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2H/1H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of 2H, 13C, 15N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the 2H, 13C, 15N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ∼81 % of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ∼17 %. Air humidity contributed an unspecified amount. Additionally, we monitored 13C and 15N-enrichment in bone collagen and found strong linear correlations with the 2H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers' collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.

International audience; Of the most abundant elemental constituents of organic matter, hydrogen has received the least attention due to its exchangeability with ambient water. The development of steam equilibration using two isotopically distinct waters can enable the user to calculate the proportion of exchangeable H and generate a δ2H value of the non-exchangeable H. Three fossil faunal assemblages from Europe were selected in order to investigate collagen δ2H with respect to body mass, diet and to compare chronological periods in the Quaternary. δ15N values in each suite of fauna show trophic level enrichment, a trend which was also observed in δ2H values. In the case of predatory species with lower δ15N values than other carnivore species, the use of hydrogen isotopes could help to infer the consumption of prey with lower δ15N values rather than a significant dietary contribution of plant foods. For example, a cave bear which exhibits low δ2H values are consistent with low δ15N and therefore point to an herbivorous diet and not omnivory. The combination of δ2H, with δ15N and δ13C, from archaeological and ecological studies of humans and fauna could help reconstruct prey preference; which is especially important when trying to distinguish the quantity of marine versus freshwater resources in diets. Relative to dietary influences, climate variability seems to be a minor control on δ2H in the faunal assemblages studied here.

Summary: Hydrogen (H) isotope ratio (δ2H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2H variability observed in plant compounds not being fully elucidated. We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ2H values of plant organic compounds. The experiments demonstrate that information recorded in the δ2H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where 2H‐fractionations occur in the biosynthesis of plant organic compounds and how these 2H‐fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems. Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.

An international project developed, quality-tested, and determined isotope-delta values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-delta scales. The RMs span a range of delta H-2(VSMOW-SLAP) values from -210.8 to +397.0 mUr or parts per thousand, for delta C-13(VPDB-LSVEC) from -40.81 to +0.49 mUr and for delta N-15(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C-16 n-alkanes, n-C-20-fatty acid methyl esters (FAMEs), glycines, and L-valines, together with polyethylene powder and string, one n-C-17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a H-2-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of delta H-2 values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain C-13 and carbon-bound organic H-2-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

RATIONALE The condition of the pyrolysis reactor is very important for obtaining stable, precise hydrogen isotopic ratios using gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC/TC/IRMS). However, few studies of the conditioning process have been conducted, and little is known about the best methods for high-precision hydrogen isotope analysis. METHODS We investigated δ2H variations and observed the changes in carbon coating using six different conditioning methods for the pyrolysis alumina tube: (i) no treatment; (ii) conditioning with 4 μL hexane; (iii) conditioning with 2 μL hexane; (iv) conditioning with 2 μL hexane followed by backflushing overnight; (v) conditioning with 10 s of backflushing with methane; (vi) conditioning with 3 s of backflushing with methane. RESULTS Conditioning the alumina tube can improve the pyrolysis efficiency of organic compounds because a moderate amount of carbon acts as a catalyst in high-temperature regions of the alumina tube. Carbon actually flows in the tube and is difficult to confine to the high-temperature region. Insufficient amounts of carbon in the high-temperature regions lead to incomplete pyrolysis of organic compounds and lower δ2H values due to kinetic fractionation of hydrogen isotopes. In contrast, excess hexane or methane can lead to higher δ2H values, probably due to enrichment of deuterium in the hydrocarbon residue. CONCLUSIONS The δ2H values obtained by Method 6 are closest to the TC/EA δ2H values and are more precise than those obtained by other methods, perhaps because this method introduces a moderate, consistent amount of carbon with each sample injection. Copyright © 2012 John Wiley & Sons, Ltd.

RATIONALE In recent experiments, we found that compound-specific δ2H values can vary as a result of changing the gas chromatography temperature program under common pyrolysis conditions. To achieve better precision, it is necessary to examine the details and find a solution to this problem when using gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC-TC-IRMS) for hydrogen isotope analysis. METHODS A test was designed to find the possible temperature effect under four different GC temperature ramp rates using n-alkanes (n-C21, n-C27, and n-C31) and fatty acids (n-C12, n-C18, and n-C24). The common 'hexane' method was used initially to condition the pyrolysis reactor. Experiments were then carried out using the 'methane condition' method because it was considered to improve pyrolysis efficiency. RESULTS Under the 'hexane condition' the measured hydrogen isotope ratios of the n-alkanes and n-fatty acids became more positive with increasing GC temperature ramp rate. The ion current intensity of hydrogen also generally increased. However, when the 'methane condition' method was used, the measured δ2H values of the n-alkanes and n-fatty acids showed little change under different GC temperature ramp rates. CONCLUSIONS Higher pyrolysis efficiency could reduce the tailing of the H2 peak and the related isotopic variations at increased GC temperature ramp rates. In addition, too slow a temperature ramp rate could broaden the peak width and thus increase the background effect and possible isotopic fractionations in the split interface; this could also influence the hydrogen isotope values. We therefore suggest that the appropriate temperature ramp rate is an important factor in improving the precision in analyzing compound-specific hydrogen isotopes. Copyright © 2012 John Wiley & Sons, Ltd.

Hydrogen isotope ratios (D/H) of lipid biomarkers extracted from aquatic sediments were measured to determine whether they can be used as a proxy for D/H of environmental water. Values of δD were determined by using a recently developed isotope-ratio-monitoring gas chromatograph-mass spectrometer system (irmGCMS) and were confirmed by conventional hydrogen isotopic measurements (i.e., combustion followed by reduction) on individual compounds isolated by preparative capillary gas chromatography. Diverse lipids (alkanes, n-alkanols, sterols, and pentacyclic triterpenols) were analyzed to examine hydrogenisotopic controls on lipids of varying origin and biosynthetic pathway. For algal sterols (24-methylcholest- 3β-ol, 24-ethylcholest-5,22-dien-3β-ol, and 4,23,24-trimethylcholesterol, or dinosterol), the fractionation between sedimentary lipids and environmental water was -201 ± 10‰ and was similar in both marine and freshwater sites. In a sediment from a small lake in a forested catchment, triterpenols from terrestrial sources were enriched in D by 30‰ relative to algal sterols. Apparent fractionation factors for n-alkyl lipids were smaller than those of triterpenols and were more variable, probably reflecting multiple sources for these compounds. We conclude that hydrogen-isotopic analyses of algal sterols provide a viable means of reconstructing D/H of environmental waters. Results are less ambiguous than reconstructions based on analyses of kerogen or other operationally defined organic matter fractions.

In recent experiments, we found that compound-specific δ(2)H values can vary as a result of changing the gas chromatography temperature program under common pyrolysis conditions. To achieve better precision, it is necessary to examine the details and find a solution to this problem when using gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC-TC-IRMS) for hydrogen isotope analysis.A test was designed to find the possible temperature effect under four different GC temperature ramp rates using n-alkanes (n-C(21), n-C(27), and n-C(31)) and fatty acids (n-C(12), n-C(18), and n-C(24)). The common 'hexane' method was used initially to condition the pyrolysis reactor. Experiments were then carried out using the 'methane condition' method because it was considered to improve pyrolysis efficiency.Under the 'hexane condition' the measured hydrogen isotope ratios of the n-alkanes and n-fatty acids became more positive with increasing GC temperature ramp rate. The ion current intensity of hydrogen also generally increased. However, when the 'methane condition' method was used, the measured δ(2)H values of the n-alkanes and n-fatty acids showed little change under different GC temperature ramp rates.Higher pyrolysis efficiency could reduce the tailing of the H(2) peak and the related isotopic variations at increased GC temperature ramp rates. In addition, too slow a temperature ramp rate could broaden the peak width and thus increase the background effect and possible isotopic fractionations in the split interface; this could also influence the hydrogen isotope values. We therefore suggest that the appropriate temperature ramp rate is an important factor in improving the precision in analyzing compound-specific hydrogen isotopes.

The broad scope of isotopic applications of bone collagen ranges from modern ecological and physiological investigations to learning about living conditions of animals in the past (Ambrose & DeNiro, 1989; Chisholm et al., 1982; DeNiro & Epstein 1978, 1981; DeNiro & Weiner, 1988; Hare et al., 1991; Hedges & Law, 1989; Leyden et al., 2006; Lis et al., 2008; Reynard & Hedges, 2008). Bone collagen represents one of the best preserved proteins in fossilized animal remains that in some cases has been chemically preserved for up to 120,000 years (Bocherens et al., 1999). Collagen is protected from degradation by being encapsulated into the bone mineral bioapatite. In comparison to other biopolymers found in the archaeological record, bone collagen is relatively abundant, easily extracted, and offers a long-term record of the life of humans and animals (Collins et al., 2002). Stable isotope ratios in the living biomass directly relate to the stable isotope ratios of life-supporting substrates in the environment. Animals integrate stable isotopes into their biomass through the air we breathe, water we drink and food we eat. Despite the apparent permanence of mineral constituents of bone, the organic components undergo constant turnover through the life of a vertebrate and its collagen content represents a ‘running average’ of months, years, or a lifetime, depending on the specific bone and the animal. Bone collagen thus offers a valuable insight into the environmental conditions during part of or the entire life of an animal (LeeThorp, 2008; Tuross et al., 2008). Carbon and nitrogen stable isotopes of bone collagen have been in broad use in determining diets, paleodiets, trophic levels, and paleoenvironments associated with modern and fossilized bone samples (Ambrose & DeNiro 1989; Chisholm et al., 1982; Hare et al., 1991; Schwarcz, 2000; Walker & DeNiro, 1986; Walter & Leslie, 2009), while other isotopes are only recently finding useful applications. Hydrogen is unique among all elements in terms of the doubling of the atomic mass from the light hydrogen nuclide protium 1H to the less abundant, but heavier nuclide deuterium 2H (or traditionally also abbreviated as D). This large mass difference between the two stable hydrogen isotopes creates strong fractionation effects (i.e. the preferential use or participation of one of the two isotopes in chemical and physical processes) and generates an extremely wide natural isotopic range of hydrogen stable isotope ratios (Sessions et al., 1999). Available hydrogen stable isotope data from bone collagen suggest greater variability than the most of the earlier studies had presumed, a situation that would compromise the diagnostic value of individual forensic collagen δD values for many species.

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the d values of these reference materials should bracket the isotopic range of samples with unknown d values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW-SLAP) and carbonates (NBS 19 and L-SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA-IRMS). At present only L-glutamic acids USGS40 and USGS41 satisfy these requirements for d 13 C and d 15 N, with the limitation that L-glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on-line (i.e. continuous flow) hydrogen reductive gas chromatography-isotope ratio mass-spectrometry (GC-IRMS), (ii) five nicotines for oxidative C, N gas chromatography-combustion-isotope ratio mass-spectrometry (GC-C-IRMS, or GC-IRMS), and (iii) also three acetanilide and three urea reference materials for on-line oxidative EA-IRMS for C and N. Isotopic off-line calibration against international stable isotope measurement standards at Indiana University adhered to the ‘principle of identical treatment’. The new reference materials cover the following isotopic ranges: d 2 Hnicotine � 162 to � 45%, d 13 Cnicotine � 30.05 to R7.72%, d 15 Nnicotine � 6.03 to R33.62%; d 15 Nacetanilide R1.18 to R40.57%; d 13 Curea � 34.13 to R11.71%, d 15 Nurea R0.26 to R40.61% (recommended d values refer to calibration with NBS 19, L-SVEC, IAEA-N-1, and IAEA-N-2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC-IRMS that are available with different d 15 N values. Comparative d 13 C and d 15 N on-line EA-IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA-IRMS reference materials. Published in 2009 by John Wiley & Sons, Ltd. Semi-automated stable isotope ratios analysis (SIRA) in continuous flow by on-line elemental analysis (EA) or gas chromatography (GC) interfaced with isotope ratio massspectrometry (IRMS) has become a routine and invaluable approach to characterize and trace organic compounds. It is commonly used in studies of ecology, environmental sciences, food science, forensics, geochemistry, and medical sciences. Rapid advances in instrumentation have reduced sample size requirements and increased sample throughput, for example in the GC-IRMS of hydrogen, 1 carbon 1,2 and nitrogen, 2‐4 and in the EA-IRMS of hydrogen, 5 carbon 6 and

Hydrogen isotopic analysis of organic materials has been widely applied in studies of paleoclimate, animal migration, forensics, food and flavor authentication, and the origin and diagenesis of organic matter. Hydrogen bound to carbon (C-H) generally retains isotopic information about the water present during organic matter synthesis and associated biosynthetic fractionations, but hydrogen bound to other elements (O, S, or N) can readily exchange with atmospheric water vapor and reflects recent exposure to water or vapor. These two pools must be separated to obtain meaningful information from isotope ratios of organic materials. Previously published analytical methods either replace exchangeable H chemically or control its isotopic composition, usually by equilibration with water or waters of known isotopic composition. In addition, the fraction of H that is exchangeable can vary among samples and is itself of scientific interest. Here we report an improved and automated double-equilibration approach.Samples are loaded in a 50-position autosampler carousel in an air-tight aluminum equilibration chamber. Water vapor of known isotopic composition is pumped through the chamber at 115 degrees C for at least 6 h. After flushing with dry N(2) and being cooled, the carousel is rapidly transferred from the equilibration chamber to a He-purged autosampler attached to a pyrolysis elemental analyzer connected to an isotope ratio mass spectrometer. By equilibrating two aliquots of each sample with two isotopically distinct waters, it is possible to calculate both (1) the D/H ratio of non-exchangeable H, and (2) the fraction of H that is exchangeable. Relative to previous double-equilibration techniques, this approach offers significant reductions in sample size and labor by allowing simultaneous equilibration of several tens of samples.

Compound-specific deltaD and delta13C analyses of gas mixtures are useful indicators of geochemical and environmental factors. However, the relative concentrations of individual components in gas mixtures (e.g., H2, CO2, methane, ethane, propane, i-butane, n-butane) may vary over several orders of magnitude. The determination of hydrogen and carbon compound-specific stable isotope ratios requires that the hydrogen and carbon dioxide produced from each separated component has a concentration adjusted to match the dynamic range of the stable isotope mass spectrometer. We present a custom-built gas sampling and injection system (GASIS) linked with a Delta Plus XP mass spectrometer that provides flexibility, ease of operation, and economical use of small gas samples with wide ranges of analyte concentrations. The overall on-line GC-ox/red-IRMS (Gas Chromatography - oxidation/reduction - Isotope Ratio Mass Spectrometry) system consists of (i) a customized GASIS inlet system and (ii) two alternative reactors, namely an oxidative Cu-Ni-Pt reactor at 950 degrees C for production of CO2 and a reductive graphitized Al2O3 reactor at 1420 degrees C for production of H2. In addition, the system is equipped with (iii) a liquid nitrogen spray-cooling unit for cryo-GC-focusing at -20 degrees C, and (iv) a Nafion dryer for removal of water vapor from product CO2. The three injection loops of the GASIS inlet allow flexibility in the volume of injected analyte gas (e.g., from 0.06 to 500 microL) in order to measure reproducible deltaD and delta13C values for gases at concentrations ranging from 100% down to 10 ppm. We calibrate our GC-ox/red-IRMS system with two isotopically distinct methane references gases that are combusted off-line and characterized using dual-inlet IRMS.