A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry

We have developed a method to analyze stable carbon isotope ((13)C/(12)C) ratios in a variety of carbohydrates using high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS). The chromatography is based on strong anion-exchange columns with low strength NaOH eluents. An eluent concentration of 1 mM resulted in low background signals and good separation of most of the typical plant neutral carbohydrates. We also show that more strongly bound carbohydrates such as acidic carbohydrates can be separated by inclusion of NO(3) (-) as an inorganic pusher ion in the eluent. Analyses of neutral carbohydrate concentrations and their stable carbon isotope ratios are shown for plant materials and marine sediment samples both at natural abundance and for (13)C-enriched samples. The main advantage of HPLC/IRMS analysis over traditional gas chromatography based methods is that no derivatization is needed resulting in simple sample treatment and improved accuracy and reproducibility.     

Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters

The use of isotope ratio infrared spectroscopy (IRIS) for the stable hydrogen and oxygen isotope analysis of water is increasing. While IRIS has many advantages over traditional isotope ratio mass spectrometry (IRMS), it may also be prone to errors that do not impact upon IRMS analyses. Of particular concern is the potential for contaminants in the water sample to interfere with the spectroscopy, thus leading to erroneous stable isotope data. Water extracted from plant and soil samples may often contain organic contaminants. The extent to which contaminants may interfere with IRIS and thus impact upon data quality is presently unknown. We tested the performance of IRIS relative to IRMS for water extracted from 11 plant species and one organic soil horizon. IRIS deviated considerably from IRMS for over half of the samples tested, with deviations as large as 46‰ (δ²H) and 15.4‰ (δ¹⁸O) being measured. This effect was reduced somewhat by using activated charcoal to remove organics from the water; however, deviations as large as 35‰ (δ²H) and 11.8‰ (δ¹⁸O) were still measured for these cleaned samples. Interestingly, the use of activated charcoal to clean water samples had less effect than previously thought for IRMS analyses. Our data show that extreme caution is required when using IRIS to analyse water samples that may contain organic contaminants. We suggest that the development of new cleaning techniques for removing organic contaminants together with instrument‐based software to flag potentially problematic samples are necessary to ensure accurate plant and soil water analyses using IRIS. Copyright © 2010 John Wiley & Sons, Ltd.     

Detailed assessment of isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters

As an alternative to isotope ratio mass spectrometry (IRMS), the isotope ratio infrared spectroscopy (IRIS) approach has the advantage of low cost, continuous measurement and the capacity for field‐based application for the analysis of the stable isotopes of water. Recent studies have indicated that there are potential issues of organic contamination of the spectral signal in the IRIS method, resulting in incorrect results for leaf samples. To gain a more thorough understanding of the effects of sample type (e.g., leaf, root, stem and soil), sample species, sampling time and climatic condition (dry vs. wet) on water isotope estimates using IRIS, we collected soil samples and plant components from a number of major species at a fine temporal resolution (every 2 h for 24–48 h) across three locations with different climatic conditions in the Heihe River Basin, China. The hydrogen and oxygen isotopic compositions of the extracted water from these samples were measured using both an IRMS and an IRIS instrument. The results show that the mean discrepancies between the IRMS and IRIS approaches for δ¹⁸O and δD, respectively, were: –5.6‰ and ?75.7‰ for leaf water; –4.0‰ and ?23.3‰ for stem water; –3.4‰ and ?28.2‰ for root water; ?0.5‰ and –6.7‰ for xylem water; –0.06‰ and ?0.3‰ for xylem flow; and ?0.1‰ and 0.3‰ for soil water. The order of the discrepancy was: leaf > stem ≈ root > xylem > xylem flow ≈ soil. In general, species of the same functional types (e.g., woody vs. herbaceous) within similar habitats showed similar deviations. For different functional types, the differences were large. Sampling at nighttime did not remove the observed deviations. Copyright © 2011 John Wiley & Sons, Ltd.     

Isotopic ((13)C) analysis of dissolved organic carbon in stream water using an elemental analyzer coupled to a stable isotope ratio mass spectrometer

A technique for measurement of the stable isotope composition of dissolved organic carbon (DOC) in stream water, using an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (IRMS), is described. Stream water samples were concentrated by rotary evaporation, acidified to remove dissolved inorganic carbon (DIC), and dried in silver cups prior to analysis. Precision was evaluated with standards (alanine and humic acid), and with stream water samples with varying (13)C enrichment. Standards and samples were also prepared in sealed quartz tubes for high-temperature combustion (HTC) and analyzed by dual inlet for comparison. The delta(13)C values of natural abundance standards and samples measured by the two techniques differed by 相似文献   

Application of simultaneous thermal analysis mass spectrometry and stable carbon isotope analysis in a carbon sequestration study

The simultaneous analysis of evolved gases and the determination of stable isotope composition (delta13C) as part of a thermal analysis experiment have been used to (a) distinguish bulk chemical hosts for carbon (C) and nitrogen (N) within a soil and (b) track labelled C within a soil sequestration experiment. C3 and C4 dung was applied to a pasture soil, and soil samples taken for analysis. The results of thermogravimetry-differential scanning calorimetry-quadrupole mass spectrometry-isotope ratio mass spectrometry (TG-DSC-QMS-IRMS) show that the proportion of more refractory C (lignin-like) is greater for the dungs than for the soil organic matter (SOM), and that this increases with time within the soil. Analysis of evolved gases shows that nitrogen is associated with the decomposition of more refractory C, and is not so strongly associated with the labile C component. IRMS analysis distinguished C3 and C4 dung, and allowed the amount of C from these sources to be estimated for the soil samples. Most dung C enters the refractory SOM fraction. This paper demonstrates the potential of TG-DSC-QMS-IRMS in the investigation of SOM.     

Simultaneous determination of the quantity and isotopic signature of dissolved organic matter from soil water using high-performance liquid chromatography/isotope ratio mass spectrometry

We assessed the accuracy and utility of a modified high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) system for measuring the amount and stable carbon isotope signature of dissolved organic matter (DOM) <1 μm. Using a range of standard compounds as well as soil solutions sampled in the field, we compared the results of the HPLC/IRMS analysis with those from other methods for determining carbon and (13)C content. The conversion efficiency of the in-line wet oxidation of the HPLC/IRMS averaged 99.3% for a range of standard compounds. The agreement between HPLC/IRMS and other methods in the amount and isotopic signature of both standard compounds and soil water samples was excellent. For DOM concentrations below 10 mg C L(-1) (250 ng C total) pre-concentration or large volume injections are recommended in order to prevent background interferences. We were able to detect large differences in the (13)C signatures of soil solution DOM sampled in 10 cm depth of plots with either C3 or C4 vegetation and in two different parent materials. These measurements also demonstrated changes in the (13)C signature that demonstrate rapid loss of plant-derived C with depth. Overall the modified HLPC/IRMS system has the advantages of rapid sample preparation, small required sample volume and high sample throughput, while showing comparable performance with other methods for measuring the amount and isotopic signature of DOM.     

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

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

Position‐specific carbon isotope analysis of trichloroacetic acid by gas chromatography/isotope ratio mass spectrometry

Trichloroacetic acid (TCAA) is an important environmental contaminant present in soils, water and plants. A method for determining the carbon isotope signature of the trichloromethyl position in TCAA using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was developed and tested with TCAA from different origins. Position‐specific isotope analysis (PSIA) can provide direct information on the kinetic isotope effect for isotope substitution at a specific position in the molecule and/or help to distinguish different sources of a compound. The method is based on the degradation of TCAA into chloroform (CF) and CO₂ by thermal decarboxylation. Since thermal decarboxylation is associated with strong carbon isotope fractionation (ε = ?34.6 ± 0.2‰) the reaction conditions were optimized to ensure full conversion. The combined isotope ratio of CF and CO₂ at the end of the reaction corresponded well to the isotope ratio of TCAA, confirming the reliability of the method. A method quantification limit (MQL) for TCAA of 18.6 µg/L was determined. Samples of TCAA produced by enzymatic and non‐enzymatic chlorination of natural organic matter (NOM) and some industrially produced TCAA were used as exemplary sources. Significant different PSIA isotope ratios were observed between industrial TCAA and TCAA samples produced by chlorination of NOM. This highlights the potential of the method to study the origin and the fate of TCAA in the environment. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of intracellular homocysteine by stable isotope dilution liquid chromatography/tandem mass spectrometry

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.     

Deuterium nuclear magnetic resonance spectroscopy and stable carbon isotope ratio analysis/mass spectrometry of certain monofloral honeys

Quantitative deuterium nuclear magnetic resonance spectroscopy (NMR) has been used in conjunction with stable carbon isotope ratio analysis/mass spectrometry to refine the detection of sugars that have been added to monofloral honeys. The 13C content of sugars indicates the type of photosynthetic metabolism of the plant that synthesized them; the deuterium content is more characteristic of secondary metabolism and of environmental factors. Consequently, determination of the 13C content of honeys and of proteins extracted from the honeys can be used to detect the addition of C4 plant sugars (cane or corn), but it does not reveal the addition of C3 plant sugars such as beet sugar. Deuterium NMR gives useful information for some monofloral honeys. NMR measurement is performed on ethanol obtained from fermentation of the honey and extracted by distillation. The isotopic composition of the ethanol indicates the nature of the sugars from which it was derived. Various types of monofloral honeys were studied, and the results obtained with commercially available honeys demonstrate the usefulness of isotopic analysis and the need to compile a database of authentic honeys to validate or affirm certain results.     

High-temperature pyrolysis/gas chromatography/isotope ratio mass spectrometry: simultaneous measurement of the stable isotopes of oxygen and carbon in cellulose

Stable isotope analysis of cellulose is an increasingly important aspect of ecological and palaeoenvironmental research. Since these techniques are very costly, any methodological development which can provide simultaneous measurement of stable carbon and oxygen isotope ratios in cellulose deserves further exploration. A large number (3074) of tree-ring α-cellulose samples are used to compare the stable carbon isotope ratios (δ(13)C) produced by high-temperature (1400°C) pyrolysis/gas chromatography (GC)/isotope ratio mass spectrometry (IRMS) with those produced by combustion GC/IRMS. Although the two data sets are very strongly correlated, the pyrolysis results display reduced variance and are strongly biased towards the mean. The low carbon isotope ratios of tree-ring cellulose during the last century, reflecting anthropogenic disturbance of atmospheric carbon dioxide, are thus overestimated. The likely explanation is that a proportion of the oxygen atoms are bonding with residual carbon in the reaction chamber to form carbon monoxide. The 'pyrolysis adjustment', proposed here, is based on combusting a stratified sub-sample of the pyrolysis results, across the full range of carbon isotope ratios, and using the paired results to define a regression equation that can be used to adjust all the pyrolysis measurements. In this study, subsamples of 30 combustion measurements produced adjusted chronologies statistically indistinguishable from those produced by combusting every sample. This methodology allows simultaneous measurement of the stable isotopes of carbon and oxygen using high-temperature pyrolysis, reducing the amount of sample required and the analytical costs of measuring them separately.     

Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is described for relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantification was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein alpha(S1)-casein, for which three phosphopeptides were found after tryptic digestion. Reproducibility of the relative quantification of seven independent replicates was found to be 11% SD. The dynamic range covered two orders of magnitude, and quantification was linear for mixtures of 0 to 100% alpha(S1)-casein and dephospho-alpha(S1)-casein (R(2) = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation.