Wood from Hardwood Angiosperms and Coniferous Gymnosperms Shows Distinctive Lignin Peaks in Direct Analysis in Real Time (DART) Mass Spectra.

A data set was constructed consisting of 3021 mass spectra randomly selected from all available families in the ForeST© (Forensic Spectra of Trees) database of mass spectra for wood analyzed by Direct Analysis in Real Time ionization coupled with time-of-flight mass spectrometry (DART-TOFMS). Clear and reproducible differences were observed between the lignin peaks for hardwood angiosperms and coniferous gymnosperms, with DART-TOFMS spectra of angiosperms showing significantly higher relative abundances for peaks associated with syringyl subunits. Application of the method to processed wood samples demonstrated that these differences can be used to provide support for enforcing trade laws by accurately identifying the source of finished wood products from hardwood angiosperms and coniferous gymnosperms.

Opportunities in phytochemistry, ecophysiology and wood research via laser ablation direct analysis in real time imaging-mass spectrometry.

Analysis of wood transects in a manner that preserves the spatial distribution of the metabolites present is highly desirable to among other things: (1) facilitate ecophysiology studies that reveal the association between chemical make-up and environmental factors or climatic events over time; and (2) investigate the mechanisms of the synthesis and trafficking of small molecules within specialised tissues. While a variety of techniques could be applied to achieve these goals, most remain challenging and impractical. Laser ablation direct analysis in real time imaging-mass spectrometry (LADI-MS) was successfully used to survey the chemical profile of wood, while also preserving the small-molecule spatial distributions. The tree species Entandrophragma candollei Harms, Millettia laurentii DeWild., Pericopsis elata (Harms) Meeuwen, Dalbergia nigra (Vell.) Benth. and Dalbergia normandii Bosser & R.Rabev were analysed. Several compounds were associated with anatomical features. A greater diversity was detected in the vessels and parenchyma compared with the fibres. Analysis of single vessels revealed that the chemical fingerprint used for timber identification is mainly determined by vessel content. Laser ablation direct analysis in real time imaging-mass spectrometry offers unprecedented opportunities to investigate the distribution of metabolites within wood samples, while circumventing the issues associated with previous methods. This technique opens up new vistas for the discovery of small-molecule biomarkers that are linked to environmental events.

Forensic identification of the keratin fibers of South American camelids by ambient ionization mass spectrometry: Vicuña, alpaca and guanaco.

RATIONALE: The keratin fleece of the endangered vicuña (Vicugna vicugna) commands a high value in international markets, and this trade has caused illegal poaching and a substantial decrease in vicuña populations. Morphological analysis of hairs does not have the resolution to determine the species of origin of camelid natural fibers. In addition, commerce in camelid fleece also includes the legal trade of alpaca (Vicugna pacos) and guanaco (Lama guanicoe) wool. METHODS: The keratin fiber spectra of vicuña (n = 19), guanaco (n = 20) and alpaca (n = 20) were collected using X-ray fluorescence (XRF) spectrometry, Horizontal attenuated total reflectance Fourier transform infrared (HATR-FTIR) spectroscopy and direct analysis in real time time-of-flight mass spectrometry (DART-TOFMS). Analysis with each technique evaluated the data to determine if the three taxa could be separated using either descriptive or multivariate statistics. RESULTS: XRF analysis showed that the elements detected and their relative concentrations were similar in all three species, whereas HATR-FTIR analysis could identify alpaca fleece but could not differentiate vicuña from guanaco. Ions detected by ambient ionization using DART-TOFMS, in either positive- or negative-ion mode, gave the best results and showed that each taxonomic group is distinctive. Multivariate analysis of the mass spectra created robust models which resolved each species (LOOCV = 99.9%). The analyses of eight validation samples were correctly assigned to the appropriate species and demonstrated the reliability of DART-TOFMS to infer taxonomic source. CONCLUSIONS: The DART-TOFMS spectra of unmodified keratin fibers infer that the chemotype of each species is heavily influenced by fatty acids, cholesterol and its analogs, and that these ions are useful in separating the fleece of vicuña, alpaca and guanaco. We posit that the etiological source of these chemotype differences is consistent with genetic modulations and is less influenced by diet. Accurate taxonomic identification of fleece is important to identify violations and assists in the protection of rare species.

Assessing utility of handheld laser induced breakdown spectroscopy as a means of Dalbergia speciation.

Many species of Dalbergia are prized hardwoods, generally referred to as 'Rosewood,' and used in high-end products due to their distinctive hue and scent. Despite more than 58 species of Dalbergia being listed as endangered in Appendix 1 of The Convention on International Trade in Endangered Species of Fauna and Flora (CITES), the illegal logging and trade of this timber is ongoing. In this work, a handheld laser induced breakdown spectrometer (LIBS) was used to analyze seven Dalbergia species and two other exotic hardwood species to evaluate the ability of handheld LIBS for rapid classification of Dalbergia in the field. The KNN model of the classification presented 80% to 90% sensitivity for discriminating between Dalbergia species in the training set. PLS-DA models were based on a binary decision tree structure. Cumulatively, the PLS-DA decision tree model showed greater than 97% sensitivity and 99% selectivity for prediction of Dalbergia species included in the training set. The data presented in the following study are promising for the use of handheld LIBS devices and both KNN and PLS-DA models for applications in customs screenings at the port of entry of hard woods, among others.

Identification of rhinoceros keratin using direct analysis in real time time-of-flight mass spectrometry and multivariate statistical analysis.

RATIONALE: Trade in rhinoceros horn is regulated or banned internationally in recognition of its impact on wild populations worldwide. Enforcement of the laws and regulations depends on successfully identifying when violations occur, which is complicated by the presence of alternative/imitation rhinoceros horn keratin (e.g., bovid horn keratin). In this study, we assess the potential for Direct Analysis in Real Time (DART) ionization paired with Time-Of-Flight Mass Spectrometry (DART-TOFMS) to classify different keratin types from four taxonomic groups: rhinoceros, bovid, domestic horse, and pangolin. METHODS: The spectra of 156 keratin samples from all five rhinoceros species (horn keratin), eight genera of bovids (horn keratin), domestic horses (hoof keratin), and all extant species of pangolins (scale keratin) were collected. Fisher ratio analysis identified the most important ions that characterized each class and these ions were used for the training model, which consisted of 143 spectra. Kernel Discriminant Analysis (KDA) was used to classify the different groups. RESULTS: The spectra collected for each taxonomic group are distinctive. The chemotypes demonstrate that the spectra of rhinoceros, bovids, and domestic horse are similar to each other, whereas the chemotypes of pangolins show a different chemical profile. The model built by KDA resolved each taxonomic group: 95% of samples were correctly assigned using leave-one-out cross validation. The 13 blind samples not used in model development were all correctly classified to taxonomic source. CONCLUSIONS: DART-TOFMS appears to be a reliable approach for taxonomic identification of keratin. This analysis can be carried out with a small sliver of keratin, with minimal sample preparation, inexpensively and quickly, making it a potential valuable tool for identification of rhinoceros horn and other keratin types.

Source identification of western Oregon Douglas-fir wood cores using mass spectrometry and random forest classification.

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.

Metabolic chemotypes of CITES protected Dalbergia timbers from Africa, Madagascar, and Asia.

RATIONALE: The genus Dalbergia includes approximately 250 species worldwide. Of these, 58 species are of economic importance and listed under CITES. Identification of illegal transnational timber trade is a challenge because logs or boards lack the typical descriptors used for species identification such as leaves and flowers; therefore, frequently the lowest taxonomic determination of these tree byproducts is genus. In this study, we explore the use of Direct Analysis in Real Time (DART) Time-Of-Flight Mass Spectrometry (TOFMS) in making species determinations of protected Dalbergia trees from Africa, Madagascar, and Asia. METHODS: Metabolic profiles were collected using DART TOFMS from the heartwood of seven species and the sapwood of 17 species of Dalbergia. Also included in this study are 85 Dalbergia heartwood samples from Madagascar that were only identified to genus. In all, 21 species comprising 235 specimens were analyzed, the metabolic chemotypes were interpreted, and the spectra were analyzed using chemometric tools. RESULTS: Dalbergia cochinchinensis and Dalbergia spp. from Madagascar (both CITES Appendix II) could be differentiated from each other and from the non-protected Dalbergia latifolia and Dalbergia melanoxylon. CONCLUSIONS: DART TOFMS is a valuable high-throughput tool useful for making phytochemical classifications of Dalbergia spp. The data produced allows the protected Dalbergias from Madagascar to be distinguished and can differentiate closely related rosewood trees.

A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures.

A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

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

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluating agarwood products for 2-(2-phenylethyl)chromones using direct analysis in real time time-of-flight mass spectrometry.

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Analysis of select Dalbergia and trade timber using direct analysis in real time and time-of-flight mass spectrometry for CITES enforcement.

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Forensic analysis of black coral (Order Antipatharia).

Fourier-transform infrared spectroscopy (FTIR), discriminate analysis, X-ray fluorescence spectrometry (XRF), and stereoscopic microscopy were used to separate black coral forensic evidence items from similarly appearing items manufactured from plastics, bovid keratin, and mangrove wood. In addition, novel observations were made of bromine and iodine relationships in black coral that have not been previously reported.

Forensic species identification of elephant (Elephantidae) and giraffe (Giraffidae) tail hair using light microscopy.

Here we present methods for distinguishing tail hairs of African elephants (Loxodonta africana), Asian elephants (Elephas maximus), and giraffes (Giraffa camelopardalis) from forensic contexts. Such hairs are commonly used to manufacture jewelry artifacts that are often sold illegally in the international wildlife trade. Tail hairs from these three species are easily confused macroscopically, and morphological methods for distinguishing African and Asian tail hairs have not been published. We used cross section analysis and light microscopy to analyze the tail hair morphology of 18 individual African elephants, 18 Asian elephants, and 40 giraffes. We found that cross-sectional shape, pigment placement, and pigment density are useful morphological features for distinguishing the three species. These observations provide wildlife forensic scientists with an important analytical tool for enforcing legislation and international treaties regulating the trade in elephant parts.

Analysis of fiber blends using horizontal attenuated total reflection Fourier transform infrared and discriminant analysis.

We have investigated the utility of a horizontal attenuated total reflection Fourier transform infrared spectrometer (HATR/FT-IR) for the analysis of fiber and textile blends. The identification of a blended textile can be accomplished by subtracting a reference spectrum of the textile's most abundant component, leading to a difference spectrum that infers the identity of the second constituent of the blended textile. Mathematical post-processing of the spectra employing discriminant analysis provided a useful statistical tool to confirm the fiber blend components.