Characterization of vanillin carbon isotope delta reference materials.

Stable carbon isotope ratio measurements are used to investigate the provenance of vanillin. In this study, a variety of commercial vanillin samples and vanilla products were analyzed to provide a frame of reference for the variability of carbon isotope delta values in various vanillin samples, with the results ranging from -20.6 to -36.7‰ relative to the Vienna Peedee Belemnite (VPDB). We present information on the development of two synthetic vanillin reference materials, VANA-1 and VANB-1, prepared in 0.75 g units in glass vials, to be used for the calibration of carbon isotope delta measurements of vanillin and other easily combustible organic materials. Characterization of 40 vials each of VANA-1 and VANB-1 was performed by three laboratories over several measurement sequences. The certified carbon isotope delta values are -31.30 ± 0.06‰ (VANA-1) and -25.85 ± 0.05‰ (VANB-1). These uncertainties, for the 95% confidence level, include considerations for measurement uncertainty, coherence of the reference materials used for calibration, batch homogeneity, and stability during storage and transportation. The results are traceable to the VPDB through a set of nine reference materials (IAEA-CH-6, USGS65, IAEA-600, NBS22, USGS61, IAEA-603, IAEA-610, IAEA-611, and IAEA-612). For up to date certified values, users should refer to doi.org/10.4224/crm.2022.vana-1 and doi.org/10.4224/crm.2022.vanb-1.

Multi-isotopes in human hair: A tool to initiate cross-border collaboration in international cold-cases.

Unidentified human remains have historically been investigated nationally by law enforcement authorities. However, this approach is outdated in a globalized world with rapid transportation means, where humans easily move long distances across borders. Cross-border cooperation in solving cold-cases is rare due to political, administrative or technical challenges. It is fundamental to develop new tools to provide rapid and cost-effective leads for international cooperation. In this work, we demonstrate that isotopic measurements are effective screening tools to help identify cold-cases with potential international ramifications. We first complete existing databases of hydrogen and sulfur isotopes in human hair from residents across North America by compiling or analyzing hair from Canada, the United States (US) and Mexico. Using these databases, we develop maps predicting isotope variations in human hair across North America. We demonstrate that both δ2H and δ34S values of human hair are highly predictable and display strong spatial patterns. Multi-isotope analysis combined with dual δ2H and δ34S geographic probability maps provide evidence for international travel in two case studies. In the first, we demonstrate that multi-isotope analysis in bulk hair of deceased border crossers found in the US, close to the Mexico-US border, help trace their last place of residence or travel back to specific regions of Mexico. These findings were validated by the subsequent identification of these individuals through the Pima County Office of the Medical Examiner in Tucson, Arizona. In the second case study, we demonstrate that sequential multi-isotope analysis along the hair strands of an unidentified individual found in Canada provides detailed insights into the international mobility of this individual during the last year of life. In both cases, isotope data provide strong leads towards international travel.

Site-specific carbon isotope measurements of vanillin reference materials by nuclear magnetic resonance spectrometry.

Vanillin, one of the world's most popular flavor used in food and pharmaceutical industries, is extracted from vanilla beans or obtained (bio)-synthetically. The price of natural vanillin is considerably higher than that of its synthetic alternative which leads increasingly to counterfeit vanillin. Here, we describe the workflow of combining carbon isotope ratio combustion mass spectrometry with quantitative carbon nuclear magnetic resonance spectrometry (13C-qNMR) to obtain carbon isotope measurements traceable to the Vienna Peedee Belemnite (VPDB) with 0.7‰ combined standard uncertainty (or expanded uncertainty of 1.4‰ at 95% confidence level). We perform these measurements on qualified Bruker 400 MHz instruments to certify site-specific carbon isotope delta values in two vanillin materials, VANA-1 and VANB-1, believed to be the first intramolecular isotopic certified reference material (CRMs).

Discontinuity in the Realization of the Vienna Peedee Belemnite Carbon Isotope Ratio Scale.

By convention, carbon isotope ratios are expressed relative to VPDB defined by the calcite standard NBS19 in the 1980s. [See T. Coplen, Pure Appl. Chem. 1994, 66, 273-276.] To improve the realization of the VPDB scale, a second fixed point (lithium carbonate, LSVEC) was introduced in 2006 [T. Coplen et al. Anal. Chem. 2006, 78, 2439-2441], which is now known to be isotopically unstable. [Assonov, S. Rapid Commun. Mass Spectrom., 2018, 32, 827-830.] With the high-quality reference materials made available in 2020, it is now possible to realize the VPDB scale with high confidence. [Assonov, S. et al. Rapid Commun. Mass Spectrom., 2020, 34, e8867; Assonov, S. Rapid Commun. Mass Spectrom. 2021, 35, e9014; Qi, H. et al. Rapid Commun. Mass Spectrosc. 2021, 35, e9006.] Here, we report the analysis of 25 reference materials using isotope ratio combustion mass spectrometry, show the discontinuity between the values measured against the new IAEA reference materials and the values currently assigned to these reference materials on the VPDB2006, and provide a link bringing these materials onto the new VPDB2020.

Assessing geographic controls of hair isotopic variability in human populations: A case-study in Canada.

Studying the isotope variability in fast-growing human tissues (e.g., hair, nails) is a powerful tool to investigate human nutrition. However, interpreting the controls of this isotopic variability at the population scale is often challenging as multiple factors can superimpose on the isotopic signals of a current population. Here, we analyse carbon, nitrogen, and sulphur isotopes in hair from 590 Canadian resident volunteers along with demographics, dietary and geographic information about each participant. We use a series of machine-learning regressions to demonstrate that the isotopic values in Canadian residents' hair are not only influenced by dietary choices but by geographic controls. First, we show that isotopic values in Canadian residents' hair have a limited range of variability consistent with the homogenization of Canadian dietary habits (as in other industrialized countries). As expected, some of the isotopic variability within the population correlates with recorded individual dietary choices. More interestingly, some regional spatial patterns emerge from carbon and sulphur isotope variations. The high carbon isotope composition of the hair of eastern Canadians relative to that of western Canadians correlates with the dominance of corn in the eastern Canadian food-industry. The gradient of sulphur isotope composition in Canadian hair from coast to inland regions correlates with the increasing soil pH and decreasing deposition of marine-derived sulphate aerosols in local food systems. We conclude that part of the isotopic variability found in the hair of Canadian residents reflects the isotopic signature associated with specific environmental conditions and agricultural practices of regional food systems transmitted to humans through the high consumption rate of intra-provincial food in Canada. Our study also underscores the strong potential of sulphur isotopes as tracers of human and food provenance.

Carbon isotope measurements of foods containing sugar: A survey.

Over 200 sugars, high sugar content foods, and sugar-free alternative products found in Canadian supermarkets were analyzed for carbon isotope (δ13C) values, adding to the record of food items with reported δ13C values, which can aid in food authentication and human diet studies. The δ13C values of the products were mostly consistent with the photosynthetic pathway of the plant from which the main ingredients were derived. With the exception of those sugars and syrups derived from known C3 sources, all of the sugars and syrups and most of the sugar added to food products were from C4 plants such as sugar cane or corn syrup. Many sweeteners are chemically manufactured, and most reflect an intermediate δ13C value. A mixing model estimated the contribution of C3- and C4-based ingredients in foods with high sugar content, which may be used to evaluate the percentage of added sugar from C3- or C4-sources.

Carbon Isotopic Measurements of Nanotubes to Differentiate Carbon Sources.

Stable carbon isotope (δ(13C)) analysis can provide information concerning the starting materials and the production process of a material. Carbon nanotubes (CNTs) are produced using a variety of starting materials, catalysts, and production methods. The use of δ(13C) as a tool to infer the nature of starting materials to gain insight into the mechanics of CNT growth was evaluated. The production process of NRC's SWCNT-1 was traced via the δ(13C) measurement of the available starting materials, intermediate products, and the final product. As isotopic fractionation is likely negligible at high temperatures, the δ(13C) value of the starting materials was reflected in the δ(13C) value of the final CNT product. For commercially available CNTs, the estimated δ(13C) values of identified starting materials were related to the δ(13C) signatures of CNTs. Using this information and the δ(13C) values of CNTs, the nature of unknown carbon sources was inferred for some samples. The use of δ(13C) analysis may be used as a tracer to differentiate between those processes that use relatively 13C-depleted carbon source(s) such as carbon monoxide, methane, or natural gas, and those that do not.

Three certified sugar reference materials for carbon isotope delta measurements.

RATIONALE: For isotope delta analysis, it is preferable to have at least two matrix-matched reference materials whose isotope delta values encompass those of the samples to be analyzed. The National Research Council Canada (NRC) has developed three sugar Certified Reference Materials (CRMs), BEET-1 (beet sugar), GALT-1 (galactose), and FRUT-1 (fructose), to be collectively used for carbon isotope delta measurements in sugars, and other organic materials. METHODS: All materials were homogenized and packaged in glass ampules. All three sugar materials were analyzed at the NRC using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Six additional laboratories also provided EA/IRMS measurements. Data from all laboratories were re-normalized using three international secondary reference materials (IAEA-CH-6, USGS40, and USGS62) included as blind samples in the inter-laboratory comparison, thus providing added quality control and robustness to the study. RESULTS: Re-normalized carbon isotope delta values from each laboratory were combined using a random laboratory effects statistical model with accounting of the correlations between the laboratory results due to the use of the same reference materials for calibration. The consensus δ(13 C) values and combined standard uncertainties which include effects due to characterization, homogeneity, and stability for BEET-1, GALT-1, and FRUT-1 are -26.02(7) ‰, -21.41(6) ‰, and -10.98(5) ‰, respectively, on the VPDB scale. CONCLUSIONS: Three new δ(13 C) sugar CRMs (BEET-1, GALT-1, and FRUT-1) were developed and are available from NRC. These three CRMs can be utilized as a set for daily δ(13 C) scale normalization of sugar-based or other organic materials in order to produce reliable δ(13 C) measurements.

Uncertainty evaluation in normalization of isotope delta measurement results against international reference materials.

Isotope delta measurements are normalized against international reference standards. Although multi-point normalization is becoming a standard practice, the existing uncertainty evaluation practices are either undocumented or are incomplete. For multi-point normalization, we present errors-in-variables regression models for explicit accounting of the measurement uncertainty of the international standards along with the uncertainty that is attributed to their assigned values. This manuscript presents framework to account for the uncertainty that arises due to a small number of replicate measurements and discusses multi-laboratory data reduction while accounting for inevitable correlations between the laboratories due to the use of identical reference materials for calibration. Both frequentist and Bayesian methods of uncertainty analysis are discussed.

An inter-laboratory comparative study into sample preparation for both reproducible and repeatable forensic 2H isotope analysis of human hair by continuous flow isotope ratio mass spectrometry.

Stable isotope analysis of organic materials for their hydrogen ((2)H), carbon ((13)C), nitrogen ((15)N) or oxygen ((18)O) isotopic composition using continuous flow isotope ratio mass spectrometry (CF-IRMS) is an increasingly used tool in forensic chemical analysis. (2)H isotopic analysis can present a huge challenge, especially when dealing with exhibits comprising exchangeable hydrogen such as human scalp hair. However, to yield forensic data that are fit for purpose, analysis of the (2)H isotopic composition of the same homogeneous human hair sample by any laboratory worldwide must yield the same isotopic composition within analytical uncertainty. This paper presents longitudinal (2)H isotope data for four human hair samples of different provenance, measured by three different laboratories whose sample preparation was based on a two-stage H exchange equilibration method. Although each laboratory employed varying means to comply with the generic features of the sample preparation protocol such as the (2)H isotopic composition of exchange waters or drying down of samples prior to analysis, within each laboratory the Principle of Identical Treatment (P.I.T.) was applied for each individual experiment. Despite the variation in materials and procedures employed by the three laboratories, repeatable and reproducible 'true' (2)H isotope values (δ(2)H(hair,true)) were determined by each laboratory for each of the four stock samples of human scalp hair. The between-laboratory differences for obtained δ(2)H(hair,true) values ranged from 0.1 to 2.5 ‰. With an overall 95% confidence interval of ±2.8 ‰, these differences were not significantly different, which suggests that the general method of two-stage exchange equilibration carried out at ambient temperature is suitable for accurately and reproducibly determining 'true' δ(2)H-values for hair and other proteins provided that certain key conditions are met.

Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666.

Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from -17.4 to -22.4 per thousand. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.

An approach for assessing total instrumental uncertainty in compound-specific carbon isotope analysis: implications for environmental remediation studies.

Determination of compound-specific carbon isotope values by continuous flow isotope ratio mass spectrometry is impacted by variation in several routine operating parameters of which one of the most important is signal size, or linearity. Experiments were carried out to evaluate the implications of these operating parameters on both reproducibility and accuracy of delta13C measurements. A new method is described for assessing total instrumental uncertainty of routine compound-specific delta13C analysis, incorporating both accuracy and reproducibility. These findings have important implications for application of compound-specific isotope analysis in environmental geochemistry and in particular for the rapidly developing field of isotopic investigation of biodegradation and remediation of organic chemicals in contaminant hydrogeology.

Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site.

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.

Carbon isotopic fractionation during aerobic vinyl chloride degradation.

Vinyl chloride (VC) is a carcinogenic contaminant commonly found in groundwater. Much research has focused on anaerobic reductive dechlorination of VC, and recently on aerobic VC degradation. In this study, the stable carbon isotope enrichment factor associated with aerobic VC assimilation was determined for Mycobacterium sp. strains JS60, JS61, and JS617 and Nocardioides sp. strain JS614. The enrichment factors ranged from -8.2+/-0.1 to -7.0+/-0.3 % and did not change as a function of biomass concentration. The measured enrichment factors for aerobic VC degradation were smaller than those reported for anaerobic VC degradation. Enrichment factors can also be expressed in terms of kinetic isotope effects (KIEs), 12k/13k, which result from the difference in reaction rates of bonds containing light and heavy isotopes. The KIEs for aerobic VC degradation (1.01+/-0.001) were smaller than those for anaerobic VC degradation (1.03+/-0.007). From the perspective of bond breakage during a chemical reaction, the larger KIE associated with anaerobic VC degradation as compared to aerobic VC degradation agrees with KIE theory. This theory predicts that larger fractionations can be expected in reactions where heavier atoms are involved (i.e., C-Cl bond for anaerobic versus C=C for aerobic) and in reactions involving large changes in vibrational frequencies of the molecule between its ground state and transition state (i.e., C-Cl cleavage versus C=C epoxidation). The significant fractionation observed during aerobic VC degradation suggests that stable carbon isotope measurements may be used as a tool to distinguish between biodegraded and nonbiodegraded VC.