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.     

Critical assessment of the applicability of gas chromatography‐combustion‐isotope ratio mass spectrometry to determine amino sugar dynamics in soil

Amino sugars in soils have been used as markers of microbial necromass and to determine the relative contribution of bacterial and fungal residues to soil organic matter. However, little is known about the dynamics of amino sugars in soil. This is partly because of a lack of adequate techniques to determine ‘turnover rates’ of amino sugars in soil. We conducted an incubation experiment where ¹³C‐labeled organic substrates of different quality were added to a sandy soil. The objectives were to evaluate the applicability of compound‐specific stable isotope analysis via gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) for the determination of ¹³C amino sugars and to demonstrate amino sugar dynamics in soil. We found total analytical errors between 0.8 and 2.6‰ for the δ¹³C‐values of the soil amino sugars as a result of the required δ¹³C‐corrections for isotopic alterations due to derivatization, isotopic fractionation and analytical conditions. Furthermore, the δ¹³C‐values of internal standards in samples determined via GC‐C‐IRMS deviated considerably from the δ¹³C‐values of the pure compounds determined via elemental analyzer IRMS (with a variation of 9 to 10‰ between the first and third quartile among all samples). This questions the applicability of GC‐C‐IRMS for soil amino sugar analysis. Liquid chromatography‐combustion‐IRMS (LC‐C‐IRMS) might be a promising alternative since derivatization, one of the main sources of error when using GC‐C‐IRMS, is eliminated from the procedure. The high ¹³C‐enrichment of the substrate allowed for the detection of very high ¹³C‐labels in soil amino sugars after 1 week of incubation, while no significant differences in amino sugar concentrations over time and across treatments were observed. This suggests steady‐state conditions upon substrate addition, i.e. amino sugar formation equalled amino sugar decomposition. Furthermore, higher quality substrates seemed to favor the production of fungal‐derived amino sugars. Copyright © 2009 John Wiley & Sons, Ltd.     

Reconstructing bulk isotope ratios from compound‐specific isotope ratios

Carbon isotope analysis by bulk elemental analysis coupled with isotope ratio mass spectrometry has been the mainstay of δ¹³C analyses both at natural abundance and in tracer studies. More recently, compound‐specific isotope analysis (CSIA) has become established, whereby organic constituents are separated online by gas or liquid chromatography before oxidation and analysis of CO₂ for constituent δ¹³C. Theoretically, there should be concordance between bulk δ¹³C measurements and carbon‐weighted δ¹³C measurements of carbon‐containing constituents. To test the concordance between the bulk and CSIA, fish oil was chosen because the majority of carbon in fish oil is in the triacylglycerol form and ～95% of this carbon is amenable to CSIA in the form of fatty acids. Bulk isotope analysis was carried out on aliquots of oil extracted from 55 fish samples and δ¹³C values were obtained. Free fatty acids (FFAs) were produced from the oil samples by saponification and derivatised to fatty acid methyl esters (FAMEs) for CSIA by gas chromatography/combustion/isotope ratio mass spectrometry. A known amount of an internal standard (C15:0 FAME) was added to allow analyte quantitation. This internal standard was also isotopically calibrated in both its FFA (δ¹³C = ?34.30‰) and FAME (δ¹³C = ?34.94‰) form. This allowed reporting of FFA δ¹³C from measured FAME δ¹³C values. The bulk δ¹³C was reconstructed from CSIA data based on each FFA δ¹³C and the relative amount of CO₂ produced by each analyte. The measured bulk mean δ¹³C (SD) was ?23.75‰ (1.57‰) compared with the reconstructed bulk mean δ¹³C of ?23.76 (1.44‰) from CSIA and was not significantly different. Further analysis of the data by the Bland‐Altman method did not show particular bias in the data relative to the magnitude of the measurement. Good agreement between the methods was observed with the mean difference between methods (range) of 0.01‰ (?1.50 to 1.30). Copyright © 2010 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.     

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.     

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.     

Can gas chromatography combustion isotope ratio mass spectrometry be used to quantify organic compound abundance?

Quantifying the concentrations of organics such as phospholipid fatty acids (PLFAs) and n‐alkanes and measuring their corresponding ¹³ C/¹² C isotope ratios often involves two separate analyses; (1) quantification by gas chromatography flame ionisation detection (GC‐FID) or gas chromatography/mass spectrometry (GC/MS), and (2) ¹³ C‐isotope abundance analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC‐C‐IRMS). This requirement for two separate analyses has obvious disadvantages in terms of cost and time. However, there is a history of using the data output of isotope ratio mass spectrometers to quantify various components; including the N and C concentrations of solid materials and CO₂ concentrations in gaseous samples. Here we explore the possibility of quantifying n‐alkanes extracted from sheeps' faeces and fatty acid methyl esters (FAMEs) derivatised from PLFAs extracted from grassland soil, using GC‐C‐IRMS. The results were compared with those from GC‐FID analysis of the same extracts. For GC‐C‐IRMS the combined area of the masses for all the ions (m/z 44, 45 and 46) was collected, referred to as 'area all', while for the GC‐FID analysis the peak area data were collected. Following normalisation to a common value for added internal standards, the GC‐C‐IRMS 'area all' values and the GC‐FID peak area data were directly compared. Strong linear relationships were found for both n‐alkanes and FAMEs. For the n‐alkanes the relationships were 1:1 while, for the FAMEs, GC‐C‐IRMS overestimated the areas relative to the GC‐FID results. However, with suitable reference material 1:1 relationships were established. The output of a GC‐C‐IRMS system can form the basis for the quantification of certain organics including FAMEs and n‐alkanes. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

An online method combining a Gasbench II with continuous flow isotope ratio mass spectrometry to determine the content and isotopic compositions of minor amounts of carbonate in silicate rocks

An online method using continuous flow isotope ratio mass spectrometry (CF‐IRMS) interfaced with a Gasbench II device was established to analyze carbon and oxygen isotopic compositions and to estimate the content of minor amounts of carbonate in silicate rocks. The mixtures of standard materials and high‐purity quartz are firstly used to calibrate different quantities of carbonate in silicates. The results suggest that the accuracy and precision of the online analysis are both better than those obtained using an offline method. There is a positive correlation between the carbonate weight and the Mass44 ion beam intensity (or peak area). When the weight of carbonate in the mixtures is greater than 70 µg (equal to ～1800 mV Mass44 ion beam intensity), the δ¹³C and δ¹⁸O values of samples usually have accuracy and precision of ±0.1‰ and ±0.2‰ (1σ), respectively. If the weight is less than 70 µg, some limitations (e.g., not perfectly linear) are encountered that significantly reduce the accuracy and precision. The measured δ¹⁸O values are systematically lower than the true values by ?0.3 to ?0.7‰; the lower the carbonate content, the lower the measured δ¹⁸O value. For samples with lower carbonate content, the required phosphoric acid doses are higher and more oxygen isotope exchanges with the water in the phosphoric acid. To guarantee accurate results with high precision, multiple analyses of in‐house standards and an artificial MERCK sample with δ¹³C values from ?35.58 to 1.61‰ and δ¹⁸O from 6.04 to 18.96‰ were analyzed simultaneously with the unknown sample. This enables correction of the measured raw data for the natural sample based on multiple‐point normalization. The results indicate that the method can be successfully applied to a range of natural rocks. Copyright © 2010 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.     

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.     

Determination of human muscle protein fractional synthesis rate: an evaluation of different mass spectrometry techniques and considerations for tracer choice

In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring‐¹³C₆]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC‐C‐IRMS) has thus far been considered the ‘gold’ standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography‐tandem MS (LC‐MS/MS) and GC‐tandem MS (GC‐MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N‐acetyl‐n‐propyl, phenylisothiocyanate, or N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC‐MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer‐tracee‐ratio measurements (delta tracer‐tracee‐ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC‐C‐IRMS, LC‐MS/MS, and GC‐MS/MS (MTBSTFA), respectively. FSR determined with LC‐MS/MS compared well with GC‐C‐IRMS and so did the GC‐MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC‐MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC‐C‐IRMS, and (3) If GC‐MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous δ15N, δ13C and δ34S measurements of low‐biomass samples using a technically advanced high sensitivity elemental analyzer connected to an isotope ratio mass spectrometer

Conventional simultaneous CNS stable isotope abundance measurements of solid samples usually require high sample amounts, up to 1 mg carbon, to achieve exact analytical results. This rarely used application is often impaired by high C:S element ratios when organic samples are analyzed and problems such as incomplete conversion into sulphur dioxide occur during analysis. We introduce, as a technical innovation, a high sensitivity elemental analyzer coupled to a conventional isotope ratio mass spectrometer, with which CNS‐stable isotope ratios can be determined simultaneously in samples with low carbon content (<40 µg C corresponding to ～100 µg dry weight). The system includes downsized reactors, a temperature program‐controlled gas chromatography (GC) column and a cryogenic trap to collect small amounts of sulphur dioxide. This modified application allows for highly sensitive measurements in a fully automated operation with standard deviations better than ±0.47‰ for δ¹⁵N and δ³⁴S and ±0.12‰ for δ¹³C (n = 127). Samples collected from one sampling site in a Baltic fjord within a short time period were measured with the new system to get a first impression of triple stable isotope signatures. The results confirm the potential of using δ³⁴S as a stable isotope tracer in combination with δ¹⁵N and δ¹³C measurements to improve discrimination of food sources in aquatic food webs. Copyright © 2009 John Wiley & Sons, Ltd.     

High-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry

Compound‐specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion‐exchange separation. In this work, high‐temperature reversed‐phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT‐LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C₁₈ (up to 180 °C), Acquity C₁₈ (up to 200 °C), Triart C₁₈ (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ¹³C measurements, demonstrating the suitability of these columns for HT‐LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C₁₈ columns was observed to result in shortened analysis time. The detection limit of HT‐RPLC/IRMS obtained for caffeine was 30 mg L^–1 (corresponding to 12.4 nmol carbon on‐column). Temperature‐programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ¹³C measurements compared with flow injection analysis without a column. With temperature‐programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ¹³C values, leading to an important extension of the application range of CSIA. Copyright © 2011 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.     

Hair protein and amino acid 13C and 15N abundances take more than 4 weeks to clearly prove influences of animal protein intake in young women with a habitual daily protein consumption of more than 1 g per kg body weight

A high protein or meat intake might be a risk factor for metabolic disorders. Stable isotopic abundances (SIA) of hair can be used as biomarkers for animal protein intake due to characteristic isotopic patterns of food proteins. We investigated if an additional meat intake (M, 200 g pork fillet/day) or an omission of meat and meat products (NOM) can influence the natural ¹⁵N and ¹³C SIA within 4 weeks in hair and plasma of young women. The daily protein intake (means ± SD) was 1.40 ± 0.29, 2.25 ± 0.35, and 1.15 ± 0.26 g/kg at baseline, during M, and during NOM, respectively. At baseline the animal protein intake correlated with bulk SIA of hair (¹⁵N: R² = 0.416; ¹³C: R² = 0.664; n = 14). However, isotope ratio mass spectrometry (IRMS) analyses have not shown that hair and plasma SIA were changed significantly after M or NOM. Possible reasons were discussed. Urinary SIA were significantly lower after M than after NOM (¹⁵N: p = 0.039; ¹³C: p = 0.006) and close to those of pork fillet. Characteristic patterns of SIA were measured in individual amino acids (AA) by gas chromatography/combustion isotope ratio mass spectrometry (GC/C/IRMS). The results confirmed considerable differences in SIA between AA (δ¹⁵N, up to 22‰; δ¹³C, up to 31‰). Plots of ¹⁵N versus ¹³C abundances in hair revealed characteristic differences between indispensable and dispensable AA. The intervention‐dependent changes of AA‐specific SIA were not as clear as expected. Although the AA‐specific SIA may reveal more detailed characteristics of physiological conditions, further methodological research is required. We suggest that the SIA of leucine can be potential markers of protein intake. The reliability of SIA as biomarkers of protein intake still have to be tested in longer lasting intervention studies in humans. The results may have implications in the assessment for possible benefits and risks of protein consumption. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbon and oxygen isotope microanalysis of carbonate

Technical modification of the conventional method for the δ¹³C and δ¹⁸O analysis of 10–30 µg carbonate samples is described. The CO₂ extraction is carried out in vacuum using 105% phosphoric acid at 95°C, and the isotopic composition of CO₂ is measured in a helium flow by gas chromatography/isotope ratio mass spectrometry (GC/IRMS). The feed‐motion of samples to the reaction vessel provides sequential dropping of only the samples (without the sample holder) into the acid, preventing the contamination of acid and allowing us to use the same acid to carry out very large numbers of analyses. The high accuracy and high reproducibility of the δ¹³C and δ¹⁸O analyses were demonstrated by measurements of international standards and comparison of results obtained by our method and by the conventional method. Our method allows us to analyze 10 µg of the carbonate with a standard deviation of ±0.05‰ for δ¹³C and δ¹⁸O. The method has been used successfully for the analyses of the oxygen and carbon isotopic composition of the planktonic and benthic foraminifera in detailed palaeotemperature reconstructions of the Okhotsk Sea. Copyright © 2009 John Wiley & Sons, Ltd.     

Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy

The carbon isotope composition of leaf bulk organic matter was determined on the tropical tree Elaeis guineensis Jacq. (oil palm) in North Sumatra (Indonesia) to get a better understanding of the changes in carbon metabolism during the passage from heterotrophy to autotrophy of the leaves. Leaf soluble sugar (sucrose, glucose and fructose) contents, stomatal conductance and dark respiration, as well as leaf chlorophyll and nitrogen contents, were also investigated. Different growing stages were sampled from leaf rank ?6 to rank 57. The mean values for the δ¹³C of bulk organic matter were ?29.01 ± 0.9‰ for the leaflets during the autotrophic stage, ?27.87 ± 1.08‰ for the petioles and ?28.17 ± 1.09‰ for the rachises, which are in the range of expected values for a C₃ plant. The differences in δ¹³C among leaf ranks clearly revealed the changes in the origin of the carbon source used for leaf growth. Leaves were ¹³C‐enriched at ranks below zero (around ?27‰). During this period, the ‘spear’ leaves were completely heterotrophic and reserves from storage organs were mobilised for the growth of these young emerging leaves. ¹³C‐depletion was then observed when the leaf was expanding at rank 1, and there was a continuous decrease during the progressive passage from heterotrophy until reaching full autotrophy. Thereafter, the δ¹³C remained more or less constant at around ?29.5‰. Changes in sugar content and in δ¹³C related to leaf ranks showed an interesting similarity of the passage from heterotrophy to autotrophy of oil palm leaves to the budburst of some temperate trees or seed germination reported in the literature. Copyright © 2009 John Wiley & Sons, Ltd.