Development and evaluation of a high‐performance liquid chromatography/isotope ratio mass spectrometry methodology for δ13C analyses of amino sugars in soil

Amino sugars have been used as biomarkers to assess the relative contribution of dead microbial biomass of different functional groups of microorganisms to soil carbon pools. However, little is known about the dynamics of these compounds in soil. The isotopic composition of individual amino sugars can be used as a tool to determine the turnover of these compounds. Methods to determine the δ¹³C of amino sugars using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) have been proposed in literature. However, due to derivatization, the uncertainty on the obtained δ¹³C is too high to be used for natural abundance studies. Therefore, a new high‐performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) methodology, with increased accuracy and precision, has been developed. The repeatability on the obtained δ¹³C values when pure amino sugars were analyzed were not significantly concentration‐dependent as long as the injected amount was higher than 1.5 nmol. The δ¹³C value of the same amino sugar spiked to a soil deviated by only 0.3‰ from the theoretical value. Copyright © 2009 John Wiley & Sons, Ltd.     

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 δ values of these reference materials should bracket the isotopic range of samples with unknown δ 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 δ¹³C and δ¹⁵N, 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: δ²H_nicotine ?162 to ?45‰, δ¹³C_nicotine ?30.05 to +7.72‰, δ¹⁵N_nicotine ?6.03 to +33.62‰; δ¹⁵N_acetanilide +1.18 to +40.57‰; δ¹³C_urea ?34.13 to +11.71‰, δ¹⁵N_urea +0.26 to +40.61‰ (recommended δ 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 δ¹⁵N values. Comparative δ¹³C and δ¹⁵N 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.     

Membrane permeation continuous‐flow isotope ratio mass spectrometry for on‐line carbon isotope ratio determination

Gaseous membrane permeation (MP) technologies have been combined with continuous‐flow isotope ratio mass spectrometry for on‐line δ¹³C measurements. The experimental setup of membrane permeation‐gas chromatography/combustion/isotope ratio mass spectrometry (MP‐GC/C/IRMS) quantitatively traps gas streams in membrane permeation experiments under steady‐state conditions and performs on‐line gas transfer into a GC/C/IRMS system. A commercial polydimethylsiloxane (PDMS) membrane sheet was used for the experiments. Laboratory tests using CO₂ demonstrate that the whole process does not fractionate the C isotopes of CO₂. Moreover, the δ¹³C values of CO₂ permeated on‐line give the same isotopic results as off‐line static dual‐inlet IRMS δ¹³C measurements. Formaldehyde generated from aqueous formaldehyde solutions has also been used as the feed gas for permeation experiments and on‐line δ¹³C determination. The feed‐formaldehyde δ¹³C value was pre‐determined by sampling the headspace of the thermostated aqueous formaldehyde solution. Comparison of the results obtained by headspace with those from direct aqueous formaldehyde injection confirms that the headspace sampling does not generate isotopic fractionation, but the permeated formaldehyde analyzed on‐line yields a ¹³C enrichment relative to the feed δ¹³C value, the isotopic fractionation being 1.0026 ± 0.0003. The δ¹³C values have been normalized using an adapted two‐point isotopic calibration for δ¹³C values ranging from ?42 to ?10‰. The MP‐GC/C/IRMS system allows the δ¹³C determination of formaldehyde without chemical derivatization or additional analytical imprecision. Copyright © 2009 John Wiley & Sons, Ltd.     

Gas chromatography/combustion/isotope ratio mass spectrometric analysis of the stable carbon composition of tetrols

The stable carbon isotope compositions of tetrols, erythritol and threitol were determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Using four tetrols with various δ¹³C values derivatized by methylboronic acid, the carbon isotope analysis method achieved excellent reproducibility and high accuracy. There was no carbon isotopic fractionation during the derivatization processes. The differences in the carbon isotopic compositions of methylboronates between the measured and calculated ranged from ?0.20 to 0.12‰, within the specification of the GC/C/IRMS system. It was demonstrated that δ¹³C values of tetrols could be calculated by a simple mass balance equation between tetrols, methylboronic acid, and methylboronates. The analogous 2‐methyltetrols, marker compounds of photooxidation products of atmospheric isoprene, should have similar behavior using the same derivatization reagent. This method may provide insight on sources and sinks of atmospheric isoprene. Copyright © 2009 John Wiley & Sons, Ltd.     

Single-step transesterification with simultaneous concentration and stable isotope analysis of fatty acid methyl esters by gas chromatography-combustion-isotope ratio mass spectrometry

Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is increasingly applied to food and metabolic studies for stable isotope analysis (δ¹³C), with the quantification of analyte concentration often obtained via a second alternative method. We describe a rapid direct transesterification of triacylglycerides (TAGs) for fatty acid methyl ester (FAME) analysis by GC‐C‐IRMS demonstrating robust simultaneous quantification of amount of analyte (mean r² = 0.99, accuracy ±2% for 37 FAMEs) and δ¹³C (±0.13‰) in a single analytical run. The maximum FAME yield and optimal δ¹³C values are obtained by derivatizing with 10% (v/v) acetyl chloride in methanol for 1 h, while lower levels of acetyl chloride and shorter reaction times skewed the δ¹³C values by as much as 0.80‰. A Bland‐Altman evaluation of the GC‐C‐IRMS measurements resulted in excellent agreement for pure oils (±0.08‰) and oils extracted from French fries (±0.49‰), demonstrating reliable simultaneous quantification of FAME concentration and δ¹³C values. Thus, we conclude that for studies requiring both the quantification of analyte and δ¹³C data, such as authentication or metabolic flux studies, GC‐C‐IRMS can be used as the sole analytical method. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of the stable carbon isotopic compositions of 2‐methyltetrols in ambient aerosols from the Changbai Mountains

Isoprene is one of the most important non‐methane hydrocarbons (NMHCs) in the troposphere: it is a significant precursor of O₃ and it affects the oxidative state of the atmosphere. The diastereoisomeric 2‐methyltetrols, 2‐methylthreitol and 2‐methylerythritol, are marker compounds of the photooxidation products of atmospheric isoprene. In order to obtain valuable information on the δ¹³C value of isoprene in the atmosphere, the stable carbon isotopic compositions of the 2‐methyltetrols in ambient aerosols were investigated. The 2‐methyltetrols were extracted from filter samples and derivatized with methylboronic acid, and the δ¹³C values of the methylboronate derivatives were determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The δ¹³C values of the 2‐methyltetrols were then calculated through a simple mass balance equation between the 2‐methyltetrols, methylboronic acid and the methylboronates. The δ¹³C values of the 2‐methyltetrols in aerosol samples collected at the Changbai Mountain Nature Reserves in eastern China were found to be ?24.66 ± 0.90‰ and ?24.53 ± 1.08‰ for 2‐methylerythritol and 2‐methylthreitol, respectively. Based on the measured isotopic composition of the 2‐methyltetrols, the average δ¹³C value of atmospheric isoprene is inferred to be close to or slightly heavier than ?24.66‰ at the collection site during the sampling period. Copyright © 2010 John Wiley & Sons, Ltd.     

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates

The natural ¹³C/¹²C isotope composition (δ¹³C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the ¹²C/¹³C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific δ¹³C of individual carbohydrates. Here, we examined two complementary methods for measuring the δ¹³C value of sucrose, glucose and fructose, that is, off‐line high‐performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA‐IRMS) analysis, and gas chromatography‐combustion (GC‐C)‐IRMS. We also used these methods to determine the in vitro ¹²C/¹³C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC～EA‐IRMS, and being sensitive to derivatization conditions, the GC‐C‐IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the ¹²C/¹³C isotope effect is rather small and it is not affected by the use of heavy water (D₂O). Copyright © 2009 John Wiley & Sons, Ltd.     

Stable isotopic analysis of pyrogenic organic matter in soils by liquid chromatography–isotope‐ratio mass spectrometry of benzene polycarboxylic acids

Pyrogenic organic matter (PyOM), the incomplete combustion product of organic materials, is considered stable in soils and represents a potentially important terrestrial sink for atmospheric carbon dioxide. One well‐established method of measuring PyOM in the environment is as benzene polycarboxylic acids (BPCAs), a compound‐specific method, which allows both qualitative and quantitative estimation of PyOM. Until now, stable isotope measurement of PyOM carbon involved measurement of the trimethylsilyl (TMS) or methyl (Me) polycarboxylic acid derivatives by gas chromatography–combustion–isotope ratio mass spectrometry (GC‐C‐IRMS). However, BPCA derivatives can contain as much as 150% derivative carbon, necessitating post‐analysis correction for the accurate measurement of δ¹³ C values, leading to increased measurement error. Here, we describe a method for δ¹³ C isotope ratio measurement and quantification of BPCAs from soil‐derived PyOM, based on ion‐exchange chromatography (IEC‐IRMS). The reproducibility of the δ¹³ C measurement of individual BPCAs by IEC‐IRMS was better than 0.35‰ (1σ). The δ¹³ C‐BPCA analysis of PyOM in soils, including at natural and artificially enriched ¹³ C‐abundance, produced accurate and precise δ¹³ C measurements. Analysis of samples that differed in δ¹³ C by as much as 900‰ revealed carryover of <1‰ between samples. The weighted sum of individual δ¹³ C‐BPCA measurements was correlated with previous isotopic measurements of whole PyOM, providing complementary information for bulk isotopic measurements. We discuss potential applications of δ¹³ C‐BPCA measurements, including the study of turnover rates of PyOM in soils and the partitioning of PyOM sources based on photosynthetic pathways. Copyright © 2011 John Wiley & Sons, Ltd.     

Inter‐laboratory comparison of elemental analysis and gas chromatography/combustion/isotope ratio mass spectrometry. II. δ15N measurements of selected compounds for the development of an isotopic Grob test

An inter‐laboratory exercise was carried out by a consortium of five European laboratories to establish a set of compounds, suitable for calibrating gas chromatography/combustion/isotope ratio mass spectrometry (GC‐C‐IRMS) devices, to be used as isotopic reference materials for hydrogen, carbon, nitrogen and oxygen stable isotope measurements. The set of compounds was chosen with the aim of developing a mixture of reference materials to be used in analytical protocols to check for food and beverage authentication. The exercise was organized in several steps to achieve the certification level: the first step consisted of the a priori selection of chemical compounds on the basis of the scientific literature and successive GC tests to set the analytical conditions for each single compound and the mixture. After elimination of the compounds that turned out to be unsuitable in a multi‐compound mixture, some additional oxygen‐ and nitrogen‐containing substances were added to complete the range of calibration isotopes. The results of δ¹³C determinations for the entire set of reference compounds have previously been published, while the δD and δ¹⁸O determinations were unsuccessful and after statistical analysis of the data the results did not reach the level required for certification. In the present paper we present the results of an inter‐laboratory exercise to identify and test the set of nitrogen‐containing compounds present in the mixture developed for use as reference materials for the validation of GC‐C‐IRMS analyses in individual laboratories. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of [U‐13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

The use of stable isotope labelled glucose provides insight into glucose metabolism. The ¹³C‐isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques the samples must be derivatized prior to analysis, which makes sample preparation more labour‐intensive and increases the uncertainty of the measured isotopic composition. A novel method for the determination of isotopic enrichment of glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been developed. Using this technique, for which hardly any sample preparation is needed, we showed that both the enrichment and the concentration could be measured with very high precision using only 20 µL of plasma. In addition, a comparison with GC/MS and GC/IRMS showed that the best performance was achieved with the LC/IRMS method making it the method of choice for the measurement of ¹³C‐isotopic enrichment in plasma samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Compound‐specific hydrogen isotope analysis of 1,2‐dichloroethane: potential for delineating source and fate of chlorinated hydrocarbon contaminants in groundwater

A Delta Plus XL continuous flow gas chromatography/high‐temperature conversion‐isotope ratio mass spectrometer system (GC‐TC‐IRMS) with a liquid nitrogen trap installed at the end of the micropyrolysis oven was used to measure hydrogen isotope (δ²H) values of 1,2‐dichloroethane (1,2‐DCA). The 1,2‐DCA δ²H values were within uncertainty of the δ²H value for the same 1,2‐DCA analyzed using off‐line sample preparation and conventional dual inlet mass spectrometry, verifying that this system can accurately measure 1,2‐DCA δ²H values. After 71 reproducible and accurate 1,2‐DCA δ²H measurements had been obtained, the standard deviation on the mean of the cumulative 1,2‐DCA δ²H measurements was greater than ±5‰. The cumulative load of chlorine at this point was ～5.5 × 10^?6 moles, which may be the limit to the quantity of chlorine that can be input before the reproducibility of 1,2‐DCA δ²H measurements is compromised. This study is the first to our knowledge to demonstrate a method for obtaining accurate and reproducible compound‐specific δ²H values for chlorinated hydrocarbons at dissolved concentrations typical of field conditions. Paired δ²H and δ¹³C values suggest that dual parameter isotopic measurements can distinguish between different contaminant sources, as well as providing additional constraints on degradation pathways and contaminant remediation. Copyright © 2007 John Wiley & Sons, Ltd.     

Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl‐glycerol dibiphytanyl glycerol tetraethers (2Gly‐GDGTs) are associated with methanotrophic ANME‐1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly‐GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ¹³C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ¹³C values of sugars cleaved from 2Gly‐GDGTs in two marine sediment samples, one containing predominantly ANME‐1 archaea and the other benthic archaea, were obtained and compared with the δ¹³C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in ¹³C relative to the corresponding major biphytane. This ¹³C enrichment was significantly larger in the putative major glycolipids from ANME‐1 archaea (～15‰) than in those from benthic archaea (<7‰). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms. Copyright © 2010 John Wiley & Sons, Ltd.     

A simple rapid method to precisely determine 13C/12C ratios of plant methoxyl groups

Stable isotope ratios of individual plant components have become a valuable tool for the determination of the geographical origin and authenticity of foodstuff. A recently published method with considerable potential in this context is the measurement of the deuterium/hydrogen (D/H) isotope ratios of plant matter methoxyl groups. The method entailed cleavage of methyl ethers or esters with hydriodic acid (HI) to form gaseous methyl iodide (CH₃I) and then measurement of the δ²H value of this gas. Here, as a follow up to a previous study, we describe a method for the rapid and precise δ¹³C analysis of plant matter methoxyl groups using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions optimized, the reproducibility of the measurement of standards undertaken, and the precision of the method defined. The reproducibility of the δ¹³C value determined for a CH₃I standard on 20 consecutive measurements was found to be 0.17‰. The method was also tested on four methoxyl‐rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, expressed as the average standard deviation, for these compounds was found to be better than 0.13‰. The described procedure which is simple and rapid, allowing preparation and analysis of a sample within 1 h, produces accurate and reproducible isotopic measurements. We suggest that this validated δ¹³C method when employed together with the recently published δ²H method for two‐dimensional stable isotope studies of organic matter containing methoxyl groups will be of considerable value, e.g. for proving the authenticity of foodstuff. Copyright © 2009 John Wiley & Sons, Ltd.     

Calcite‐CO2 mixed into CO2‐free air: a new CO2‐in‐air stable isotope reference material for the VPDB scale

In order to generate a reliable and long‐lasting stable isotope ratio standard for CO₂ in samples of clean air, CO₂ is liberated from well‐characterized carbonate material and mixed with CO₂‐free air. For this purpose a dedicated acid reaction and air mixing system (ARAMIS) was designed. In the system, CO₂ is generated by a conventional acid digestion of powdered carbonate. Evolved CO₂ gas is mixed and equilibrated with a prefabricated gas comprised of N₂, O₂, Ar, and N₂O at close to ambient air concentrations. Distribution into glass flasks is made stepwise in a highly controlled fashion. The isotopic composition, established on automated extraction/measurement systems, varied within very small margins of error appropriate for high‐precision air‐CO₂ work (about ±0.015‰ for δ¹³C and ±0.025‰ for δ¹⁸O). To establish a valid δ¹⁸O relation to the VPDB scale, the temperature dependence of the reaction between 25 and 47°C has been determined with a high level of precision. Using identical procedures, CO₂‐in‐air mixtures were generated from a selection of reference materials; (1) the material defining the VPDB isotope scale (NBS 19, δ¹³C = +1.95‰ and δ¹⁸O = ?2.2‰ exactly); (2) a local calcite similar in isotopic composition to NBS 19 (‘MAR‐J1’, δ¹³C = +1.97‰ and δ¹⁸O = ?2.02‰), and (3) a natural calcite with isotopic compositions closer to atmospheric values (‘OMC‐J1’, δ¹³C = ?4.24‰ and δ¹⁸O = ?8.71‰). To quantitatively control the extent of isotope‐scale contraction in the system during mass spectrometric measurement other available international and local carbonate reference materials (L‐SVEC, IAEA‐CO‐1, IAEA‐CO‐8, CAL‐1 and CAL‐2) were also processed. As a further control pure CO₂ reference gases (Narcis I and II, NIST‐RM 8563, GS19 and GS20) were mixed with CO₂‐free synthetic air. Independently, the pure CO₂ gases were measured on the dual inlet systems of the same mass spectrometers. The isotopic record of a large number of independent batches prepared over the course of several months is presented. In addition, the relationship with other implementations of the VPDB‐scale for CO₂‐in‐air (e.g. CG‐99, based on calibration of pure CO₂ gas) has been carefully established. The systematic high‐precision comparison of secondary carbonate and CO₂ reference materials covering a wide range in isotopic composition revealed that assigned δ‐values may be (slightly) in error. Measurements in this work deviate systematically from assigned values, roughly scaling with isotopic distance from NBS 19. This finding indicates that a scale contraction effect could have biased the consensus results. The observation also underlines the importance of cross‐contamination errors for high‐precision isotope ratio measurements. As a result of the experiments, a new standard reference material (SRM), which consists of two 5‐L glass flasks containing air at 1.6 bar and the CO₂ evolved from two different carbonate materials, is available for distribution. These ‘J‐RAS’ SRM flasks (‘Jena‐Reference Air Set’) are designed to serve as a high‐precision link to VPDB for improving inter‐laboratory comparability. a Copyright © 2005 John Wiley & Sons, Ltd.     

Compound‐specific δ18O analyses of neutral sugars in soils using gas chromatography–pyrolysis–isotope ratio mass spectrometry: problems,possible solutions and a first application

Although gas chromatography–pyrolysis–isotope ratio mass spectrometry (GC‐Py‐IRMS) has allowed us to make online compound‐specific δ¹⁸O measurements for about the last ten years, this technique has hardly been applied. We tested different pyrolysis reactor designs using standards (vanillin, ethylvanillin, a fatty acid methyl ester and alkanes) in order to optimize the GC‐Py‐IRMS δ¹⁸O measurements. The method was then applied to methylboronic acid (MBA) sugar derivatives (pentoses, 6‐deoxyhexoses and hexoses). Plant‐ and microbial‐derived monosaccharides were extracted hydrolytically from litter and topsoils before derivatization. The measured δ¹⁸O values of samples and co‐analyzed reference material were first drift‐corrected by use of regularly discharged pulses of CO reference gas. Secondly, they were corrected for the amount dependence of the δ¹⁸O values. Thirdly, the δ¹⁸O values were calibrated using the reference material (principle of ‘Identical Treatment’), and, finally, a correction was applied by taking the hydrolytically introduced and water‐exchangeable oxygen atoms into account. Our results suggest that the δ¹⁸O values of plant‐derived monosaccharides in litter reflect the climatic conditions of the last year, whereas δ¹⁸O values of the respective topsoils reflect the averaged climate signal of the last decades or even centuries. This demonstrates the high potential of the method for palaeoclimate reconstructions. Copyright © 2009 John Wiley & Sons, Ltd.     

Stable carbon isotopes as an indicator for soil degradation in an alpine environment (Urseren Valley,Switzerland)

Analyses of soil organic carbon (SOC) content and stable carbon isotope signatures (δ¹³C) of soils were assessed for their suitability to detect early stage soil erosion. We investigated the soils in the alpine Urseren Valley (southern central Switzerland) which are highly impacted by soil erosion. Hill slope transects from uplands (cambisols) to adjacent wetlands (histosols and histic to mollic gleysols) differing in their intensity of visible soil erosion, and reference wetlands without erosion influence were sampled. Carbon isotopic signature and SOC content of soil depth profiles were determined. A close correlation of δ¹³C and carbon content (r > 0.80) is found for upland soils not affected by soil erosion, indicating that depth profiles of δ¹³C of these upland soils mainly reflect decomposition of SOC. Long‐term disturbance of an upland soil is indicated by decreasing correlation of δ¹³C and SOC (r ≤ 0.80) which goes in parallel with increasing (visible) damage at the site. Early stage soil erosion in hill slope transects from uplands to adjacent wetlands is documented as an intermediate δ¹³C value (?27.5‰) for affected wetland soil horizons (0–12 cm) between upland (aerobic metabolism, relatively heavier δ¹³C of ?26.6‰) and wetland isotopic signatures (anaerobic metabolism, relatively lighter δ¹³C of ?28.6‰). Carbon isotopic signature and SOC content are found to be sensitive indicators of short‐ and long‐term soil erosion processes. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

A method for 13C‐labeling of metabolic carbohydrates within French bean leaves (Phaseolus vulgaris L.) for decomposition studies in soils

The molecular composition of plant residues is suspected to largely govern the fate of their constitutive carbon (C) in soils. Labile compounds, such as metabolic carbohydrates, are affected differently from recalcitrant and structural compounds by soil‐C stabilisation mechanisms. Producing ¹³C‐enriched plant residues with specifically labeled fractions would help us to investigate the fate in soils of the constitutive C of these compounds. The objective of the present research was to test ¹³C pulse chase labeling as a method for specifically enriching the metabolic carbohydrate components of plant residues, i.e. soluble sugars and starch. Bean plants were exposed to a ¹³CO₂‐enriched atmosphere for 0.5, 1, 2, 3 and 21 h. The major soluble sugars were then determined on water‐soluble extracts, and starch on HCl‐hydrolysable extracts. The results show a quick differential labeling between water‐soluble and water‐insoluble compounds. For both groups, ¹³C‐labeling increased linearly with time. The difference in δ¹³C signature between water‐soluble and insoluble fractions was 7‰ after 0.5 h and 70‰ after 21 h. However, this clear isotopic contrast masked a substantial labeling variability within each fraction. By contrast, metabolic carbohydrates on the one hand (i.e. soluble sugars + starch) and other fractions (essentially cell wall components) on the other hand displayed quite homogeneous signatures within fractions, and a significant difference in labeling between fractions: δ¹³C = 414 ± 3.7‰ and 56 ± 5.5‰, respectively. Thus, the technique generates labeled plant residues displaying contrasting ¹³C‐isotopic signatures between metabolic carbohydrates and other compounds, with homogenous signatures within each group. Metabolic carbohydrates being labile compounds, our findings suggest that the technique is particularly appropriate for investigating the effect of compound lability on the long‐term storage of their constitutive C in soils. Copyright © 2009 John Wiley & Sons, Ltd.     

Amino acid δ13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry: paleodietary implications from intra‐individual comparisons

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (δ¹³C) of individual amino acids in hair proteins and bone collagen using the LC‐IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound‐specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context by the δ¹³C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used as a proxy for bone collagen at the amino acid level, this validates compound‐specific isotope studies using hair as a model for palaeodietary reconstruction. Our results suggest that a small offset observed in the bulk δ¹³C values of the hair and bone samples may be attributed to two factors: (i) amino acid compositional differences between hair and bone proteins, and (ii) differential turnover rates of the tissues and the amino acid pools contributing to their synthesis. This application proposes that hair may be a useful complementary or alternative source of compound‐specific paleodietary information. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous analysis of 13C‐glutathione as its dimeric form GSSG and its precursor [1‐13C]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine ¹³C‐glutathione as its dimeric form (GSSG) and its precursor [1‐¹³C]glycine in a small volume of erythrocytes in one single analysis. After having transformed ¹³C‐glutathione into its dimeric form GSSG, we determined both the intra‐erythrocytic concentrations and the ¹³C‐isotopic enrichment of GSSG and glycine in 150 µL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of µmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 µmol/mL. The ¹³C‐isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3‰) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright © 2009 John Wiley & Sons, Ltd.