Simultaneous, High-Precision Measurements of δ2H and δ13C in Nanomole Quantities of Acetate Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry.

Stable hydrogen isotope compositions (2H/1H ratios) have been an invaluable tool for studying biogeochemical processes in nature, but the diversity of molecular targets amenable to such analysis is limited. Here, we demonstrate a new technique for measuring δ2H of biomolecules via Orbitrap mass spectrometry (MS) using acetate as a model analyte. Acetate was chosen as a target molecule because its production and consumption are central to microbial carbon cycling, yet the mechanisms behind acetate turnover remain poorly understood. δ2H of acetate could provide a useful constraint on these processes; however, it remains uncharacterized in nature due to analytical challenges. Electrospray ionization (ESI)-Orbitrap MS circumvents these challenges and delivers methyl-specific H-isotope compositions of acetate with nanomole sensitivity, enough to enable analyses of environmental samples. This approach quantifies the methyl-specific δ2H and molecular-average δ13C of acetate simultaneously while achieving <3 and <0.5‰ uncertainty, respectively. Using optimized ionization and Orbitrap parameters, this level of precision is obtained within 15 min using only 15 nmol of acetate. As a demonstration of our analytical approach, we cultured three acetogenic bacteria and found a large 2H-fractionation between acetate and water (>310‰ depletion) associated with the Wood-Ljungdahl pathway, while fermentation expressed a muted (∼80‰) fractionation. With its high precision and sensitivity, Orbitrap MS is a promising tool for investigating these signals in nature after offline purification. Furthermore, the ESI-Orbitrap method presented here could be applied to other molecules amenable to ESI, including central metabolites and sugars, greatly expanding the molecular targets used in hydrogen isotope biogeochemistry.

Stable isotopes of H, C and N in mice bone collagen as a reflection of isotopically controlled food and water intake.

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.

Linking variation in intrinsic water-use efficiency to isohydricity: a comparison at multiple spatiotemporal scales.

Species-specific responses of plant intrinsic water-use efficiency (iWUE) to multiple environmental drivers associated with climate change, including soil moisture (θ), vapor pressure deficit (D), and atmospheric CO2 concentration (ca ), are poorly understood. We assessed how the iWUE and growth of several species of deciduous trees that span a gradient of isohydric to anisohydric water-use strategies respond to key environmental drivers (θ, D and ca ). iWUE was calculated for individual tree species using leaf-level gas exchange and tree-ring δ13 C in wood measurements, and for the whole forest using the eddy covariance method. The iWUE of the isohydric species was generally more sensitive to environmental change than the anisohydric species was, and increased significantly with rising D during the periods of water stress. At longer timescales, the influence of ca was pronounced for isohydric tulip poplar but not for others. Trees' physiological responses to changing environmental drivers can be interpreted differently depending on the observational scale. Care should be also taken in interpreting observed or modeled trends in iWUE that do not explicitly account for the influence of D.

2 H-fractionations during the biosynthesis of carbohydrates and lipids imprint a metabolic signal on the δ2 H values of plant organic compounds.

Hydrogen (H) isotope ratio (δ2 H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2 H 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 δ2 H values of plant organic compounds. The experiments demonstrate that information recorded in the δ2 H 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 2 H-fractionations occur in the biosynthesis of plant organic compounds and how these 2 H-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.

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, L-Valines, Polyethylenes, and Oils.

An international project developed, quality-tested, and determined isotope-δ 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-δ scales. The RMs span a range of δ(2)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(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, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-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 δ(2)H 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 (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

An evaluation of alumina reaction tube conditioning for high-precision 2H/1H isotope measurements via gas chromatography/thermal conversion/isotope ratio mass spectrometry.

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 δ(2)H 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 δ(2)H values due to kinetic fractionation of hydrogen isotopes. In contrast, excess hexane or methane can lead to higher δ(2)H values, probably due to enrichment of deuterium in the hydrocarbon residue. CONCLUSIONS: The δ(2)H values obtained by Method 6 are closest to the TC/EA δ(2)H 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.

Variation of compound-specific hydrogen isotope ratios under changing temperature program in gas chromatography/thermal conversion/isotope ratio mass spectrometry.

RATIONALE: 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. METHODS: 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. 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 δ(2)H 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 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.

Carbon isotope signature of dissolved inorganic carbon (DIC) in precipitation and atmospheric CO2.

This paper describes results of chemical and isotopic analysis of inorganic carbon species in the atmosphere and precipitation for the calendar year 2008 in Wroclaw (SW Poland). Atmospheric air samples (collected weekly) and rainwater samples (collected after rain episodes) were analysed for CO2 and dissolved inorganic carbon (DIC) concentrations and for δ13C composition. The values obtained varied in the ranges: atmospheric CO2: 337-448 ppm; δ13CCO2 from -14.4 to -8.4‰; DIC in precipitation: 0.6-5.5 mg dm(-3); δ13CDIC from -22.2 to +0.2‰. No statistical correlation was observed between the concentration and δ13C value of atmospheric CO2 and DIC in precipitation. These observations contradict the commonly held assumption that atmospheric CO2 controls the DIC in precipitation. We infer that DIC is generated in ambient air temperatures, but from other sources than the measured atmospheric CO2. The calculated isotopic composition of a hypothetical CO2 source for DIC forming ranges from -31.4 to -11.0‰, showing significant seasonal variations accordingly to changing anthropogenic impact and atmospheric mixing processes.

Nicotine, acetanilide and urea multi-level 2H-, 13C- and 15N-abundance reference materials for continuous-flow isotope ratio mass spectrometry.

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 delta values of these reference materials should bracket the isotopic range of samples with unknown delta 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 delta13C and delta15N, 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: delta2H(nicotine) -162 to -45 per thousand, delta13C(nicotine) -30.05 to +7.72 per thousand, delta15N(nicotine) -6.03 to +33.62 per thousand; delta15N(acetanilide) +1.18 to +40.57 per thousand; delta13C(urea) -34.13 to +11.71 per thousand, delta15N(urea) +0.26 to +40.61 per thousand (recommended delta 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 delta15N values. Comparative delta13C and delta15N on-line EA-IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA-IRMS reference materials.

Simplified batch equilibration for D/H determination of non-exchangeable hydrogen in solid organic material.

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.