 2012;Espinoza et al. 2014), Dalbergia (Lancaster & Espinoza 2012;Espinoza et al. 2014), and Quercus (Cody et al. 2012). ...
... To improve species identification of ebony woods, we are currently applying a complementary chemical profiling technique based on DART TOFMS. This chemical profiling method uses species-specific wood chemical composition in order to identify species (Cody et al. 2005) and has been successfully applied to several tree genera, such as Aquilaria (Lancaster & Espinoza 2012;Espinoza et al. 2014), Dalbergia (Lancaster & Espinoza 2012;Espinoza et al. 2014), and Quercus (Cody et al. 2012). ...
Article
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The typical black coloured ebony wood ( Diospyros , Ebenaceae) is desired as a commercial timber because of its durable and aesthetic properties. Surprisingly, a comprehensive wood anatomical overview of the genus is lacking, making it impossible to fully grasp the diversity in microscopic anatomy and to distinguish between CITES protected species native to Madagascar and the rest. We present the largest microscopic wood anatomical reference database for ebony woods and reconstruct evolutionary patterns in the microscopic wood anatomy within the family level using an earlier generated molecular phylogeny. Wood samples from 246 Diospyros species are described based on standardised light microscope observations. For the ancestral state reconstruction, we selected eight wood anatomical characters from 88 Ebenaceae species (including 29 Malagasy Diospyros species) that were included in the most recently reconstructed family phylogeny. Within Diospyros , the localisation of prismatic crystals (either in axial parenchyma or in rays) shows the highest phylogenetic value and appears to have a biogeographical signal. The molecular defined subclade Diospyros clade IX can be clearly distinguished from other ebony woods by its storied structure. Across Ebenaceae, Lissocarpa is distinguishable from the remaining genera by the combined presence of scalariform and simple vessel perforation plates, and Royena typically has silica bodies instead of prismatic crystals. The local deposition of prismatic crystals and the presence of storied structure allow identifying ebony wood species at the subgeneric level, but species-level identification is not possible. In an attempt to improve the identification accuracy of the CITES protected Malagasy woods, we applied computer vision algorithms based on microscopic images from our reference database (microscopic slides from ca. 1000 Diospyros specimens) and performed chemical profiling based on DART TOFMS.
... As a precious spice, agarwood is not only widely respected by global religions such as Buddhism and Christianity but also valuable for health care and collection. As a traditional medicine, this resincontaining wood can be used to make antiemetics, sedatives and digestive drugs (Naef, 2011;Xu et al., 2013;Espinoza et al., 2014). However, the current endangered status of wild agarwood resources and the low efficiency of artificial production makes it difficult to meet market demand (Zhang et al., 2010;Espinoza et al., 2014;Siah et al., 2016). ...
... As a traditional medicine, this resincontaining wood can be used to make antiemetics, sedatives and digestive drugs (Naef, 2011;Xu et al., 2013;Espinoza et al., 2014). However, the current endangered status of wild agarwood resources and the low efficiency of artificial production makes it difficult to meet market demand (Zhang et al., 2010;Espinoza et al., 2014;Siah et al., 2016). ...
... For DART TOMFS, there are few studies demonstrating origin analysis; however, since exogenous factors influence the chemical fingerprint, the origin might be determined based on this variation. Wild agarwood was distinguished from cultivated agarwood (Aquilaria spp.) based on single chemical fingerprints per specimen, with inferences to a geographical region between China, Borneo, Vietnam, and Thailand [23]. DART TOFMS was also used for classifying multiple sampling sites within Bolivia for two Cedrela species but this proved to be difficult, likely due to the limited sample number within the proposed set-up [24]. ...
... Relative abundances of ions appear to contribute more to country differentiation compared to frequency differences. This study further complements work showing that mass spectral data can be used for distinguishing individuals from disparate geographic regions (see Finch et al., 2017 andEspinoza et al., 2014). This study is a proof-of-concept, to assess the potential of growth ring chemical fingerprints to distinct individuals and geographical provenances. ...
Article
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Background and Objectives: The origin of traded timber is one of the main questions in the enforcement of regulations to combat the illegal timber trade. Substantial efforts are still needed to develop techniques that can determine the exact geographical provenance of timber and this is vital to counteract the destructive effects of illegal logging, ranging from economical loss to habitat destruction. The potential of chemical fingerprints from pith-to-bark growth rings for individual comparison and geographical provenance determination is explored. Materials and Methods: A wood sliver was sampled per growth ring from four stem disks from four individuals of Pericopsis elata (Democratic Republic of the Congo) and from 14 stem disks from 14 individuals of Terminalia superba (Côte d'Ivoire and Democratic Republic of the Congo). Chemical fingerprints were obtained by analyzing these wood slivers with Direct Analysis in Real Time Time-Of-Flight Mass Spectrometry (DART TOFMS). Results: Individual distinction for both species was achieved but the accuracy was dependent on the dataset size and number of individuals included. As this is still experimental, we can only speak of individual comparison and not individual distinction at this point. The prediction accuracy for the country of origin increases with increasing sample number and a random sample can be placed in the correct country. When a complete disk is removed from the training dataset, its rings (samples) are correctly attributed to the country with an accuracy ranging from 43% to 100%. Relative abundances of ions appear to contribute more to differentiation compared to frequency differences. Conclusions: DART TOFMS shows potential for geographical provenancing but is still experimental for individual distinction; more research is needed to make this an established method. Sampling campaigns should focus on sampling tree cores from pith-to-bark, paving the way towards a chemical fingerprint database for species provenance.
... In particular, resources with specific traits are generally most attractive to poachers (Jachmann et al., 1995;Cotterill, 2005;Steinmetz et al., 2010;Gong et al., 2017;Marin et al., 2018), and this is also evident for natural resources (Challender and MacMillan, 2014;Liu et al., 2019a). Poaching from natural, uncultivated resources is more prevalent because traditionally, people believe that individuals grown in nature contain more specific properties or compounds than cultivated ones (Espinoza et al., 2014;Liu et al., 2019a), and thus, the former sell for higher prices than the latter. Flaunting wealth by buying natural-origin products is another way of incentivizing poaching from nature. ...
... Poaching natural plants, animals, and their products can negatively affect population sizes and the survival of species that are protected. If these stolen resources are illegally traded, then pinpointing their origins (sites, populations, herds, breeds, or varieties, etc.) and revealing their genetic background (cultivated vs. natural) (Manel et al., 2002;Espinoza et al., 2014;Huang et al., 2014;Hoelzel, 2015;Pimm et al., 2015;Wasser et al., 2015;Ng et al., 2017;Marin et al., 2018;Liu et al., 2019a) are important for local resource conservation. In this study, we used both microsatellite and SNP markers to study the genetic background of illegally traded A. sinensis trees. ...
Article
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Because of high market demand, agarwood-producing trees are frequently illegally traded in Asia and are usually priced high if they are cut from natural forests. In China, some of these traded Aquilaria sinensis trees are declared to be from natural population(s) and are said to produce agarwood “easily and fast”. To distinguish the origins and genetic differences of natural populations from cultivated ones, we used genetic markers, microsatellites and single nucleotide polymorphisms (SNP) to compare them to trees from both cultivated and known natural origin trees. Our microsatellite results revealed that the illegally traded trees were genetically close to cultivated trees, indicating that they were of cultivated origin. By separating SNPs into genic (in genic regions) and nongenic categories representing functional and non-functional SNPs, our results revealed that the genic SNP markers did not detect more genetic differences between the illegally traded A. sinensis trees and cultivated ones than the nongenic SNP markers did, indicating that they are not functionally discernable from the cultivated trees. Our study revealed that sources labelled as natural by poachers might not have natural origins, which is especially true for agarwood-producing species given their limited natural populations and their long and extensive cultivation history. Our results may reduce the public’s desire for natural agarwood from A. sinensis and other agarwood-producing species and benefit the legal agarwood trade.
... 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]. ...
... 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. ...
Article
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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.
... Researchers from Japan revealed that agarwood is a mixture of monoterpenes, sesquiterpenes and its chromone derivatives (Ishihara, Tsuneya, & Uneyama, 1993;Espinoza et al., 2014). In their studies, Gas Chromatography -Mass Spectrometry (GC-MS) technique was used to characterise the chemical composition of agarwood high (Kanankoh) and low (Jinkoh) quality. ...
... In 2013, Espinanoza et al. found 2-(2-phenylethyl) chromones using direct analysis in real time and time of-flight mass spectrometry (DART-TOFMS). On the other hand, dry Gas -Chromatography (GC) and preparative Thin Layer Chromatograph (TLC) were performed by Yoshii et al. and found out that agarospirol, agarol and agarofurans in their agarwood samples (Espinoza et al., 2014). Several researchers agreed that Gas Chromatography -Flame Ionization Detector (GC-FID) and GC-MS are the most common analytical methods to identify the agarwood compounds (Marriott, Shellie, & Cornwell, 2001;Pripdeevech, Khummueng, & Park, 2011;Tajuddin & Yusoff, 2010). ...
Article
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Agarwood or gaharu, due to its unique scent and quality is considered one of the most expensive wood in the world. This paper uses solvent trap, Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) to establish agarwood quality, and is part of ongoing research. The result showed that monoterpenes hydrocarbon, sesquiterpenes hydrocarbon and oxygenated sesquiterpene contribute to agarwood smoke. Among many compounds extracted, 2-hydrdoxy-benzaldehyde, β-selinene and guaia-1(10),11-dien-15,2-olide were found dominant in monoterpenes hydrocarbon, sesquiterpenes hydrocarbon and oxygenated sesquiterpenes respectively. This finding pointed to the utility of solvent trap in extracting chemical compounds of agarwood smoke. This study is also useful for further research on establishing the grade of agarwood.
... (Cody et al. 2012) and Aquilaria spp. (Lancaster & Espinoza, 2012b;Espinoza et al. 2014). Here we hypothesize that DART TOFMS will be able to identify selected Araucariaceae wood samples including the wood of the critically endangered rare "living fossil" Wollemia nobilis. ...
... alba L.), Dalbergia spp. (Wiemann & Espinoza 2017) and samples of wild and cultivated agarwood (Aquilaria spp.) (Cody et al. 2012;Lancaster & Espinoza 2012a,b;Espinoza et al. 2014Espinoza et al. , 2015McClure et al. 2015). Softwoods are "more uniform in their anatomy than hardwoods and present many more problems of accurate identification" (Gasson 2011). ...
Article
Determining the species source of logs and planks suspected of being Araucaria araucana (Molina) K.Koch (CITES Appendix I) using traditional wood anatomy has been difficult, because its anatomical features are not diagnostic. Additionally, anatomical studies of Araucaria angustifolia (Bertol.) Kuntze, Araucaria heterophylla (Salisb.) Franco, Agathis australis (D.Don) Lindl., and Wollemia nobilis W.G.Jones, K.D.Hill & J.M.Allen have reported that these taxa have similar and indistinguishable anatomical characters from A. araucana. Transnational shipments of illegal timber obscure their geographic provenance, and therefore identification using wood anatomy alone is insufficient in a criminal proceeding. In this study we examine the macroscopic appearance of selected members of the Araucariaceae and investigate whether analysis of heartwood chemotypes using Direct Analysis in Real Time (DART) Time-of-Flight Mass Spectrometry (TOFMS) is useful for making species determinations. DART TOFMS data were collected from 5 species (n =75 spectra). The spectra were analyzsed statistically using supervised and unsupervised classification algorithms. Results indicate that A. araucana can be distinguished from the look-alike taxa. Another statistical inference of the data suggests that Wollemia nobilis is more similar and within the same clade as Agathis australis. We conclude that DART TOFMS spectra can help in making species determination of the Araucariaceae even when the geographic provenance is unknown.
... Direct Analysis in Real Time (DART) (see Cody et al. [6] ) Time-Of-Flight Mass Spectrometry (TOFMS) has shown promise in the analysis of wood and plants. Previous research using DART TOFMS spectra was able to distinguish between two species of American oak (Quercus alba L. and Quercus rubra L.) [7] , between four species of agarwood (Aquilaria spp.) [8,9] , and between Dalbergia timbers from Africa, Madagascar and Asia. [10] Recent research focused on the identification of plant species (Mitragyna speciosa (Korth.) ...
... This gas has a temperature of 350°C leading to ionization and the emission of compounds from the wood entering the mass analyser. Spectra were acquired in positive ion mode with the DART ion source parameters and mass spectrometer settings as defined in Evans et al. [12] , McClure et al. [10] , Lancaster & Espinoza [8] and Espinoza et al. [9] The text files of the mass-calibrated, centroided mass spectra were exported using TSS Unity (Shrader Software Solutions, Inc., Grosse Poine Park, MI, USA) data reduction software and used for further analysis. ...
Article
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Rationale: The genus Pericopsis includes four tree species of which only Pericopsis elata (Harms) Meeuwen is of commercial interest. Enforcement officers might have difficulties discerning this CITES-listed species from some other tropical African timber species. Therefore, we tested several methods to separate and identify these species rapidly in order to enable customs officials to uncover illegal trade. In this study, two classification methods using DART TOFMS data to discern between several species are presented. Methods: Metabolome profiles were collected using Direct Analysis in Real Time (DART(TM) ) ionization coupled with Time-of-Flight Mass Spectrometry (TOFMS) analysis of heartwood specimens of all four Pericopsis species and Haplormosia monophylla (Harms) Harms, Dalbergia melanoxylon Guill. & Perr. Harms, and Milicia excelsa (Welw.) C.C.Berg. In total, 95 specimens were analysed and the spectra evaluated. Kernel Discriminant Analysis (KDA) and Random Forest classification were used to discern the species. Results: DART TOFMS spectra obtained from wood slivers and post-processing analysis using KDA and Random Forest classification separated Pericopsis elata from the other Pericopsis taxa and its lookalike timbers Haplormosia monophylla, Milicia excelsa, and Dalbergia melanoxylon. Only 50 ions were needed to achieve the highest accuracy. Conclusions: DART TOFMS spectra of the taxa were reproducible and the results of the chemometric analysis provided comparable accuracy. Haplormosia monophylla was visually distinguished based on the heatmap and was excluded from further analysis. Both classification methods, KDA and Random Forest, were capable of distinguishing Pericopsis elata from the other Pericopsis taxa, Milicia excelsa, and Dalbergia melanoxylon timbers that are commonly traded.
... This approach is rapid, but generally incapable of identifying trees to species or predicting their geographic origin [6,7]. Chemical [8,9] and genetic [10,11] approaches are increasingly used to provide more accurate species identifications [4], but determining geographic origin continues to be a daunting task [12][13][14][15]. Here, we demonstrate an efficient use of next generation sequencing (NGS) data to predict the geographic source of white oak species (Quercus subg. ...
Article
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Background The application of genomic data and bioinformatics for the identification of restricted or illegally-sourced natural products is urgently needed. The taxonomic identity and geographic provenance of raw and processed materials have implications in sustainable-use commercial practices, and relevance to the enforcement of laws that regulate or restrict illegally harvested materials, such as timber. Improvements in genomics make it possible to capture and sequence partial-to-complete genomes from challenging tissues, such as wood and wood products. Results In this paper, we report the success of an alignment-free genome comparison method, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${d}_2^{\ast },$$\end{document}d2∗, that differentiates different geographic sources of white oak (Quercus) species with a high level of accuracy with very small amount of genomic data. The method is robust to sequencing errors, different sequencing laboratories and sequencing platforms. Conclusions This method offers an approach based on genome-scale data, rather than panels of pre-selected markers for specific taxa. The method provides a generalizable platform for the identification and sourcing of materials using a unified next generation sequencing and analysis framework. Electronic supplementary material The online version of this article (10.1186/s12864-018-5253-1) contains supplementary material, which is available to authorized users.
... This results in a unique chemical pattern based on secondary metabolites (metabolic or chemical fingerprint), which is used to identify the species in question. DART-TOFMS has proven to be successful to discern between several timber species (Deklerck et al. 2017;Espinoza et al. 2014;Lancaster and Espinoza 2012;McClure et al. 2015;Musah et al. 2015) or even keratin types (Price et al. 2018). However, little is known about the effect of parameter settings in the data processing on species classification accuracy. ...
Article
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Using chemical fingerprints for timber species identification is a relatively new, but promising technique. However, little is known about the effect of pre-processing spectral data parameter settings on the timber species classification accuracy. Therefore, this study presents an extensive and automated analysis method using the random forest machine learning algorithm on a set of highly valuable timber species from the Meliaceae family. Metabolome profiles were collected using direct analysis in real-time (DART™) ionisation coupled with time-of-flight mass spectrometry (TOFMS) analysis of heartwood specimens for 175 individuals (representing 10 species). In order to analyse variability in classification accuracy, 110 sets of data pre-processing parameter combinations consisting of mass tolerance for binning and relative abundance cut-off thresholds were tested. Furthermore, for each set of parameters (designated “binning/threshold setting”), a random search for one hyperparameter of interest was performed, i.e. the number of variables (in this case ions) drawn randomly for each random forest analysis. The best classification accuracy (82.2%) was achieved with 47 variables and a binning and threshold combination of 40 mDa and 4%, respectively. Entandrophragma angolense is mostly confused with Entandrophragma candollei and Khaya anthotheca, and several Swietenia species are confused with each other due to the high similarity of their chemical fingerprints. Entandrophragma cylindricum, Entandrophragma utile, Khaya ivorensis, Lovoa trichilioides and Swietenia macrophylla are easy to discriminate and show less misclassifications. The choice of parameter settings, whether it is in the data pre-processing (binning and threshold) or classification algorithm (hyperparameters), results in variability in classification accuracy. Therefore, a preliminary parameter screening is proposed before constructing the final model when using the random forest algorithm for classification. Overall, DART-TOFMS in combination with random forest is a powerful tool for species identification.
... The presence of the diagnostic TPEC ions at m/z 349.129 or at m/z 319.118, are characteristic of the PECs [215]. Interestingly, cultivated agarwood and wild agarwood samples showed differences in PECs, which helped to distinguish the wild agarwood from cultivated one [216]. The characteristic fragmentation behaviors of FPECs, and cleavage of the CH 2 -CH 2 bond between the chromone moiety and phenyl moiety are used to calculate the number of methoxy or hydroxy groups, which enabled the identification of FPECs [202]. ...
Article
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Agarwood is a resin-impregnated heartwood obtained from the plants belongs to the genera, Aquilaria, Daphne, Gonystylus, Gyrinops and Wikstroemia. It is traditionally used for the production of perfume and incense stick, and pharmaceutical applications. Agarwood usually induced by the natural (traditional), conventional, and non-conventional methods. The major groups of phytochemicals identified in agarwood extracts are sesquiterpenes, 2-(2-phenylethyl)-4H-chromen-4-one derivatives (PECs), and aromatic compounds. These phytochemicals are showed various pharmalogical properties such as anti-inflammatory, cytotoxic, neuroprotective, anti-diabetic, anti-bacterial, etc. Several analytical techniques are applied to analyze the agarwood phytochemicals including sesquiterpenes, which exists mostly in the form of essential oils, and the fragrance constituents of PECs. The present review summarize the agarwood traditional uses, induction methods, phytochemical constituents, potential pharmacological activities, along with analyses methods. This review was carried out by searching various scientific databases, including Google Scholar, PubMed, Elsevier, ACS publications, Taylor and Francis, Wiley Online Library, MDPI, Springer, Thieme, and ProQuest. The present review provides a scientific basis for future studies and necessary information for the development of agarwood based therapeutic agents.
... Several analytical techniques have also been entertained as a means of separating Dalbergia species, including spectroscopy (mid and near infra-red, and nuclear magnetic resonance spectroscopy), and gas chromatography/mass spectrometry (MS) [7][8][9][10][11] and other forms of spectrometry, most notably, Direct Analysis in Real Time (DART) Time-of-Flight MS (DART/ToF). DART/ToF (AccuToF TM of JEOL Inc., hereafter JOEL ToF) is the only MS technique that has been widely tested, and it is currently accepted as the method of choice for identifying Dalbergia and other CITES listed species [12][13][14][15][16][17][18][19] . DART/JEOL ToF is not without its drawbacks, however, including potential interference by wood treatments (coatings and biocides), inability to automate analysis and, most importantly, the fact that the current DART/JEOL ToF mass spectral library derived from numerous authentic wood samples cannot be easily transferred to other ToF MS instruments due to the nature of automorphic ionization of the DART design. ...
Article
The international trade in illegally logged and environmentally endangered timber has spurred enforcement agencies to seek additional technical procedures for the identification of wood species. All Dalbergia species are listed under the Convention on International Trade in Endangered Species (CITES) which is the reason this genus was chosen for study. Multiple sources of the heartwood from different Dalbergia species were extracted and chromatographic profiles collected by gas chromatography with high resolution quadrupole Time of Flight mass spectrometry (GC/QToF). The collected data was mined to select peaks and mass ions representative of the investigated Dalbergia species, and used to develop a Microsoft Excel® template offering immediate graphical representation of the results. Using wood specimens sourced from different xylaria, this graphical fingerprint proved adept at definitive identification of Dalbergia species. The CITES Appendix I species, D. nigra, was easily distinguished from D. melanoxylon and look-alike species of other genera. Similarly, a number of other Dalbergia species were differentiated using this current approach. Kernel discrimination analysis (KDA) was applied to increase the confidence of the species identification. The mislabeling of specimens appears to be common, and the emerging technique of GC/QToF in combination with other techniques, offers improved confidence in identification. GC/QToF further provides automation, the dimension of chromatography to avoid interferences, and production of reproducible electron impact positive (EI+) spectra. The prospect of building an EI+ spectral database for future wood identification is an important feature considering the limited accessibility of authenticated wood species specimens.
... While epi-γ-Eudesmol, Jinkoh-eremol, and β-agarofuranare do not widely exist but can only be found in A. malaccensis (Fig. 10.4) (Naef 2011;Chen et al. 2012;Hashim et al. 2016). The other "main indicator" of gaharu oil is 2-(2-phenylethyl) chromium, which has been identified in different forms of 39 2-(2-phenylethyl) from different agarwood (Naef 2011;Lancaster and Espinoza 2012;Espinoza et al. 2014). Notably, the method of induction can directly affect the quality of agarwood. ...
Chapter
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Endophytic fungi are organisms that reside asymptomatically in the tissues of higher plants without initiating any disease or overt negative symptoms in the host plants. This type of fungus acts as a biostimulant and uses living plant cells as a biofactory for the production of different desirable products. While promoting the production of secondary metabolites in host plants, endophytic fungi can also contribute to improving the development, fitness, and tolerances of host plants against abiotic and biotic stresses. However, their diverse distributions and populations are influenced by several factors, for example, genetic conditions, age, and the environmental conditions of their hosts. Nowadays, different types of endophytic fungi are currently used as biological agents to induce the formation of resinous substances in plants as a response to fungal infections, such as in the case of Aquilaria trees. Agarwood or resin wood is an important high-value product that is synthesized naturally as a result of certain fungal infections of Aquilaria trees. This resin is a high-value component in the manufacture of essential oils and perfumes. This review provides up-to-date information about the process and mechanism of agarwood resin production in Aquilaria trees as well as the best practices used in the sustainable production of this high-value product in Southeast Asia.
... Paredes-Villanueva et al. (2018) suggested the influence of local conditions such as climate, soil characteristics, nutrient availability on the chemical composition of the trees may explain these classification errors. Espinoza et al. (2014) discerned wild from cultivated agarwood (Aquilaria sinensis, Aquilaria crassna and Aquilaria beccariana) with inference to country (Borneo, China, Thailand and Vietnam). Eleven of the 13 samples (85%) were correctly assigned to either cultivated or wild harvested for their respective geographic provenance. ...
Article
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Illegal logging and illegal timber trade is a global problem. Anatomical, genetic, and chemical techniques support illegal logging legislation by verifying the species and geographic origin of timber. In principle, these methods can be used to identify timber species and the origin of harvest, however, the availability of specific tests for important timber species is unclear. We review the status of these methods for the top 322 global priority timber taxa. Our results show that for species identification, reference data exist for 100% of taxa using wood anatomy, 86% using genetics, 41% for using DART TOFMS, and 6% using NIRS. For origin identification, data exist for 24% of taxa, with most studies applying genetic approaches (23%). No studies have developed forensic-ready tests for the global priority timber taxa. The review highlights that the current potential for identifying species is greater than for geographic origin and more research focused on determining the geographical origin of timber is required. Based on the current rate, it will take approx. 27 years to generate geographic data for all 322 priority taxa. Finally, we identify research opportunities to improve global timber tracing efforts. Our findings indicate more research is needed, and quickly so that scientific verification can support regulators to combat illegal logging.
... However, the isolation of these markers requires prior knowledge on genetic information of the target species (Vieira et al 2016), the cost of which remains a hurdle for economically weaker parts of the world that harbour many of the threatened species. Moreover, challenges in the collection of adequate samples for marker development further hampers this endeavour (Espinoza et al. 2014). ...
Article
Tree species in the Aquilarieae tribe of the Thymelaeaceae family produce agarwood, a natural product highly valued for its fragrance, but the species are under threat due to indiscriminate harvesting. For conservation of these species, molecular techniques such as DNA profiling have been used. In this study, we assessed cross-amplification of microsatellite markers, initially developed for three Aquilaria species (A. crassna, A. malaccensis, and A. sinensis), on ten other agarwood-producing species, including members of Aquilaria (A. beccariana, A. hirta, A. microcarpa, A. rostrata, A. rugosa, A. subintegra, and A. yunnanensis) and Gyrinops (G. caudata, G. versteegii, and G. walla), both from the Aquilarieae tribe. Primers for 18 out of the 30 microsatellite markers successfully amplified bands of expected sizes in 1 sample each of at least 10 species. These were further used to genotype 74 individuals representing all the 13 studied species, yielding 13 cross-amplifiable markers, of which only 1 being polymorphic across all species. At each locus, the number of alleles ranged from 7 to 23, indicating a rather high variability. Four markers had relatively high species discrimination power. Our results demonstrated that genetic fingerprinting can be an effective tool in helping to manage agarwood genetic resources by potentially supporting the chain-of-custody of agarwood and its products in the market.
... Li et al. [12] have constructed fourteen chromone derivatives using UPLC-ESI-QTOF-MS and multivariate statistical methods, and have presented results that may be useful for distinguishing between natural and arti cial agarwood. Espinoza et al. [13] have employed the direct analysis in real time (DART TM ) and time-of-ight mass spectrometry (TOF-MS) to distinguish between natural and arti cial agarwood based on the composition of 2-(2-phenylethyl)chromone derivatives. Ismail et al. [14] have established the PLS-DA and Random Forests Classi cation Models using 1 H-NMR-based metabolomics and employed them to discriminate between different gaharu with different grades. ...
Preprint
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Background: Agarwood is widely used as a traditional medicine all over the world. Distinction between the qualities of natural and artificial agarwood is a current hot research topic among agarwood research communities. An important sensory characteristic of agarwood lies in its incense smoke, and an analysis of incense smoke has been traditionally used to evaluate the agarwood quality since ancient times. The aim of this study is to establish a rapid detection method using electronic nose (E-nose) systems to distinguish between natural and artificial agarwood. Result: Incense smokes of 45 natural and artificial agarwood samples were analyzed by E-nose, and principal component analysis (PCA) was employed to cluster the E-nose data. The chemical markers which could be used to distinguish between artificial and natural agarwood were identified by GC-MS combined with information value and decision tree algorithm. The results showed that the smellprints of artificial agarwood contained more peaks, while those of natural agarwood had higher response intensities. The compounds that were different between the two types of agarwood were three sesquiterpenes and six chromone derivatives. The result from decision tree algorithm further showed that 6-hydroxy-2-(2-phenylethyl)chromone was the chemical marker that could be used to distinguish between artificial and natural agarwood. Nootkatone and 2-(2-phenylethyl)chromone were the chemical markers that may contributed to the clustering of the E-nose data; the two compounds can be used to evaluate the incense smoke of agarwood. Conclusion: We demonstrated that our developed E-nose-based method could rapidly distinguish between the incense smokes of artificial and natural agarwood; this method could be applied to evaluate the quality of agarwood in the future.
... Consequently, it leads up to a search for substitution/different diagnostic methods (Dormontt et al. 2015). Some of the developed methods for the identification of the species with high success rates are as follows: application of multivariate data analysis to wood anatomy (Clark 2003, Hellberg and Carcaillet 2003, Evans et al. 2008, Gasson and MacLachlan 2010, Turhan and Serdar 2013, Giménez et al. 2014, Marques et al. 2015, Esteban et al. 2017; machine vision, which allows wood anatomy experts to teach the necessary information to computer-based camera systems to distinguish tree species with similar characteristics (Hermanson and Wiedenhoeft 2011, Ravindran et al. 2018, de Andrade et al. 2020; DNA barcoding, which allows us to work on very small samples but can take up to several days (Jiao et al. 2014); and application of mass spectroscopy based on extractives content of wood (Espinoza et al. 2014, Deklerck et al. 2017, Evans et al. 2017). In addition to these studies, another prominent solution proposal is the use of near-infrared spectroscopy (NIRS) with chemometric methods, which evaluates the data obtained by analysing the chemical structure of the wood with statistical approaches, multivariate analysis (Pasquini 2003, Brereton 2007, Burns and Ciurczak 2008, Tsuchikawa and Kobori 2015. ...
Article
Identification of wood species with fast, reliable and non-destructive methods is highly important for forestry and wood-related industries. Near-infrared spectra of anatomically similar pine species (Pinus sylvestris L. and Pinus nigra J.F. Arnold) were taken and analysed by partial least squared discriminant analysis (PLS-DA) for comparing the efficiency of preprocessing methods. Raw data were subjected to multiple scatter correction (MSC), standard normal variate (SNV), Savitzky–Golay for derivatives (1st and 2nd Dr) and smoothing (Sm) and combination of these preprocessing methods (1st Dr, 1st Dr + SNV, 1st Dr + MSC, Sm + 1st Dr and Sm + 2nd Dr). The success of the models was determined by the accuracies of test sets that did not participate in the calibration phase. In this study, it was determined that not all the preprocessing methods improve the model performance. Smoothing with 1st derivatives (Sm + 1st Dr) enhanced 14.3% improvement and have the best performance (95%) for classification of pine species. For understanding modelled relationship, mean spectra and selectivity ratio were used. It was found that discrimination was held by the differences at their absorption, and the most important variables for wood classification were noted around 4000–7000 cm⁻¹. https://www.tandfonline.com/eprint/WZGH2QSZEZKSA6TBMZQN/full?target=10.1080/17480272.2021.2012821
... Cold treatment with or without brine induced six 2-(2-phenylethyl) chromones and derivatives which is contrary to the report of Naziz et al. [54]. These 2-(2-phenylethyl) chromones and derivatives have received much attention in recent years due their role in influencing quality of agarwood [57], chemical isolation [5,[58][59][60], pharmacological effects [61][62][63] and their biosynthesis pathways [19,28]. In all, nine 2-(2-phenylethyl) chromones and derivatives were detected in F while four, six and one were detected in FS, D and DS, respectively (Fig. 7). ...
Article
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Background Agarwood is a highly sought-after resinous wood for uses in medicine, incense, and perfume production. To overcome challenges associated with agarwood production in Aquilaria sinensis , several artificial agarwood-induction treatments have been developed. However, the effects of these techniques on the metabolome of the treated wood samples are unknown. Therefore, the present study was conducted to evaluate the effects of four treatments: fire drill treatment (F), fire drill + brine treatment (FS), cold drill treatment (D) and cold drill + brine treatment (DS)) on ethanol-extracted oil content and metabolome profiles of treated wood samples from A. sinensis . Results The ethanol-extracted oil content obtained from the four treatments differed significantly (F < D < DS < FS). A total of 712 metabolites composed mostly of alkaloids, amino acids and derivatives, flavonoids, lipids, phenolic acids, organic acids, nucleotides and derivatives, and terpenoids were detected. In pairwise comparisons, 302, 155, 271 and 363 differentially accumulated metabolites (DAM) were detected in F_vs_FS, D_vs_DS, F_vs_D and FS_vs_DS, respectively. The DAMs were enriched in flavonoid/flavone and flavonol biosynthesis, sesquiterpenoid and triterpenoid biosynthesis. Generally, addition of brine to either fire or cold drill treatments reduced the abundance of most of the metabolites. Conclusion The results from this study offer valuable insights into synthetically-induced agarwood production in A. sinensis .
... The application of high-resolution Direct Analysis in Real Time Time-of-Flight Mass Spectrometry (DART-TOFMS) for timber species identification has shown it to be a fast and robust method that requires little to no sample preparation. Chemical fingerprinting with DART-TOFMS can confidently classify timber to species-level [30,31], and the utility of chemical fingerprinting of heartwood for provenance is emerging [32][33][34][35]. There is clear potential for its use in the field of identifying timber provenance and more studies are required to understand the limitations of current methods for future development. ...
Article
In Pterocarpus, three out of 46 tree species (P. erinaceus, P. santalinus, and P. tinctorius) are listed as endangered and protected under Appendix II of CITES, and the status of several Pterocarpus species suggests that more will find a place on CITES in the future. As unsustainable forest exploitation has increased, regulations for timber traceability have also increased with the creation of such laws as the 2005 Lacey Act in the United States (Lacey Act, 2005) and the European Union Timber Regulation (EUTR) in 2013. These laws were implemented to ensure that members of the timber trade operated in accordance with guidelines and practiced due diligence, with the goal of halting illegal timber imports (FAO, 2016). Unfortunately, the illegal timber trade remains prevalent and has progressed into the third largest transnational crime (Mavrellis, 2017). To combat the prevalence of illegal timber, researchers have utilized Direct Analysis in Real Time Time-of-Flight Mass Spectrometry (DART-TOFMS). With the success found using DART-TOFMS for species identification, we investigated whether the method could be used to identify the original geographic region of timber through analysis of 211 samples taken from 12 Pterocarpus species taken from the Neotropics, Africa, South Asia, and Southeast Asia. We found that analysis using DART-TOFMS could identify the broad geographical provenances of the timber with an accuracy of 98%. This research demonstrated that DART-TOFMS can be used to detect the phytochemical trends that exist between timber species occupying broad geographic regions.
... PCA is an unsupervised learning algorithm that does not make use of the information about class membership and is a good method to determine whether there are real patterns in the data. 28 Additionally, OPLS-DA is a supervised learning algorithm that uses the class membership information to achieve the greatest separation between classes in the training set, the procedure was used for cross validation when fitting to decide the significance of a component. 29 Results of OPLS-DA and PCA of the data obtained from the mass spectra of 62 agarwood samples are shown in Figure 3 and Supporting Information, respectively. ...
Article
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Introduction Agarwood is a highly valuable fragrant resinous wood which is widely used as traditional Chinese medicines, perfumes, incense and decorations. Due to its high economic value and excessive demand, this leads to a rising price and proliferation of fake commodities. Thus, strict authenticity identification and quality evaluation of agarwood are of great significance. Objective To establish a simple, rapid and non‐destructive technique for identifying the authenticity of agarwood. Methods Liquid extraction surface analysis mass spectrometry (LESA‐MS) was firstly proposed to identify the authenticity of 62 agarwood samples without sample preparation. In addition, multivariate statistical models and thin‐layer chromatography (TLC) method were used to analyse and verify the results of LESA‐MS. Results Representative compounds of agarwood were detected by LESA‐MS. A characteristic 2‐(2‐phenylethyl)chromone compound (m/z 319.1) was treated as a key chemical marker to identify agarwood and its counterfeits rapidly. Several other chromones ions were identified and used as additional evidence for authentic samples. A total of 62 samples were visually discriminated as two groups by principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS‐DA), and the specific characteristic marker was highlighted. Moreover, the qualitative results of the conventional TLC method were in agreement with the LESA‐MS approach. Conclusion The proposed LESA‐MS method was successfully applied in the direct qualitative analysis of agarwood from different sources. This study indicated great feasibility and practicality of LESA‐MS in the rapid identification of agarwood, and provided a non‐destructive and meaningful preliminary screening tool for the agarwood industry.
... Agarwood is a valuable commodity produced from Aquilaria and Gyrinops species and wild populations are increasingly vulnerable to extinction. Analysis of wood samples using direct analysis in real time (DART™) ionisation and time-of-flight mass spectrometry (TOFMS), identified agarwood specific chromones that enabled probable origin (wild vs cultivated) to be assigned [52]. ...
Article
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Forensic botany is a diverse discipline that spans many aspects of plant sciences, particularly taxonomy, field botany, anatomy, and ecology. Internationally, there is a significant opportunity to expand the application of forensic botany in criminal investigations, especially war crimes, genocide, homicide, sexual violence, serious physical assault, illegal trade in endangered species and wildlife crime. In civil proceedings, forensic botany may, for example, be called upon in trade disputes such as accidental contamination of commodities. Despite the potential, there are barriers to the wider application of forensic botany in criminal cases; there is a widespread need to improve the efficiency of botanical trace evidence identification. This could partly be addressed by embracing innovations in image recognition and by accessing the huge quantity of specimens and images housed in natural history collections worldwide. Additionally, the recent advances in DNA sequencing technologies and the expansion of environmental DNA (eDNA) and forensic ecogenomics, offers opportunities to more rapidly provide species-level identifications. The impact of taphonomic processes upon vegetation, and vice versa, remains poorly understood; improved understanding of these interactions and their ecological impacts may be invaluable in improving clandestine burial search protocols.
... DART is increasingly employed in law enforcement, however, usage is not yet prevalent in wildlife forensics and wildlife crime detection. Nevertheless, it has been used to identify and differentiate between timber species in trade (Cody et al. 2012;Lancaster et al. 2012;Espinoza et al. 2014) and to identify rhinoceros horn (Price et al. 2018). ...
Article
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In recent years lion bones have been legally traded internationally to Asian markets from captive-bred sources in South Africa. There are also indications of increasing instances of illegal international trade in wild lion bones. The existence of parallel captive and wild supplies of lion bone are a cause of law enforcement concern regarding the potential for the laundering of illegally sourced bones through legal trade, and present a problem for the assessment of the conservation impact of wild lion bone trade due to the difficulty of determining what market-share wild and captive-bred lion bones account for. Captive-bred and wild lion bone are visually indistinguishable and no reliable method currently exists for distinguishing them. We present a preliminary study that explores the use of DART mass spectrometry as a method to differentiate between captive-bred and wild lion bones. We find that DART is able to differentiate between a batch of captive-bred South African lion bone and a batch of wild lion bone and suggest that DART mass spectrometry shows strong potential as a tool for the regulation and investigation of lion bone trade. Further testing is needed to prove the suitability of this technique. Therefore, we suggest that further research focuses on testing the capability of DART to differentiate between contemporary wild and captive-bred lion bone originating from South Africa, and attempts to identify chemical markers in bone that can be used as indicators of captive-bred origin.
... The harvesting of wild agarwood is destructive because it involves the felling of the tree (Jensen 2009;Faizal et al. 2016 Agarwood is a complex combination of various volatile aromatic compounds, and the development of synthetic agarwood is complicated and expensive (Espinoza et al. 2014). Agarwood has been used for thousands of years; it is mentioned in the Old Testament, and it is used in the Islamic, Buddhist and Hindu traditions as incense (Sampson and Page 2018). ...
... Artificial agarwood or agar oil formation is quite impossible as it is a composition of numerous volatile and semi-volatile compounds. However, the chemical composition of cultivated agarwood is almost similar with naturally inoculated agarwood (Espinoza et al., 2014). Cost involvement in different processing stages i.e. nursing of tree (up to 10 year), carrying, logging, chipping, collection of inoculants and establishment were 500, 50, 50, 100, 100 and 50 BDT per tree, respectively. ...
Article
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p>Abstract not available Bangladesh J. Agril. Res. 42(1): 191-196, March 2017</p
... Artificial agarwood or agar oil formation is quite impossible as it is a composition of numerous volatile and semi-volatile compounds. However, the chemical composition of cultivated agarwood is almost similar with naturally inoculated agarwood (Espinoza et al., 2014). Cost involvement in different processing stages i.e. nursing of tree (up to 10 year), carrying, logging, chipping, collection of inoculants and establishment were 500, 50, 50, 100, 100 and 50 BDT per tree, respectively. ...
... NOTE: DART TOFMS is not regularly used for provenance identification at the time of this publication. However, although the variation in the results is subject of further investigations, the protocol below can be used as a start for provenance identification as well (Espinoza et al. 2014). ...
Method
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Today we have five types of timber tracking tools available. Each has its own strengths and limitations (see the Timber Tracking Tool Infogram), but together they offer a broad range of methods that can assist us in identifying the botanical as well as the geographic origin (provenance) of most kinds of timber samples, even those smaller than 1 cm³. With this guide we want to provide an overview of the current best-practice methods used to analyse data derived from different wood identification methods, while presenting their respective strengths and limitations. We give advice on data analysis, from the development of reference data, through to the verification of identity and provenance of unknown samples against the reference database. We end with an expert view on combining methods for wood identification and discuss how timber identification possibilities could expand in the future.
... To fight illegal trade and spot signatures of illicit timber, various HRMS-based methods have been developed to track the species, country of origin and even the region of the timber. For example, Espinoza et al. (2014) utilized direct analysis in real time (DART) ionization coupled to HRMS, to analyze agarwood samples obtained from five countries. In these analyses, 85% of the samples were correctly assigned to either cultivated or wild harvested agarwood for their respective geographic provenance. ...
Article
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Among the different techniques for mass analysis, ultra‐high‐resolution Fourier transform ion cyclotron resonance (FTICR) is the method of choice for highly complex samples, as it offers unrivaled mass accuracy and resolving power, combined with a high degree of flexibility in hybrid instruments as well as for ion activation techniques. FTICR instruments are readily embraced by the biological and biomedical research communities and applied over a wide range of applications for the analysis of biomolecules such as carbohydrates, lipids, nucleic acids, and proteins. In the field of natural organic matter (NOM) analysis, petroleum‐related studies currently dominate FTICR‐MS applications. Recently, however, there is a growing interest in developing high‐performance MS methods for the characterization of NOM samples from natural aquatic and terrestrial environments. Here, we present an overview of FTICR‐MS techniques for complex, non‐petroleum NOM samples, including data analysis and novel tandem mass spectrometry (MS/MS) methods for structural classifications. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.
... Direct Analysis in Real Time (DART) Mass Spectrometry [34][35][36][37][38] is a form of ambient ionization mass spectrometry [39] that has found broad use in chemotaxonomic classification based upon profiling small-molecule metabolites of plants, animals, insects, and bacteria [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] . ...
Article
Rationale Seventeen different dried yeast strains, including 12 strains of Saccharomyces cerevisiae and five strains of Saccharomyces pastorianus, were analyzed by Direct Analysis in Real Time Time‐of‐Flight Mass Spectrometry (DART‐TOFMS). The resulting mass spectra were used for rapid species and strain differentiation based upon small‐molecule metabolomic profiles. Methods Yeast strains purchased from local shops were suspended in a 1:1 water/methanol solution. Solutions were sampled by dipping the sealed end of melting point capillaries into each vial. Six replicates were measured in positive‐ion and negative‐ion mode for each strain by using an automated linear rail with the DART source operated with helium gas and a gas heater temperature of 350°C. Averaged and centroided mass spectra were exported for analysis with chemometric software. Results Negative‐ion DART mass spectra exhibited less chemical background and more distinctive components than positive‐ion DART mass spectra. An on‐line search of the Yeast Metabolome Database provided candidate metabolites for selection as features for chemometric analysis. Negative‐ion DART mass spectra could distinguish both species and all strains. The DART analysis was also able to identify potential metabolomic differences between top‐fermenting and bottom‐fermenting yeast, between beer and baking yeast, and between red wine and champagne yeast. Conclusions All strains could be distinguished by their negative‐ion DART mass spectra with 97.7% validation accuracy. Clear differences were observed between dry and liquid forms and Saccharomyces strains with different applications to baking or beverage fermentation. Possible differences in metabolite profiles were suggested, but not confirmed, by accurate mass data.
... This leads to the ionization and emissions of compounds from the wood, which then enter the mass analyser. Spectra were acquired in positive ion mode with DART ion source parameters settings (as indicated in Lancaster and Espinoza 2012;Espinoza et al. 2014;McClure et al. 2015): electrode 1 150 V; electrode 2 250 V. The mass spectrometer settings were: Orifice 1, 120 °C, 30 V; ring lens 5 V; Orifice 2 5 V; ion guide RF 600 V; ion guide bias voltage 33 V. ...
Article
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The resistance of wood against fungal decay, in short the natural durability, is one of the main criteria for defining the potential use of a wood species. Wood collections, or xylaria, offer the unique opportunity to screen many specimens and species for the latter purpose yet sample size is often limited and standardized tests are often not possible neither desired given the historical and cultural value of these specimens. Two different methods to determine the natural durability are tested and presented here, more specifically the mini-block test and chemical fingerprinting by Direct Analysis in Real-Time Time-Of-Flight Mass Spectrometry (DART TOFMS). Fungal decay by Trametes versicolor was determined for 577 mini-blocks collected from xylarium specimens and 602 mini-blocks from commercial species, not belonging to the xylarium collection, were included as a benchmark. Mass loss percentages of the different species are similar to reported values, supporting the use of the mini-block test when standardized testing is hardly feasible. Furthermore, as expected there is also a significantly negative relationship between density and the mass loss percentages from the mini-block test (r-Spearman = − 0.65***). Finally, partial least square-based prediction of recorded mass loss by using the DART TOFMS chemical fingerprints is promising (R2-adjusted = 0.40***), yet the accuracy differs between species.
Chapter
Direct analysis in real time (DART), in comparison with other techniques such as liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS), matrix-assisted laser desorption ionization (MALDI), enables rapid analysis without derivatization or additional sample handling and sample preparation. This chapter introduces miscellaneous examples applying DART, which are unique and valuable. The DART ion source has been used to analyze an extremely wide range of analytes, including prescription, over-the counter, veterinary, illicit, and counterfeit drugs, in dose form or in body fluids or tissue; explosives and arson accelerants; chemical weapons, agents, and their signatures; synthetic organic or organometallics compounds. It is also used to analyze environmentally important compounds; inks and dyes; and foods, spices, and beverages. An important benefit of DART is that materials can be analyzed directly, even if on surfaces such as glass, thin-layer chromatography (TLC) plates, concrete, paper, or currency, without requiring wipes or solvent extraction.
Article
Thirteen previously undescribed 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones named tetrahydrochromone A–M, together with nine known ones, were isolated from artificial agarwood (induced by holing) originating from Aquilaria sinensis (Lour.) Gilg. The structures of these compounds were unambiguously determined based on extensive NMR spectroscopic analyses, and the absolute configuration was resolved by CD analyses, X-ray crystallographic, chemical and Mosher's method. Tetrahydrochromone A, B, K–M, and Oxidoagarochromone An exhibited inhibitory activity against AChE with the percentage inhibition range from 17.5% to 47.9% (with Tacrine as the positive control; inhibition ratio: 66.7%) when tested at 50 μg/mL. Tetrahydrochromone A−E, F−J feature one methoxy and three hydroxys linked at the cyclohexene ring rather than usual four hydroxys, and tetrahydrochromone K–M represent the first examples of 7,8-epoxy tetrahydrochromones.
Article
Pterocarpus santalinus and Pterocarpus tinctorius are commonly used species of the genus Pterocarpus in the wood trade. Although both of them have been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) since 2019, it is still critical to identify them in terms of plant taxonomy. Currently, high-temperature heating is an accepted treatment method for high-density wood species such as Pterocarpus to improve dimensional stability and restore previous drying defects partially. It has proved challenging to identify the high-temperature (e.g., 120 °C) heated wood from these two species. Thus, this study approaches species identification of two Pterocarpus of high-temperature (e.g., 120 °C) heated solid wood samples using headspace–gas chromatography–mass spectrometry (HS–GC–MS). Besides, a computational analytical method named similarity network fusion (SNF) was proposed to aggregate data in two different types, respectively, derived from the HS–GC–MS and direct analysis in real time–mass spectrometry (DART–MS) to explore the feasibility of improving the efficiency and accuracy of wood species discrimination. The SNF exhibits more significant differences and higher predictive accuracy (100%) between P. santalinus and P. tinctorius than that based on the HS–GC–MS data (77.78%) or DART–MS (66.67%) alone. These results demonstrated the capability of the HS–GC–MS technique in the analysis of high-temperature heated solid wood and the potential of multidimensional or comprehensive data sets based on the SNF algorithm in the field of wood species identification.
Article
The increasing use of atmospheric pressure mass spectrometry has led to the development of many ambient ionization sources, for which sampling versatility and low cost are desired features. One such recent ambient ionization method is direct analysis in real time mass spectrometry (DART-MS), which has proven to be well suited to the analysis of native samples of both simple and complex natures. We describe a home-built DART source (EZ-DART) with versatile sampling capabilities, low power requirements, and low assembly cost which can be easily interfaced to mass spectrometers equipped with an atmospheric pressure inlet. The operating temperature range (22-250°C) enables the acquisition of both temperature programmed desorption-based DART mass spectra and the collection of multistep collision-induced dissociation (CID) mass spectra. We present here the validation of the EZ-DART source and a demonstration of its performance in a number of relevant applications. Additionally, we introduce the new DART application of reagent assisted desorption ionization (RADI) for the targeting of specific chemical functionality in complex organic mixtures through a host-guest chemical system.
Article
Covering: Up to the end of 2019. Agarwood is a resinous portion of Aquilaria trees, which is formed in response to environmental stress factors such as physical injury or microbial attack. It is very sought-after among the natural incenses, as well as for its medicinal properties in traditional Chinese and Ayurvedic medicine. Interestingly, the chemical constituents of agarwood and healthy Aquilaria trees are quite different. Sesquiterpenes and 2-(2-phenethyl)chromones with diverse scaffolds commonly accumulate in agarwood. Similar structures have rarely been reported from the original trees that mainly contain flavonoids, benzophenones, xanthones, lignans, simple phenolic compounds, megastigmanes, diterpenoids, triterpenoids, steroids, alkaloids, etc. This review summarizes the chemical constituents and biological activities both in agarwood and Aquilaria trees, and their biosynthesis is discussed in order to give a comprehensive overview of the research progress on agarwood.
Chapter
Phytochemicals are chemicals that are produced by plants. These phytochemicals find applications as flavors, fragrances, traditional medicines, pharmaceuticals, dyes, and pest control agents. Direct analysis in real time-mass spectrometry (DART-MS) parameters have to be optimized to realize effective phytochemical analysis. DART-MS was used for the nontargeted metabolite analysis of oolong tea during the manufacturing and fermentation process. Taxoids expressed in cell cultures of Taxus wallichiana were profiled by DART-MS by placing the calli directly between the DART source and the mass spectrometer in the presence of ammonium hydroxide. DART-MS is the simplest and fastest mass spectrometric technique for phytochemical analysis as it uses an extractionless technique, and sample to results is only minutes away. Gas chromatography, liquid chromatography, thin-layer chromatography (TLC), and capillary electrophoresis (CE) serve as sample inlets to DART-MS, thus harnessing the separation power of these techniques with the fast analysis capacity of DART-MS.
Article
Structural analysis of biomolecules is essential to natural product discovery, especially for precious biomaterials such as agarwood. However, one of the greatest challenges to the characterization of natural products is the profound cost in time and manpower to the structural elucidation of these highly diverse compounds. Here, we demonstrate a multi-modal mass spectrometric strategy, integrating matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) and mass spectral molecular networking, to uncover agarwood natural products of Aquilaria sinensis trees. A simple workflow for preparing wood sections for MALDI-MSI analysis was demonstrated. Notably, tens of natural products in the agarwood region in wood stem section of A. sinensis were spatially revealed by MALDI-MSI. For the first time, such a great number of plant specialized metabolites is obtained by a single wood section MSI. Guided by the spatially resolved features, mass spectral molecular networking was subsequently applied for structural analysis of the agarwood natural products, in which three major classes of 2-(2-phenylethyl)chromones and their analogues were putatively characterized. These results suggest an efficient strategy to the dereplication of plant natural products.
Article
Illegal logging and trafficking of endangered timber species has attracted the world's major organized crime groups, with associated deforestation and serious social damage. The inability of traditional methodologies and DNA analysis to readily perform wood identification to the species level for monitoring has stimulated research on chemotyping techniques. In this study, simple wood extraction of endangered rosewoods (Dalbergia spp), amenable to use in the field, produced colorful hues that were suggestive of wood species. A more definitive study was conducted to develop wood species identification procedures using high-resolution quadrupole time-of-flight (QTOF) mass spectrometers interfaced with liquid chromatography (LC), gas chromatography (GC), and Direct Analysis in Real Time (DART). The time consuming process of extracting “identifying” mass spectral ions for species identification, contentious due to their ubiquitous nature, was supplanted by application of machine learning processes. The unbiased software mining of raw data from multiple analytical batches, followed by statistical Random Forest analysis, enabled discrimination between both anatomically and chemotypically similar Dalbergia species. Statistical Principal Component Analysis (PCA) scatterplots with 95% confidence ellipses were visually compelling in showing a differential clustering of Dalbergia from other commonly traded and lookalike wood species. The information rich raw data from GC or LC analyses offered a corroborative, legally defensible, and widely available confirmatory tool in the identification of timber species.
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Species determination of the various life stages of flies (order: Diptera) is challenging, particularly for the immature forms, because analogous life stages of different species are difficult to differentiate morphologically. It is demonstrated here that DART high-resolution mass spectrometry (DART-HRMS) combined with supervised Kohonen Self-Organizing Maps (SOM) enables accomplishment of species-level identification of larvae, pupae and adult life stages of carrion flies. DART-HRMS data for each life stage were acquired from analysis of ethanol suspensions representing Calliphoridae, Phoridae and Sarcophagidae families, without additional sample preparation. After preprocessing, the data were subjected to a combination of minimum Redundancy Maximal Relevance (mRMR) and Sparse Discriminant Analysis (SDA) methods to select the most significant variables for creating accurate SOM models. The resulting data were divided into training and validation sets, and then analyzed by the SOM method to define the proper discrimination models. The 5-fold venetian blind cross-validation misclassification error was below 7% for all life stages, and the validation samples were correctly identified in all cases. The multiclass SOM model also revealed which chemical components were the most significant markers for each species, with several of these being amino acids. The results show that processing of DART-HRMS data using artificial neural networks (ANNs) based on the Kohonen SOM approach enables rapid discrimination and identification of fly species even for the immature life stages. The ANNs can be continuously expanded to include a larger number of species, and can be used to screen DART-HRMS data from unknowns to rapidly determine species identity.
Chapter
The utility of direct analysis in real time-high resolution mass spectrometry (DART-HRMS) for quantification of a variety of compounds has been explored, but the number of reports of validated methods using this technique is limited. Furthermore, despite the increasing use in crime labs of DART-HRMS for the detection and identification of drugs of abuse, very few published reports have appeared describing how the method can be exploited for the analysis of small molecules of interest within complex matrices such as plant tissues. Herein we describe the steps to be taken to establish a validated quantification method for psychoactive compounds within complex plant matrices through its application to the detection and quantification of atropine in Datura stramonium seeds. Six calibration standard series are analyzed eight times over a period of several days to create a calibration curve. The resulting calibration curve is tested using six quality control samples and finally utilized to determine the concentration of atropine in a D. stramonium seed extract. The linear range for quantification of atropine in this study was found to be comparable to that reported previously using GC, LC, HPLC, and UHPLC-MS methods. Furthermore, the method can be applied to the quantification of other biomarkers in plant materials, despite the complexity of the plant matrix. The speed of the analysis (<10 min for duplicate analysis of 20 samples) and the ability to integrate peaks using accurate masses for specificity are advantages of the DART-HRMS quantification approach.
Chapter
Endophytic fungi are organisms that reside asymptomatically in the tissues of higher plants without initiating any disease or overt negative symptoms in the host plants. This type of fungus acts as a biostimulant and uses living plant cells as a biofactory for the production of different desirable products. While promoting the production of secondary metabolites in host plants, endophytic fungi can also contribute to improving the development, fitness, and tolerances of host plants against abiotic and biotic stresses. However, their diverse distributions and populations are influenced by several factors, for example, genetic conditions, age, and the environmental conditions of their hosts. Nowadays, different types of endophytic fungi are currently used as biological agents to induce the formation of resinous substances in plants as a response to fungal infections, such as in the case of Aquilaria trees. Agarwood or resin wood is an important high-value product that is synthesized naturally as a result of certain fungal infections of Aquilaria trees. This resin is a high-value component in the manufacture of essential oils and perfumes. This review provides up-to-date information about the process and mechanism of agarwood resin production in Aquilaria trees as well as the best practices used in the sustainable production of this high-value product in Southeast Asia.
Article
Pterocarpus santalinus , listed in CITES Appendix II, is an endangered timber species as a result of illegal harvesting due to its high value and commercial demand. The growing demand for P. santalinus and timbers with the morphologically similar Pterocarpus tinctorius has resulted in confusion as well as identification problems. Therefore, it is of vital importance to explore reliable ways to accurately discriminate between P. santalinus and P. tinctorius . In this study, the method of direct analysis in real time and fourier transform ion cyclotron resonance mass spectrometry (DART-FTICR-MS), combined with multivariate statistical analysis, was used to extract chemical information from xylarium wood specimens and to explore the feasibility of distinguishing these two species. Significant differences were observed in their DART-FTICR-MS spectra. Orthogonal partial least square-discriminant analysis (OPLS-DA) showed the highest prediction, with an accuracy of 100%. These findings demonstrate the feasibility of authenticating wood types using DART-FTICR-MS coupled with multivariate statistical analysis.
Article
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This study is to characterize and profi le the chemical constituents of Aquilaria species, mainly Aqularia malaccensis, Aqularia sinensis, and Aqularia crassna. This precious wood has long been used traditionally in religious ceremonies in the form of incense and oil. There are variations among the chemical group that has been identifi ed in this species, mostly sesquiterpenoids, chromones and volatile aromatic compounds. Studies on the chemistry of agarwood smoke are discussed. Emphasis is given to structural and analytical aspects through this study. The agarwood grading system totally depended on its physical characteristics; there was no scientifi c approach based on the chemical profi le which was used to classify the different grades of agarwood. A different type of analytical technique was used to isolate single pure compounds from chip wood of agarwood, and solvent refluxing was the most popular among them. The polar solvent was found to be used to obtain the polar compounds and chromium, while most of the sesquiterpene was obtained from the non-polar solvent. For extraction of agarwood, hydrodistillation was the most common technique to extract the oil and followed by supercritical fluid extraction, while steam distillation and Soxhlet distillation was rarely used. Different analytical instrumentation were used for the investigation of the chemical profi le of agarwood such as GC-FID, GC-MS, GC-MS-OLF which were the most common research tools. The chemical profi le of the wood from the different species and countries all looked different except the Vietnamese Aquilaria sinensis, and A. agallocha which seemed to have a very closed chemical profi le. There were remarkable similarities in the chemical profi le of A. agallocha India (wood ), A. agallocha Vietnam (wood), and A. sinensis Vietnam (wood) with the minor absence of some compounds from A. agallocha India (wood). From the literature, we could say that the 6-methoxy-2-[2-(4 -methoxypheny1)-ethyl]chromone and2-(2 (4methoxyphenyl)ethyl)chromone was the most dominate chromones found in all species.
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Thirty-four samples of Red Oak (Quercus rubra) and fifty samples of White Oak (Quercus alba) were analyzed by pyrolytic direct analysis in real time (DART) ionization coupled with time-of-flight (TOF) mass spectrometry. Although significant differences were not observed in the positive-ion mass spectra, the negative-ion mass spectra showed clear differences. Principal component analysis (PCA) and linear discriminant analysis (LDA) were calculated for the relative abundances of 11 peaks in the negative-ion mass spectra including peaks tentatively assigned as representing deprotonated acetic, malic, gallic, dimethoxycinnamic, and ellagic acids. Leave one out cross validation (LOOCV) was 100% successful in classifying the samples for both PCA and LDA.
Article
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A new species of Aquilaria is described from Vietnam and keys to the flowering and fruiting specimens of all species from this region are provided. DNA sequences of the nrITS1-5.8S-ITS2 region seem to confirm its status as a distinct species.
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Two new 2-(2-phenylethyl)chromones, (5S(*),6R(*),7S(*))-5,6,7-trihydroxy-2-(3-hydroxy-4-methoxyphenethyl)-5,6,7,8-tetrahydro-4H-chromen-4-one (1) and (5S(*),6R(*),7R(*))-5,6,7-trihydroxy-2-(3-hydroxy-4-methoxyphenethyl)-5,6,7,8-tetrahydro-4H-chromen-4-one (2), were isolated from the Chinese eaglewood of Aquilaria sinensis (Lour.) Gilg. Their structures were established by detailed MS and NMR spectroscopic analysis, as well as comparison with the literature data.
Article
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A method is described for the rapid identification of biogenic, volatile organic compounds (VOCs) emitted by plants, including the analysis of the temperature dependence of those emissions. Direct analysis in real time (DART) enabled ionization of VOCs from stem and leaf of several eucalyptus species including E. cinerea, E. citriodora, E. nicholii and E. sideroxylon. Plant tissues were placed directly in the gap between the DART ionization source skimmer and the capillary inlet of the time-of-flight (TOF) mass spectrometer. Temperature-dependent emission of VOCs was achieved by adjusting the temperature of the helium gas into the DART ionization source at 50, 100, 200 and 300 degrees C, which enabled direct evaporation of compounds, up to the onset of pyrolysis of plant fibres (i.e. cellulose and lignin). Accurate mass measurements facilitated by TOF mass spectrometry provided elemental compositions for the VOCs. A wide range of compounds was detected from simple organic compounds (i.e. methanol and acetone) to a series of monoterpenes (i.e. pinene, camphene, cymene, eucalyptol) common to many plant species, as well as several less abundant sesquiterpenes and flavonoids (i.e. naringenin, spathulenol, eucalyptin) with antioxidant and antimicrobial properties. The leaf and stem tissues for all four eucalypt species showed similar compounds. The relative abundances of methanol and ethanol were greater in stem wood than in leaf tissue suggesting that DART could be used to investigate the tissue-specific transport and emissions of VOCs.
Article
We propose a novel method called robust kernel principal component analysis (RKPCA) to decompose a partially corrupted matrix as a sparse matrix plus a high or full-rank matrix whose columns are drawn from a nonlinear low-dimensional latent variable model. RKPCA can be applied to many problems such as noise removal and subspace clustering and is so far the only unsupervised nonlinear method robust to sparse noises. We also provide theoretical guarantees for RKPCA. The optimization of RKPCA is challenging because it involves nonconvex and indifferentiable problems simultaneously. We propose two nonconvex optimization algorithms for RKPCA: alternating direction method of multipliers with backtracking line search and proximal linearized minimization with adaptive step size. Comparative studies on synthetic data and nature images corroborate the effectiveness and superiority of RKPCA in noise removal and robust subspace clustering.
Article
Ecological and economic data are essential to the identification of tropical nontimber forest products with The potential for sustainable and profitable extraction in a managed system. We studied the demographic effect and economic returns of harvesting aromatic gabaru wood from fungus-infected trees of Aquilaria malaccensis Lam. at Gunung Patting National Park, Indonesia, to evaluate the management potential of gaharu wood. Aquilaria malaccensis trees of >20 cm in diameter occurred at low preharvest densities (0.16-0.32 ha) but were distributed across five of six forest types surveyed. During a recent harvest, 75% of trees were felled, with harvest intensities ranging from 50% to 100% among forest types. Overall, 50% of trees contained gabaru wood, but trees at higher elevations contained gabaru wood more frequently (73%) than trees at lower elevation (27%,). The mean density of regeneration (juveniles >15 cm in height) near adult trees (3-7 m away) was 0.2/m(2), 200 times greater than at random in the forest (10/ha), but long-term data on growth and survivorship are needed to determine whether regeneration is sufficient for population recovery, Gabaru wood extraction from Gunung Patting was very profitable for collectors, generating an estimated gross financial return per day of US $8.80, triple the mean tillage wage. Yet, the estimated sustainable harvest of gabaru wood at natural tree densities generates a mean net present value of only$10.83/ha, much lower than that of commercial timber harvesting, the dominant forest use in Kalimantan. Returns per unit area could be improved substantially, however, by implementing known silvicultural methods to increase tree densities, increase the proportion of trees that produce gabaru wood, and shorten the time interval between successive harvests. The economic potential of gabaru wood is unusual among nontimber forest products and justifies experimental trials to develop small-scale cultivation methods.
Article
Agarwood is the resinous material harvested from threatened Aquilaria species. We investigated how many protonated 2-(2-phenylethyl)chromone ions were sufficient to make an accurate identification of agarwood. Analysis of 125 reference samples was carried out by direct analysis in real time time-of-flight mass spectrometry (DART-TOFMS). The identification criteria developed were applied to commercial samples. We developed a technique that uses DART-TOFMS to detect 2-(2-phenylethyl)chromones. Additionally, we developed a set of criteria to infer the presence of Aquilaria in commercial samples of wood chips, sawdust, incense and liquids. Additionally, we examined other fragrant woods to determine if they contained a chemical profile that could be falsely identified as agarwood. Analysis of reference and commercial samples (n = 151) established that DART-TOFMS provides reproducible mass spectra that are useful for inferring the genus of suspected agarwood samples. We identified 17 ions which were useful for authenticating agarwood. Comparison of the number of chromone ions detected by direct analyses of dry wood chips versus eluent analysis of methanol-extracted wood showed that results were similar. Lastly, analysis of 25 scented woods of other species did not give false positive results. Reliable criteria for inferring agarwood include the presence of diagnostic ions, m/z 319.118 or 349.129, in addition to ten or more ions characteristic of 2-(2-phenylethyl)chromones. Wood anatomists challenged with difficult morphological identifications can use this tool to assist in their analyses. Published 2012. This article is a US Government work and is in the public domain in the USA.
Article
A compilation of the volatile and semi-volatile constituents of agarwood, the infected fragrant heartwood of some Aquilaria species, mainly A. malaccensis, A. sinensis and A. crassna, is presented. This high-priced wood has a long tradition in religious ceremonies as incense and its essential oil is well appreciated in fine perfumery. More than 150 compounds have been identified so far, mostly sesquiterpenoids, chromones and volatile aromatic compounds. The scarce knowledge of their organoleptic properties is summarized. Studies on the chemistry of agarwood smoke are discussed. Emphasis is given to structural and analytical aspects. Copyright © 2011 John Wiley & Sons, Ltd.
Chapter
A new method for performing a nonlinear form of Principal Component Analysis is proposed. By the use of integral operator kernel functions, one can efficiently compute principal components in highdimensional feature spaces, related to input space by some nonlinear map; for instance the space of all possible d-pixel products in images. We give the derivation of the method and present experimental results on polynomial feature extraction for pattern recognition.
Article
Article
International trade of several Dalbergia wood species is regulated by The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In order to supplement morphological identification of these species, a rapid chemical method of analysis was developed. Using Direct Analysis in Real Time (DART) ionization coupled with Time-of-Flight (TOF) Mass Spectrometry (MS), selected Dalbergia and common trade species were analyzed. Each of the 13 wood species was classified using principal component analysis and linear discriminant analysis (LDA). These statistical data clusters served as reliable anchors for species identification of unknowns. Analysis of 20 or more samples from the 13 species studied in this research indicates that the DART-TOFMS results are reproducible. Statistical analysis of the most abundant ions gave good classifications that were useful for identifying unknown wood samples. DART-TOFMS and LDA analysis of 13 species of selected timber samples and the statistical classification allowed for the correct assignment of unknown wood samples. This method is rapid and can be useful when anatomical identification is difficult but needed in order to support CITES enforcement. Published 2012. This article is a US Government work and is in the public domain in the USA.
Article
A new ion source has been developed for rapid, noncontact analysis of materials at ambient pressure and at ground potential. The new source, termed DART (for "Direct Analysis in Real Time"), is based on the reactions of electronic or vibronic excited-state species with reagent molecules and polar or nonpolar analytes. DART has been installed on a high-resolution time-of-flight mass spectrometer (TOFMS) that provides improved selectivity and accurate elemental composition assignment through exact mass measurements. Although DART has been applied to the analysis of gases, liquids, and solids, a unique application is the direct detection of chemicals on surfaces without requiring sample preparation, such as wiping or solvent extraction. DART has demonstrated success in sampling hundreds of chemicals, including chemical agents and their signatures, pharmaceutics, metabolites, peptides and oligosaccharides, synthetic organics, organometallics, drugs of abuse, explosives, and toxic industrial chemicals. These species were detected on various surfaces, such as concrete, asphalt, human skin, currency, airline boarding passes, business cards, fruits, vegetables, spices, beverages, body fluids, horticultural leaves, cocktail glasses, and clothing. DART employs no radioactive components and is more versatile than devices using radioisotope-based ionization. Because its response is instantaneous, DART provides real-time information, a critical requirement for screening or high throughput.
Article
A new method for performing a nonlinear form of Principal Component Analysis is proposed. By the use of integral operator kernel functions, one can efficiently compute principal components in high-- dimensional feature spaces, related to input space by some nonlinear map; for instance the space of all possible d--pixel products in images.
• R A Blanchette
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Chemical constituents of agarwood originating from the endemic genus Aquilaria plants
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