Stable carbon, nitrogen, and oxygen isotope analysis as a potential tool for verifying geographical origin of beef

Stable isotope analysis of organic elements such as carbon and nitrogen has been employed as a powerful tool for provenance determination of food materials, because isotopic compositions of the materials reflect many factors in natural environment. In this study, we examined carbon, nitrogen, and oxygen isotope signatures of beef from Australia, Japan, and USA, in order to confirm the method as a potential tool for verifying geographical origin of beef commercially distributed in Japan. Defatted dry matter of beef from USA was characterized by higher carbon isotopic composition (-13.6 per thousand to -11.1 per thousand) than that from Japan (-19.6 per thousand to -17.0 per thousand) and Australia (-23.6 per thousand to -18.7 per thousand). That from Australia was characterized by higher oxygen isotopic composition (+15.0 per thousand to +19.4 per thousand) than that from Japan (+7.3 per thousand to +13.6 per thousand) and USA (+9.5 per thousand to +11.7 per thousand). The oxygen isotopic composition in Japanese beef showed a positive correlation with the isotopic composition of cattle drinking water, the difference in which is clearly latitude dependent. These results suggest that a comparison of carbon, nitrogen, and oxygen isotopic compositions is applicable as a potential tool to discriminate the provenance of beef not only between different countries (i.e. Australia, Japan, and USA) but also among different regions within Japan.     

Stable carbon and nitrogen isotope analysis of avian uric acid

We report results obtained using a new technique developed to measure the stable-isotope composition of uric acid isolated from bird excreta (guano). Results from a diet-switch feeding trial using zebra finches suggest that the delta(13)C of uric acid in the guano equilibrates with the diet of the bird within 3 days of a change in diet, while the equilibration time for delta(15)N may be longer. The average carbon isotope discrimination between uric acid and food before the diet switch was +0.34 +/- 1 per thousand (1sigma) while after the diet switch this increased slightly to +0.83 +/- 0.7 per thousand (1sigma). Nitrogen isotope discrimination was +1.3 +/- 0.3 per thousand (1sigma) and +0.3 +/- 0.3 per thousand (1sigma) before and after the diet switch; however, it is possible that the nitrogen isotope values did not fully equilibrate with diet switch over the course of the experiment. Analyses of other chemical fractions of the guano (organic residue after uric acid extraction and non-uric acid organics solubilised during extraction) suggest a total range of up to 3 per thousand for both delta(13)C and delta(15)N values in individual components of a single bulk guano sample. The analysis of natural samples from a range of terrestrial and marine species demonstrates that the technique yields isotopic compositions consistent with the known diets of the birds. The results from natural samples further demonstrate that multiple samples from the same species collected from the same location yield similar results, while different species from the same location exhibit a range of isotopic compositions indicative of different dietary preferences. Given that many samples of guano can be rapidly collected without any requirement to capture specimens for invasive sampling, the stable-isotope analysis of uric acid offers a new, simple and potentially powerful tool for studying avian ecology and metabolism.     

Stable isotope variation as a tool to trace the authenticity of beef

Organic beef coming principally from Germany was analysed for the hydrogen, carbon, oxygen, nitrogen and sulfur stable isotopic composition to test the possibility of tracing back the geographical origin. Since there is a well-known pattern of D/H and ¹⁸O/¹⁶O in meteoric water and in ground water, there is an existing link to tissue water in the beef. By including the stable isotope ratios of the other elements of life further information is available: soils show different isotope ratios of ¹⁵N/¹⁴N and ³⁴S/³²S depending on the geological composition, cultivation and atmospheric sulfur deposition. As organic farming is mainly obliged to use only their produced fodder, that ratio is reflected in the beef as well.Different organic beef samples from various German farms have been collected and analysed over nearly two years. To check the differentiation of foreign beef, samples from Argentina and Chile were also included in the study. The analyses of meat samples indicate that it is possible to trace back the region (e.g. Argentina and Germany) by using isotopes of oxygen and hydrogen. A local geographical differentiation can be done by using the stable isotopes of nitrogen and sulfur, as was demonstrated for three farms in Colonia Bay. An optimal differentiation also depends on the quality of further information (e.g. the season, kind of cattle breeding or the declaration of the local geographical origin). Certainly authenticity of beef is not only linked with the geographical origin but can also reflect the differentiation of organic and conventional farming. The fodder of organic cattle farming consists mainly of C₃ plants and the use of C₄ plants is more usual in conventional cattle farming. A ¹³C/¹²C ratio above –20 appears as a limit for organic farming. Increased values have to be controlled based on their authenticity.     

Stable carbon isotope ratios of methyl-branched fatty acids are different to those of straight-chain fatty acids in dairy products

Methyl-branched fatty acids (MBFAs) are the dominant form of fatty acid found in many bacteria. They are also found at low levels in a range of foodstuffs, where their presence has been linked to bacterial sources. In this study we evaluated the potential of compound-specific isotope analysis to obtain insights into the stable carbon isotope ratios (δ¹³C values in ‰) of individual MBFAs and to compare them to the stable carbon isotope ratios of straight-chain fatty acids in food. Due to their low abundance in foodstuffs, the MBFAs were enriched prior to gas chromatography coupled to isotope ratio mass spectrometric (GC–IRMS) analysis. After transesterification, urea complexation was used to suppress the 16:0 and 18:0 methyl esters that were dominant in the samples. Following that, silver-ion high performance liquid chromatography was used to separate the saturated from the unsaturated fatty acids. The resulting solutions of saturated fatty acids obtained from suet, goat’s milk, butter, and human milk were studied by GC–IRMS. The δ¹³C values of fatty acids with 12–17 carbons ranged from −25.4‰ to −37.6‰. In all samples, MBFAs were most depleted in carbon-13, followed by the odd-chain fatty acids 15:0 and 17:0. 14:0 and 16:0 contained the highest proportions of carbon-13. The results from this study illustrate that MBFAs have distinctive δ¹³C values and must originate from other sources and/or from very different substrates. These measurements support the initial hypothesis that δ¹³C values can be used to attribute MBFAs to particular sources.     

Stable isotope mass spectrometry in honey analysis

Honey is frequently subject to adulteration with relatively cheap high fructose corn syrup. Not only is this a serious consumer fraud but it is a major threat to the apicultural industry. Stable isotope ratio analysis using mass spectrometry may be used to detect honey that has been adulterated with high fructose corn syrup due to the fact that honey, usually from C₃ plants, has a different ¹³C:¹²C ratio than high fructose corn syrup which is a C₄ plant product     

Stable isotope dilution assays in mycotoxin analysis

The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC–MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.     

Stable isotope (13C, 15N and 34S) analysis of the hair of modern humans and their domestic animals

Relationships between dietary status and recent migration were examined by delta(13)C, delta(15)N and delta(34)S analysis of hair samples from 43 modern humans living in a rural community in SW England. The isotopic content of 38 'local' hair samples was compared with that of five recently arrived individuals (from Canada, Chile, Germany and the USA). Hair samples from domestic animals (i.e. mainly cats, dogs, cows and horses) were analysed to examine the difference in delta(13)C, delta(15)N and delta(34)S values between herbivores and carnivores. Generally, modern human hair data from the triple stable isotope (delta(13)C, delta(15)N and delta(34)S) provided enough information to confirm the dietary status and origin of the individual subjects. The dietary intake was generally reflected in the animal hair delta(15)N and delta(13)C values, i.e. highest in the carnivores (cats). However, a non-local origin of food sources given to domesticated omnivores (i.e. dogs) was suggested by their hair delta(34)S values.     

Stable isotope analysis of dissolved organic carbon in soil solutions using a catalytic combustion total organic carbon analyzer‐isotope ratio mass spectrometer with a cryofocusing interface

Stable carbon isotopes are a powerful tool to assess the origin and dynamics of carbon in soils. However, direct analysis of the ¹³C/¹²C ratio in the dissolved organic carbon (DOC) pool has proved to be difficult. Recently, several systems have been developed to measure isotope ratios in DOC by coupling a total organic carbon (TOC) analyzer with an isotope ratio mass spectrometer. However these systems were designed for the analysis of fresh and marine water and no results for soil solutions or ¹³C‐enriched samples have been reported. Because we mainly deal with soil solutions in which the difficult to oxidize humic and fulvic acids are the predominant carbon‐containing components, we preferred to use thermal catalytic oxidation to convert DOC into CO₂. We therefore coupled a high‐temperature combustion TOC analyzer with an isotope ratio mass spectrometer, by trapping and focusing the CO₂ cryogenically between the instruments. The analytical performance was tested by measuring solutions of compounds varying in the ease with which they can be oxidized. Samples with DOC concentrations between 1 and 100 mg C/L could be analyzed with good precision (standard deviation (SD) ≤0.6‰), acceptable accuracy, good linearity (overall SD = 1‰) and without significant memory effects. In a ¹³C‐tracer experiment, we observed that mixing plant residues with soil caused a release of plant‐derived DOC, which was degraded or sorbed during incubation. Based on these results, we are confident that this approach can become a relatively simple alternative method for the measurement of the ¹³C/¹²C ratio of DOC in soil solutions. Copyright © 2010 John Wiley & Sons, Ltd.     

Stable isotope analysis of safety matches using isotope ratio mass spectrometry--a forensic case study

Isotope ratio mass spectrometry (IRMS) was used to assess what contribution the technique could make towards the comparative analysis of matchstick samples within the 'normal' framework of a forensic investigation. A method was developed to allow the comparison of samples submitted as a result of an investigation, with the added advantage of rapid sample turn-around expected within this field. To the best of our knowledge this is the first time that wooden safety matches have been analysed using IRMS. In this particular case, bulk stable isotope analysis carrried out on a 'like-for-like' basis could demonstrate conclusively that matches seized from a suspect were different from those collected at the scene of crime. The maximum delta13C variability observed within one box was 2.5 per thousand, which, in conjunction with the error of measurement, was regarded to yield too wide an error margin as to permit differentiation of matchsticks based on 13C isotopic composition alone given that the 'natural' 13C abundance in wood ranges from -20 to -30 per thousand. However, from the delta2H values obtained for crime scene matches and seized matches of -114.5 per thousand and -65 per thousand, respectively, it was concluded that the matches seized were distinctly different from those collected at the crime scene.     

Stable isotope dilution analysis of wine fermentation products by HS-SPME-GC-MS

The aim of this study was to quantify, in a single analysis, 31 volatile fermentation-derived products that contribute to the aroma of red and white wine. We developed a multi-component method based on headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). The 31 volatile compounds analysed include ethyl esters, acetates, acids and alcohols. Although these compounds have a range of functional groups, chemical properties, volatilities, affinities for the SPME fibre, and are found in wine at various concentrations, the accuracy of the analysis was achieved with the use of polydeuterated internal standards for stable isotope dilution analyses (SIDA). Nine of the labelled standards were commercially available, while 22 were synthesised. The method was validated by a series of duplicate spiked standard additions to model, white and red wine matrices over the concentration range relevant for each compound in wine. This demonstrated that the appropriate use of SIDA helped to account for matrix effects, for instance potential sources of variation such as the relative response to the MS detector, ionic strength, ethanol content and pH of different wine matrices. The resultant calibration functions had correlation coefficients (R²) ranging from 0.995 to 1.000. Each compound could be quantified at levels below its aroma threshold in wine. Relative standard deviations were all <5%. The method was optimised for the best compromise (over the 31 compounds) of wine dilution factor, level of sodium chloride addition, SPME fibre, SPME temperature, SPME time, GC column and MS conditions. Confirmation of identity was achieved by retention time and peak shape, and measurement of at least three ions for each analyte and internal standard with the MS operating in selected ion monitoring mode to facilitate more precise quantitation with a high sampling rate. The method is a valuable research tool with many relevant applications. A novel method for the combined chiral separation and SIDA quantification of 2- and 3-methylbutanoic acid is also demonstrated.     

Stable carbon and oxygen isotopic analysis of carbon monoxide in natural waters

Techniques have been developed to allow on-line simultaneous analysis of concentration and stable isotopic compositions ((13)C and (18)O) of dissolved carbon monoxide (CO) in natural water, using continuous-flow isotope ratio mass spectrometry (CF-IRMS). The analytical system consisted sequentially of a He-sparging bottle of water, a gas dryer, CO(2)-trapping stage using both Ascarite trap and silica-gel packed gas chromatography (GC), on-line oxidation to CO(2) using the Schütze reagent, cryofocusing, GC purification using a capillary column and measurement by CF-IRMS. Each sample analysis takes about 40 minutes. The detection limit with delta(13)C standard deviation of 0.5 per thousand is 300 pmol and that with delta(18)O deviation of 1.0 per thousand is 750 pmol. Analytical blanks associated with these methods are 21+/-9 pmol. The procedures are evaluated through analyses of temporally varying concentration and isotopic compositions of CO in an artificial lake on the university campus. The delta(13)C and delta(18)O values of CO showed wide variation in accordance with diurnal variation of CO concentration, probably due to significant isotopic effects during photochemical production and microbial oxidation of CO in the aquatic environment. The delta(13)C and delta(18)O values of CO should be a useful tool in studies of the mechanism and pathways of CO production and consumption in natural waters.     

Stable isotope ratios of carbon and hydrogen to distinguish olive oil from shark squalene‐squalane

Squalene and its hydrogenated derivate squalane are widely used in the pharmaceutical and cosmetic fields. The two compounds are mainly produced from the liver oil of deep sea sharks and from olive oil distillates. Squalene and squalane from shark cost less than the same compounds derived from olive oil, and the use of these shark‐derived compounds is unethical in cosmetic formulations. In this work we investigate whether ¹³C/¹²C and ²H/¹H ratios can distinguish olive oil from shark squalene/squalane and can detect the presence of shark derivates in olive oil based products. The ¹³C/¹²C ratios (expressed as δ¹³C values) of bulk samples and of pure compounds measured using isotope ratio mass spectrometry (IRMS) were significantly lower in authentic olive oil squalene/squalane (N: 13; ?28.4 ± 0.5‰; ?28.3 ± 0.8‰) than in shark squalene/squalane samples (N: 15; ?20.5 ± 0.7‰; ?20.4 ± 0.6‰). By defining δ¹³C threshold values of ?27.4‰ and ?26.6‰ for olive oil bulk and pure squalene/squalane, respectively, illegal addition of shark products can be identified starting from a minimum of 10%. ²H/¹H analysis is not useful for distinguishing the two different origins. δ¹³C analysis is proposed as a suitable tool for detecting the authenticity of commercial olive oil squalene and squalane samples, using IRMS interfaced to an elemental analyser if the purity is higher than 80% and IRMS interfaced to a gas chromatography/combustion system for samples with lower purity, including solutions of squalane extracted from cosmetic products. Copyright © 2010 John Wiley & Sons, Ltd.     

Stable isotope ratio analysis to differentiate temporal diets of a free‐ranging herbivore

Stable isotope ratio analysis (SIRA) of carbon (δ¹³C) and nitrogen (δ¹⁵N) in tissue samples of herbivores can identify photosynthetic pathways (C₃ vs. C₄) of plants consumed. We present results from free‐ranging Rocky Mountain elk (Cervus elaphus) that highlight the ability to differentiate diets using tissue δ¹³C and δ¹⁵N. The signatures of δ¹³C and δ¹⁵N differed in tissues of varying metabolic activity: muscle, a short‐term dietary indicator (i.e., 1–2 months) and hoof, a long‐term dietary indicator (i.e., 3–12 months). We also documented that δ¹³C and δ¹⁵N values along elk hooves (proximal, middle, distal sections) elucidated temporal shifts in dietary selection. The carbon isotopes of the composite hoof were similar to those of the middle section, but the composite hoof differed in δ¹³C from the distal and proximal sections. The δ¹³C and δ¹⁵N signatures also differed among elk populations, indicating temporal dietary shifts of individuals occupying disparate native range and human‐derived agricultural landscapes. Analyses of stable isotopes in various tissues highlighted carbon and nitrogen assimilation through time and differences in the foraging ecology of a rangeland herbivore. Published in 2009 by John Wiley & Sons, Ltd.     

Direct analysis of carbon isotope variability in albumins by liquid flow-injection isotope ratio mass spectrometry

We demonstrate the high precision C isotopic analysis of a series of purified albumins by liquid chromatography-combustion isotope ratio mass spectrometry (IRMS) by using direct aqueous liquid injection. Albumins from 18 species and albumens from chicken and turkey egg were obtained from a commercial source and shown to be of > 98% purity by capillary zone electrophoresis and high-performance liquid chromatography. One microliter of an aqueous protein solution with a total of < 40-pmol protein (2. 5 µg), which contained about 150-nmol C, was injected directly into a flowing stream of high-performance liquid chromatography grade water. The solution passed through a pneumatic nebulizer, was sprayed onto a moving wire, passed through a drying oven, and was combusted in a furnace. After the water of combustion was removed, the resulting CO₂ gas was directed to a high precision IRMS instrument operated in continuous flow mode. The average precision across the 20 samples analyzed was SD(δ ¹³C)=0.45%., and the average accuracy was δ¹³C < 0.4%. compared to aliquots analyzed by conventional preparation by using combustion tubes and dual inlet analysis. The observed isotope ratio range was about ?22.5%. < δ ¹³C_PDB < ?16%. as expected for modern materials from a natural source. These results demonstrate rapid, high precision, and accurate C isotopic analysis of untreated macromolecules in an aqueous stream by liquid source IRMS.