Isotope Ratio Mass Spectrometry Analysis Research Articles

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia

AbstractNandrolone and its prohormones, including 19‐norandrost‐4‐ene‐3,17‐dione and 19‐norandrost‐4‐ene‐3β,17β‐diol, are anabolic steroids forbidden at all times in sports according to the World Anti‐Doping Code Prohibited List and its metabolite 19‐norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non‐athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than −26‰). This indicates that there is a high prevalence of nandrolone‐containing anabolic androgenic steroid preparations in Australia that have ‘endogenous’ carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

Comparison of analytical approaches for the detection of oral testosterone undecanoate administration in men.

For antidoping laboratories, the determination of an illicit testosterone (T) administration in urine samples remains a difficult process as it requires the determination of the exogenous origin by carbon isotope ratios (CIRs) of testosterone and its metabolites. As a complement to the urinary analysis, targeting testosterone esters (e.g. testosterone undecanoate [TU]) in serum samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) could represent a simpler approach compared with isotope ratio mass spectrometry (IRMS). These two approaches both lead to the direct detection of the administration of exogenous T but with a difference in effort and complexity of the analysis. To compare the detection window obtained with the two strategies, serum and the corresponding urine samples collected from an administration study with oral TU were analysed. Results showed that, at all timepoints where the intact TU was detected in serum, the CIRs of urinary steroids were also not in agreement with an endogenous origin. IRMS analysis required more effort but resulted in slightly longer detection windows than the ester analysis. Finally, this comparison study showed that, in the presence of a suspicious urinary steroid profile, the LC-MS/MS steroid esters analysis in the corresponding serum samples can be very helpful. If steroid esters are not detected, the IRMS analysis can then be conducted on the urine sample afterwards. Overall, the combination of matrices might facilitate the detection of prohibited T administration in sports, especially for athletes with naturally low T/E ratios.

Current trends and challenges in the analysis of marine environmental contaminants by isotope ratio mass spectrometry.

An increasing number of organic and inorganic pollutants are being detected in the marine environment, posing a severe threat to the ecosystem and human health, even in trace concentrations. Isotope ratio mass spectrometry (IRMS) is one of the critical methods for determining the origin and fate of environmental pollutants and characterising their transformation processes. It has been used for a relatively long time for ecological monitoring of some well-studied industrial hydrocarbons at contaminated sites. However, the method still faces many analytical challenges. This review provides a comprehensive overview of recent technical advances concerning IRMS analysis of various contaminants and discusses typical pitfalls encountered in marine environment analysis. Particular attention is given to the study of sampling techniques and sample preparation for examination, often the keys to successful research given the complexity of marine matrices and the diverse and numerous nature of contaminants. Prospects for developing IRMS to monitor pollution sources and pollutant transformation in the marine environment are outlined.

Organo-mineral associations and size-fractionated colloidal organic carbon dynamics in a redox-controlled wetland

The formation of organo-mineral associations serves as a crucial mechanism for stabilizing soil organic matter, particularly for determining the fate of soil organic carbon (OC) in redox dynamic wetlands characterized by high C content. Despite its importance, few studies have assessed the retention, transformation, and transport of colloids (1–1000 nm) and colloidal OC (COC) in those environments. This leaves a crucial knowledge gap concerning the quantities of colloids and COC and their molecular compositions, especially considering the significant role of metabolically active depressional wetlands that may play as biogeochemical hotspots for C cycling. To address this gap, we conducted a study in a Delmarva Bay depressional wetland located in Blackbird State Forest, Delaware, USA. We established a transect encompassing upland (U), transition (T), and wetland (W) zones based on seasonal hydrologic conditions. We installed piezometers at 50 cm, 100 cm, and 200 cm depths within each zone and collected pore-water samples from 11/2017 to 05/2019. We then fractionated these samples into dissolved (<2.3 nm), natural nanoparticle (NNP, 2.3–100 nm), fine colloid (100–450 nm), and particulate (450–1000 nm) fractions via sequential centrifugation and ultrafiltration, and quantified their concentration and molecular composition. We observed variations in the concentration and molecular composition of soil COC both vertically at different soil depths and horizontally along a redox gradient from the U-T-W transect. The sum of the NNP and fine colloid fractions accounted for 47±20% of the operationally defined “dissolved” (<450 nm) fraction. Isotope ratio mass spectrometry and X-ray photoelectron spectroscopy analyses further revealed that the NNP fraction is more enriched in heavier δ13C isotope and oxidized carbonyl/carboxyl C functional groups (C=O, p<0.05) with significantly higher surficial atomic percentages of C (p<0.01), N (p<0.01), and Mg:Al ratios (p<0.05), and lower atomic percentages of Al (p<0.01) and Si (p<0.01) compared to the larger particles. The formation of cation bridges and/or hydrogen bonds between carboxylic OC and phyllosilicate minerals likely dominated the mineral-OC association in the NNP fraction. The higher surface C enrichment in the particulate fraction compared to the NNP fraction suggests a patchy distribution of more plant-derived OC through organo-organic interactions in the larger fractions. Along the transect, Zone U exhibited significant enrichment of heavier δ13C isotope and lower SUVA values than the Zones T and W indicating inefficient decomposition and dissociation of SOM from minerals due to the reductive dissolution of Fe and Al oxides under anoxic conditions. Furthermore, an increase in low molecular weight microbial metabolites with reduced aromatic OC content was observed at deeper depths in Zones U and T. Our study provides new insights into the concentration and molecular composition of size-fractionated COC, highlighting the need to separately consider the NNP and fine colloid fractions from the operationally defined “dissolved” phase. This is crucial for assessing the biogeochemical cycling of OC in redox-active wetlands, a pressing need amidst the growing concerns about the impacts of climate change.

Tracing the geographical origin of grain vinegar using the stable isotope ratio mass spectrometry and chemometrics

Shanxi extra-aged vinegar (SX) and Zhenjiang vinegar (ZJ) are prominent vinegar varieties in China, but they frequently fall victim to counterfeit products due to their exclusive geographical indication labels. In this study, the carbon isotope ratio data (δ13C) obtained via elementary analyzer isotope ratio mass spectrometry (EA-IRMS) were employed to effectively distinguish between the two geographical indications of vinegar (SX and ZJ) and other vinegar samples sourced from different regions. The δ13C values were individually measured for vinegar (δ13Ctotal), the protein content extracted from vinegar (δ13Cprotein), and the sugar content extracted from vinegar (δ13Csugar). The results revealed that vinegars with exceptionally low sugar and protein contents can be precisely differentiated from the two geographical indications by directly comparing the δ13Cprotein and δ13Csugar values. Moreover, a linear discriminant analysis (LDA) discriminant analysis model was established, integrating all three δ13C isotope ratio datasets as multi-indicators. The results demonstrated that the proposed model distinctly discriminates 24 SX and 35 ZJ vinegar samples from the remaining 26 vinegar samples. This data indicated that the combination of EA-IRMS and LDA offers a potent tool for enhancing vinegar traceability and safeguarding geographical indications.

Simple software solution for N2 diversion when measuring δ18O values of nitrogen-rich samples materials using a Thermo Scientific EA-IRMS System

This method is a simple, cost-free, and reliable approach for the removal of N2 interference on a CO analyte when analysing nitrogen-rich (>0.5% w/w) samples by Elemental Analysis Isotope Ratio Mass Spectrometry. Specifically, the isobaric interference on m/z 30 is eliminated using only the open split of the Thermo Scientific ConFlo IV Universal Interface Device, improving the analytical workflow when using a static temperature Gas Chromatography (GC) column. It simplifies the N2 diversion methods described in recent decades. When applied, the method described here:•Provides sufficient baseline resolution between the N2 and CO analytes, to permit quantitative N2 diversion, using an extended length packed GC column;•Quantitatively eliminates all N2 analyte from the analytical gas stream ensuring that no N2 enters the ion source and therefore no isobaric interference is produced on m/z 30 ion trace of the CO analyte;•Allows reproducible measurement of δ18O values from nitrogen-rich sample materials without a N2 isobaric interference, where the CO analyte is measured on the analytical baseline that it was produced on in the reactor (i.e., no addition make-up helium or new baseline of pure helium for the CO analyte).

C/H/O stable isotope of rape honey and its components combined with machine learning algorithmsto identify geographical origin.

The distribution of rape honey is among the largest and most diverse of all honeys available to humankind with respect to the geographical origin. Accurate isotopic reference values for rape honey are therefore important for precise verification of honey origin and its traceability. New combined rape honey δ13 C, δ2 H, and δ18 O values in combination with values on its compounds (protein and saccharides) were used to complement existing databases to better identify the geographical origin of Chinese rape honey. Traceability methods based on elemental analyzer isotope ratio mass spectrometry and liquid chromatography isotope ratio mass spectrometry were established for geographical origin of rape honey. Rape honey harvested in the high-altitude region (QH; Qinghai) had significantly higher values (1.4 to 5.3‰ for δ13 C, 7.9 to 12.9‰ for δ2 Hprotein ) for the δ13 C of whole honey (-23.8‰), its protein (-24.4‰), fructose (-23.5‰), glucose (-23.6‰), and disaccharide (-24.7‰), and also δ2 H of the protein (103.5‰) than those in low-altitude regions (HB; Hubei, SC; Sicuan, and JS; Jingsu). The δ18 Orape honey was a useful index to differentiate whether rape honey from coastal (JS) or non-coastal (HB, SC, and QH) regions. The δ13 C, δ2 H, and δ18 O values in rape honey are affected by geographical factors, such as temperature and altitude. The δ13 Cprotein and δ13 Crape honey values were better to identify the geographical origin of rape honey than δ13 Csaccharides . The δ18 O and δ2 H values of rape honey protein were more suitable for traceability than those of rape honey. The combination of the δ13 C, δ2 H, and δ18 O values of rape honey and its extracted protein and saccharides improved the precision of three models (linear discriminant analysis, SVM, and random forest) used to discriminate rape honey from different regions in China. The SVM model obtained the best accuracy (93.2%). Stable isotopes could be significant predictors in determining the geographical origin of rape honey.

Evaluation of honey authenticity in Thailand by analysis of carbon stable isotope ratio using elemental analyser coupled to isotope ratio mass spectrometry and cavity ring‐down spectrometry

SummaryIn Thailand, honey is used in a variety of commercial products that are distributed in domestic markets and abroad. As honey in Thailand comes from many locations, it is challenging to control the production quality of commercial products. The main problem commonly found in the honey industry is adulteration with undeclared added sugars. Identification of adulterated honey is crucial for authentication, which can increase consumer confidence on honey purity. In this study, 49 honey samples from beekeepers (natural) and 15 commercial honey producers were collected from different cities across Thailand. They were analysed for stable carbon isotope ratios (expressed as 13C/12C or δ13C and reported in relation to the international reference standard unit Vienna Pee Dee Belemnite, ‰ VPDB) using Elemental Analysis Isotope Ratio Mass Spectrometry (EA‐IRMS) for developing a δ13C database of natural honey and for checking the adulteration of honey. The δ13C values detected in 49 natural honey samples varied from −23.1‰ to −29.1‰ VPDB with an average of −26.8 ± 1.2‰ VPDB. According to the AOAC standard (No. 998.12, 2019), natural honey should have δ13C values lower than −23.5‰ VPDB, with an acceptable difference in δ13C values between honey and the protein extracted from honey up to 1‰. Compared to the standard guideline, 60% of commercial honey samples collected for this study (9 out of 15 samples) were found to contain undeclared added sugars.

Interpreting δ13C Values Obtained on SOM from Ancient Maya Reservoirs and Depressions

Elemental analyzer (EA) Isotope Ratio Mass Spectrometry was used to measure ∂13C values on soil organic matter from reservoirs and depressions at the ancient Maya urban centers of Tikal, Guatemala and Yaxnohcah, Mexico. Variation in δ13C values on soil organic matter were &gt; −2.0‰, which suggests enrichment from C4 plants including maize, other tropical grasses (Poaceae), and tropical sedges (Cyperaceae), CAM plants (Clusia sp.), and cyanobacteria (blue-green algae). Cyanobacteria were likely a major contributor to the 13C enrichment of soil organic matter in Maya reservoirs and depressions, which has obfuscated our understanding of ancient Maya maize production. It is possible that the Maya used cyanobacteria as a fertilizer, which enriched agricultural field soil organic matter.

Rapid discrimination of the authenticity and geographical origin of bear bile powder using stable isotope ratio and elemental analysis.

Bear bile powder (BBP) is one of the most famous traditional Chinese medicines derived from animals. It has a long history of medicinal use and is widely used in the treatment of hepatobiliary and ophthalmic diseases. Due to its similar morphological characterizations and chemical composition compared with other bile powders, it is difficult to accurately identify its authenticity. In addition, there are very few methods that could analyze the geographical origins of BBP. In this study, elemental analysis isotope ratio mass spectrometry (EA-IRMS) and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine stable isotope ratios and elemental contents, respectively. Combined these variables with chemometrics, the discrimination models were established successfully for identifying the authenticity and geographical origins of BBP. Meanwhile, the discrimination markers were identified by calculating the variable importance for the projection (VIP) value of each variable. A total of 13 discrimination markers (δ13C, δ15N, C, Li, Mg, K, Ca, Cr, Ni, Zn, As, Se, and Sr) were used to further establish the fingerprint of BBP. According to similarity analysis, the authenticity and geographical origins of BBP could be identified without chemometrics. In conclusion, the present study established a reliable method for authenticity identification and origin traceability of BBP, which will provide references for the quality control of bile medicines.

Application of stable isotopes with machine learning techniques for identifying Aconiti Lateralis Radix Praeparata (Fuzi) geographical origins

Authentication of geographical traceability is essential to the safety of traditional Chinese medicines (TCMs). Aconiti Lateralis Radix Praeparata (Chinese: Fuzi), a well-known TCMs, the therapeutic efficacy and toxicity are clearly correlated with its geographical origin, and a rapid and effective method of origin tracing must be established. Herein, an elemental analysis-isotope ratio mass spectrometry (EA-IRMS) combined with a machine learning model is constructed for the origin traceability of Fuzi. We analyzed stable isotope information (δ13C‰, δ15N‰, δ2H‰, δ18O‰, C% and N%) for a total of 243 samples from eight major producing areas. Classification models were built using seven machine learning techniques, including random forest (RF), support vector machine (SVM), AdaBoost, etc., to investigate the feasibility of stable isotopes in Fuzi geographic identification. Results show that stable isotopes exhibit an overwhelming origin discrimination advantage. Among them, the RF model showed the best recognition performance with 94.2 % accuracy, 95.45 % sensitivity, and 99.15 % specificity in the independent test set, respectively. The results show that stable isotopes combined with machine learning techniques have great potential for the origin traceability of TCMs.

Addressing recent challenges in isotope ratio mass spectrometry: Development of a method applicable to 1-androstene-steroids, 6α-hydroxy-androstenedione, and androstatrienedione.

In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5α-androst-1-ene-3β,17β-diol, 5α-androst-1-ene-3,17-dione [1AD], 17β-hydroxy-5α-androst-1-en-3-one, 3α-hydroxy-5α-androst-1-ene-17-one [1AND], and 3β-hydroxy-5α-androst-1-ene-17-one [1EpiAND]), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione [TRD] and its main metabolite 17β-hydroxy-androsta-1,4,6-triene-3-one) and 6α- and 6β-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS instrumentation, limits of quantification were estimated at 10 ng/ml for a 20 ml urine aliquot for all analytes, except for 1AND (20 ng/ml), and combined measurement uncertainties were estimated between 0.4‰ and 0.9‰. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.

Investigations on the in vivo metabolism of 5α-androst-2-en-17-one.

The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2β,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5β-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5β-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9days after administration. The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9days. The conversion of a 5α-steroid to urinary metabolites with 5β-configuration has not been reported so far and should be further investigated.

Desulfurization of black powder for isotopic profiling using isotope ratio mass spectrometry

The stable isotope analysis of black powder is of utmost significance with regard to its further comparison and traceability in explosion-related cases. To date, only a limited number of studies have reported the stable isotope analysis of black powder using isotope ratio mass spectrometry (IRMS). However, because the sulfur content in black powder is high, sulfur deposition is extremely likely during the analysis process, thereby damaging the instrument and affecting the reproducibility of the results. Therefore, desulfurization of black powder samples prior to IRMS analysis is extremely desirable. Herein, based on the difference in the solubility of black powder components, chloroform was selected to desulfurize black powder. The results demonstrated that chloroform extraction could effectively eliminate sulfur from the black powder. The isotope fractionation effect caused by desulfurization was also studied. The changes in δ13C, δ15N, and δ18O after desulfurization were close to the standard deviation values and did not affect the analysis of black powder. Desulfurization and stable isotope analysis were performed on 12 black powder samples obtained from cases that happened in eight different areas in China, and the carbon, nitrogen, and oxygen isotope ratios in all the samples were effectively distinguished. Therefore, the established method can successfully distinguish the stable isotope ratios in black powder samples using IRMS after desulfurization while avoiding damage to the instrument and enhancing the stability of the results.

The first report on the use of deuterium-labelled water in analysing the splash phenomenon

Splash is a widely studied phenomenon, partly forming the phenomenon of erosion. Until now, tracing the origin of splash water was a highly error-prone, or impossible, task. We propose a method for tracking splash water by means of stable isotope ratio mass spectrometry (IRMS) analysis. We tested the method on a water-water model, checking the effect of drop height, water layer thickness and the effect of water evaporation. It turns out that the drop height and the thickness of the water layer have practically no effect on the origin of the water in the splash, and all the water comes from the vessel, not from the drops. The only exception is when a thin layer of water is impacted with a drop, in which case 23% of the water also comes from the drop. The proposed method could be used to study more complex systems such as water-soil.

New method for simultaneous determination of dissolved organic carbon and its stable carbon isotope ratio in liquid samples: environmental applications

ABSTRACT This study reports the development of an all-in-one elemental analyser isotope ratio mass spectrometry (EA-IRMS) system modified for simultaneous analysis of dissolved organic carbon (DOC) concentration and its stable carbon isotope footprint (δ 13CDOC) in aqueous samples. The method involves a quantitative oxidation of DOC in a 200 µL liquid sample to CO2, after sample acidification and stripping by nitrogen. The detection limit of the method for DOC quantification was 0.2 mg C/L with an analytical precision of 12 %. Uncertainty of stable isotope determinations was 2 % at 0.2 mg DOC/L, while decreasing to 0.3 % at 20 mg DOC/L. Quantitative oxidation of DOC in aqueous samples was validated by using ring test water samples and Deep Sea reference seawater. The method performances of isotope analysis were evaluated by analysing different isotopic standard solutions. The applicability of the method was tested through the analysis of different environmental types of water, showing that δ 13CDOC ranged from – 23.30 to –31.85 ‰, allowing to characterize samples of different environmental origin. The developed method offers several advantages including rapidity, use of small sample volumes and minimal sample pre-treatment, making it a valuable tool for routine DOC concentration measurements paired with isotopic characterization.

A Case Study to Establish a Basis for Evaluating Geographic Origin Claims of Timber From the Solomon Islands Using Stable Isotope Ratio Analysis

Global demand for low-cost forest products is leading manufacturers and traders to source timber and wood products from vulnerable nations and delicate ecosystems. One small island nation, the Solomon Islands, is seeing exploitation of natural resources accelerating to such a point that its natural forests may be exhausted by 2036. The main causes of natural forest loss on the archipelago are unsustainable or illegal logging practices. Various laws in consumer countries require that members of industry ensure that only legally sourced timber is placed onto their respective national markets. Those that break these laws or fail to act in a way that is compliant may be subject to harsh penalties. This study aims to establish scientific data to evaluate claims that timber has originated from the Solomon Islands. This will enable Operators to carry out due diligence analysis and permit members of Law Enforcement to conduct forensic investigations. Eighty timber core samples comprising 13 different genera of tropical trees were obtained from mature trees in two sites in the Solomon Islands (Guadalcanal and Kolombangara islands) during the period August 2019 to November 2019 using a Pickering Punch sampling device. Homogenised core samples were subject to δ18O, δ2H, δ13C, and δ34S stable isotope analysis using elemental analysis-isotope ratio mass spectrometry. Additional stable isotope data from relevant taxa and geographic origins (elevation, geographic co-ordinates) were also included in this research as an initial assessment of differences in stable isotope ratios between countries. Results show that significant differences are evident in the stable isotope ratios of the sampled taxa within the Solomon Islands (Guadalcanal and Kolombangara Islands) and between other countries. These data can be used as a basis of evaluation to evaluate origin claims of timber or wood products from the Solomon Islands, particularly Kolombangara Island. Furthermore, in the right context, these data can also be used to establish whether timber or wood products declared to be from origins other than the Solomon Islands have stable isotope ratios that are consistent with data from the Solomon Islands. If not, this would suggest foreign timber/forest products are from elsewhere and are being passed-off as originating from the Solomon Islands.

Determination of Sucrose Additives and Geographical Origin Markers in Honey Using Isotope Ratio Mass Spectrometry and Ultra High Performance Liquid Chromatography – Evoparative Light Scattering Detection

Abstract Honey can easily be adulterated with various cheaper sweeteners for higher commercial profits. Commonly used adulterants include high fructose maize syrup and refined cane sugar, which origin is C4 plants. Isotope ratio mass spectrometry (IRMS) can be used to determine the adulteration of honey with C4 plants. A significant δ13C value difference between honey and its protein provide valuable information of honey authenticity. The IRMS analysis of honey and its proteins and ultra high performance liquid chromatography – evoparative light scattering detection analysis for adulterated honey were performed to assess honey authenticity and origin. Principal component analysis of the obtained IRMS data was performed in order to determine discrimination between groups and for visualisation of observations. The results showed that isotope ratio values in combination with principal component analysis could be useful for determination of adulterant and discrimination of honey of different geographical origins. The chromatography results show ascending increase of the deliberately added sucrose, but do not fully recover the expected value.

Stable Isotope Ratio Analysis for the Comparison of Timber From Two Forest Concessions in Gabon

Consumers are becoming increasingly aware of the environmental impacts caused by deforestation and illegal logging and there is an increasing demand for supply chain transparency and traceability of wood products. Many importing and exporting nations have implemented regulations which aim to control the origin and species of traded timbers of high ecological importance and economic value. However, despite growing interest in method development for timber authentication purposes, many studies have been limited by insufficient numbers of authentic timber reference samples. Our aim was to address the differences in stable isotope ratio profile of bulk, homogenized wood samples collected from living or recently felled trees in two FSC concessions in Gabon, which are approximately 240 km apart, for the purposes of origin classification and protecting valuable forest commodities. Forty-seven timber samples comprising 10 genera of tropical trees were obtained using a Pickering Punch sampling device or chainsaw from two forest concessions in Gabon (Precious Woods Group and Compagnie des Bois du Gabon) during July 2019. Samples were subject to δ18O, δ2H, δ13C, δ15N, and δ34S stable isotope analysis using elemental analysis-isotope ratio mass spectrometry (EA-IRMS). Results show that significant differences are evident in the stable isotope ratios of Aucoumea klaineana between Precious Woods Group and Compagnie des Bois du Gabon forest concessions. Relationships are evident between climatic and geological variables and the stable isotope ratios of the samples suggesting that further degrees of origin classification may be achievable in Gabon. For other species, insufficient numbers meant the possibility to determine discriminating factors between the two concessions was limited though data from these samples may prove useful to contribute to the understanding of stable isotope variability in tropical timber. The data presented establish a basis for evaluating origin claims of forest products and timber from the Compagnie des Bois du Gabon and Precious Woods Group concessions and lay a foundation for future development of timber tracking technologies in Gabon. The technique can be used for purposes of due diligence or forensic investigation by law enforcement as part of demand-side regulations such as the EU Timber Regulation, Illegal Logging Prevention Act, or the Lacey Act.

Extraction possibilities of lipid fraction and authentication assessment of chaga (Inonotus obliquus)

Chaga (Inonotus obliquus) (Fr.) Pilát is a black perennial fungus that grows on adult birch trunks and has been traditionally used as a health promoting remedy in different countries. The lipophilic compounds of chaga were isolated applying conventional (Soxhlet), intensive (ultrasound assisted, accelerated solvent extraction) and environmentally friendly (supercritical fluid) extraction methods utilizing both polar and non-polar solvents. Soxhlet extraction showed the highest inotodiol yield (231 mg/100 g chaga) using cyclohexane. For authentication of chaga samples, isotope ratio mass spectrometry (IRMS) and multi-elemental analysis was performed. The extraction yield and profile of lipids, sterols and triterpenoids depend on the chaga origin. The amount of lipids in chaga samples varied in the range from 0.5 to 1.0%. The antimicrobial activity of chaga extracts against pathogenic and opportunistic pathogenic microorganisms was demonstrated. The results showed that extraction using supercritical CO2 delivered yields comparable to those obtained by conventional and intensive extraction techniques. Amount of target compounds (sterols and triterpenoids) obtained using supercritical CO2 extraction could represent the future for sustainable industrial-scale production of biologically active ingredients with antimicrobial activity. Authentication using IRMS allowed to differentiate between natural (fruiting bodies) and cultivated (mycelium) chaga.