Application of elemental analysis-coupled isotope ratio mass spectrometry for protein turnover analysis using deuterium labeling: Purification and analysis of hydrogen isotope ratio of non-derivatized protein-bound alanine.

RATIONALE: Heavy water can be used as a tracer for the evaluation of protein turnover. By adding heavy water (D2 O) to the precursor pool, nonessential amino acids, including alanine, can be isotopically labeled in vivo. Protein turnover can then be quantified by measuring the hydrogen isotope ratio of protein-bound alanine. METHODS: In this study, we constructed a novel method to apply deuterium labeling of alanine to the evaluation of protein turnover using elemental analysis-coupled isotope ratio mass spectrometry (EA-IRMS). We established a preparative high-performance liquid chromatography method to isolate alanine from protein hydrolysates. EA-IRMS was then used to determine the hydrogen isotope ratio of alanine isolated from hydrolysates of protein from mouse myoblast C2C12 cells that had been treated with D2 O over the course of 72 h. RESULTS: In cells treated with 4% D2 O, the deuterium enrichment of alanine increased to approximately 0.9% over time, while that of cells treated with 0.017% D2 O increased to approximately 0.006%. The rate of protein synthesis calculated by fitting the increase of deuterium excess to rise-to-plateau kinetics was similar regardless of the concentration of D2 O. When C2C12 cells treated with insulin and rapamycin were analyzed 24 h after the addition of 0.017% D2 O, protein turnover was found to be accelerated by insulin, but this effect was offset by co-treatment with rapamycin. CONCLUSION: The derivative-free measurement of the hydrogen isotope ratio of protein-bound alanine using EA-IRMS can be applied to the evaluation of protein turnover. The proposed method is an accessible option for many laboratories to perform highly sensitive IRMS-based evaluations of protein metabolic turnover.

Intramolecular isotopic evidence for bacterial oxidation of propane in subsurface natural gas reservoirs.

Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

Analysis of Minor Acidic N-Glycans in Human Serum.

Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column and were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples of the N-glycan analysis in human serum were reported, but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allow the analysis of minor acidic glycans that are typically difficult to detect.

IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts.

In skeletal muscle, myoblast differentiation results in the formation of multinucleated myofibers. Although recent studies have shown that unfolded protein responses (UPRs) play an important role in intracellular remodeling and contribute to skeletal muscle differentiation, the involvement of IRE1-XBP1 signaling, a major UPR signaling pathway, remains unclear. This study aimed to investigate the effect of the IRE1-XBP1 pathway on skeletal muscle differentiation. In C2C12 cells, knockdown of IRE1 and XBP1 in cells remarkably suppressed differentiation. In addition, apoptosis and autophagy were dramatically enhanced in the XBP1-knockdown cells, highlighting the participation of IRE1-XBP1 in cell survival maintenance with differentiation stimuli during skeletal muscle differentiation. In myogenic cells, we demonstrated that the expression of CDK5 (cyclin-dependent kinase 5) is regulated by XBP1s, and we propose that XBP1 regulates the expression of MyoD family genes via the induction of CDK5. In conclusion, this study revealed that IRE1-XBP1 signaling plays critical roles in cell viability and the expression of differentiation-related genes in predifferentiated myoblasts and during the early differentiation phase.

Simultaneous Analysis of Sulfated and Phosphorylated Glycans by Serotonin-Immobilized Column Enrichment and Hydrophilic Interaction Chromatography.

Changes in the structures and quantities of sulfated glycans play important roles in inflammatory and neurological diseases and cancer. Therefore, sulfated glycans are expected to become diagnostic markers for a variety of diseases such as Alzheimer's disease and cancer. On the other hand, structural abnormalities in the phosphorylated glycans on lysosomal enzymes cause a number of lysosomal diseases, while novel phosphorylated glycans have been found in other proteins. As with sulfated glycans, structural and quantitative changes in these phosphorylated glycans and their associations with disease are also of interest. In this article, we introduce a new method for the simultaneous analysis of sulfated and phosphorylated glycans. We first employ a serotonin-immobilized column to enrich these glycans. Glycans obtained in this manner were sequentially subjected to other analytical techniques without desalting. We employed hydrophilic interaction chromatography to distinguish the sulfate and phosphate groups of the glycans and were able to analyze sulfated and phosphorylated N-glycans expressed on thyroglobulin, ovalbumin, and myozyme. We showed that our method not only analyzes sulfated and phosphorylated glycans, but also glycans containing the GlcNAc-HPO3 residue. We comparatively analyzed sulfated glycans, phosphorylated glycans, and GlcNAc-HPO3-residue-containing glycans expressed on MKN7 cells (well-differentiated gastric cancer cells) and MKN45 cells (poorly differentiated gastric cancer cells). To the best of our knowledge, this is the first report of a method for the simultaneous and quantitative analysis of sulfated and phosphorylated glycans.

Position-specific 13 C/12 C analysis of amino acid carboxyl groups - automated flow-injection analysis based on reaction with ninhydrin.

RATIONALE: The fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off-line preparation to reveal this information. An automated position-specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ13 CCARBOXYL values). This automation makes PSIA measurements easier and routine. METHODS: An existing high-performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post-column derivatisation unit between the HPLC system and the interface. The post-column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin. RESULTS: The methodology described produced δ13 CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ13 CCARBOXYL values) between amino acids over a 1-year period. First estimates are presented for the δ13 CCARBOXYL values of a number of internationally available amino acid reference materials. CONCLUSIONS: The PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole-molecule level. When combined with on-line chromatographic separation or off-line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position-specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.

Measurement of natural carbon isotopic composition of acetone in human urine.

The natural carbon isotopic composition of acetone in urine was measured in healthy subjects using gas chromatography-combustion-isotope ratio mass spectrometry combined with headspace solid-phase microextraction (HS-SPME-GC-C-IRMS). Before applying the technique to a urine sample, we optimized the measurement conditions of HS-SPME-GC-C-IRMS using aqueous solutions of commercial acetone reagents. The optimization enabled us to determine the carbon isotopic compositions within ±0.2 ‰ of precision and ±0.3‰ of error using 0.05 or 0.2 mL of aqueous solutions with acetone concentrations of 0.3-121 mg/L. For several days, we monitored the carbon isotopic compositions and concentrations of acetone in urine from three subjects who lived a daily life with no restrictions. We also monitored one subject for 3 days including a fasting period of 24 h. These results suggest that changes in the availability of glucose in the liver are reflected in changes in the carbon isotopic compositions of urine acetone. Results demonstrate that carbon isotopic measurement of metabolites in human biological samples at natural abundance levels has great potential as a tool for detecting metabolic changes caused by changes in physiological states and disease.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Position-Specific Isotope Analysis of Xanthines: A (13)C Nuclear Magnetic Resonance Method to Determine the (13)C Intramolecular Composition at Natural Abundance.

The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.

Analytical method for simultaneous determination of bulk and intramolecular (13) C-isotope compositions of acetic acid.

RATIONALE: Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography/pyrolysis-gas chromatography/combustion-isotope ratio mass spectrometry (GC/Py-GC/C-IRMS) was developed for the simultaneous determination of the intramolecular and molecular carbon-isotopic composition (δ(13) C value) of acetic acid. METHODS: The δ(13) C values of carboxyl and methyl carbon were standardized using calibration curves constructed from the regression between the measured δ(13) C values and the δ(13) C values of working standards determined in a previous study. We applied this developed HS-SPME-GC/Py-GC/C-IRMS technique to commercial vinegars. RESULTS: In one injection analysis, the bulk and intramolecular δ(13) C values of pure acetic acid standards can be obtained. The repeatability (1σ) of the bulk δ(13) C values is within ±0.4‰, and that of the δ(13) Ccarboxyl and δ(13) Cmethyl values is within ±0.6‰. The intramolecular δ(13) C values of acetic acid in vinegars exhibit a similar pattern. The average Δδ value (δ(13) CCOOH - δ(13) CCH3 ) is 4.3 ± 2.0‰. CONCLUSIONS: The approach presented herein for the molecular and intramolecular δ(13) C determination of acetic acid avoids switching between configuration systems and thereby reduces systematic errors. It is expected to be useful for examining isotope fractionation associated with processes related to organic acid (bio)transformations.

Dietary Deficiency of Calcium and/or Iron, an Age-Related Risk Factor for Renal Accumulation of Cadmium in Mice.

The major route of cadmium (Cd) intake by non-smokers is through food ingestion. Cd is a non-essential metal absorbed through one or more transporters of essential metal ions. Expression of these transporters is affected by nutritional status. To investigate the risk factors for Cd toxicity, the effects of deficiency of essential metals on hepatic and renal accumulation of Cd were studied in mice of different ages. Mice were administered a control diet or one of the essential metal-deficient diets, administered Cd by gavage for 6 weeks, and killed; then, Cd accumulation was evaluated. Iron deficiency (FeDF) or calcium deficiency (CaDF) resulted in remarkable increases in hepatic and renal Cd accumulation compared with control-diet mice and other essential metal-deficient mice. Cd accumulation in hepatic and renal tissue was increased significantly at all ages tested in FeDF and CaDF mice. Renal Cd concentrations were higher in 4-week-old mice than in 8- and 25-week-old mice. Increase in intestinal mRNA expression of calcium transporter (CaT)1, divalent metal ion transporter-1, and metallothionein (MT)1 was also higher in 4-week-old mice than in other mice. Renal accumulation of Cd showed strong correlation with intestinal mRNA expression of CaT1 and MT1. These data suggest that CaDF and FeDF at younger ages can be a risk factor for Cd toxicity.

ALS-Linked P56S-VAPB Mutation Impairs the Formation of Multinuclear Myotube in C2C12 Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder that affects upper and lower motor neurons. Since motor neurons target skeletal muscles, the maintenance system of muscles is disturbed in ALS; however, the mechanism by which this occurs is unknown. In the present study, we investigated the effects of ALS-associated P56S-vesicle-associated membrane protein-associated protein B (VAPB) (P56S-VAPB) on the IRE1-XBP1 pathway, which is involved in the unfolded protein response (UPR) of the mouse myoblast cell line (C2C12 cells). Experiments with C2C12 cells transfected with wild-type wt-VAPB and P56S-VAPB expression vectors showed reduced myotube formation and aberrant myonuclear position in cells expressing P56S-VAPB. Activity of the IRE1-XBP1 pathway in the cells visualized with the ERAI system revealed that the pathway was disrupted in cells expressing P56S-VAPB, whereas the IRE1-XBP1 pathway activity was enhanced in the differentiation process of normal C2C12 cells. These results suggest that disruption of the IRE1-XBP1 pathway is a cause for the reduced myotube formation in P56S-VAPB-expressing cells. The expression level of the VAPB protein has been reported to be reduced in the neurons of patients with ALS. Therefore, it is expected that the IRE1-XBP1 pathway is also impaired in muscle tissues of patients with ALS, which causes a disturbance in the muscle maintenance system.

Common glycoproteins expressing polylactosamine-type glycans on matched patient primary and metastatic melanoma cells show different glycan profiles.

Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

Evaluation of commercially available reagents as a reference material for intramolecular carbon isotopic measurements of acetic acid.

RATIONALE: Recent advances in analytical techniques for the intramolecular carbon isotopic ratio measurement of some organic compounds have provided important information on carbon cycles in biochemistry, organic geochemistry and food chemistry. These advances have made it necessary to prepare intramolecular isotopic reference materials (RMs) to use for inter-laboratory calibration and/or inter-calibration among different analytical methods. METHODS: We evaluated the feasibility of preparing RMs using commercially available reagents for intramolecular carbon isotopic ratio measurement of acetic acid. The intramolecular carbon isotopic distribution of nine acetic acid and four sodium acetate reagents was determined with high precision using off-line pyrolysis combined with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We also evaluated the potential alteration in the isotopic signature of acetic acid reagents by evaporation. RESULTS: The intramolecular carbon isotopic distributions for the acetic acid and sodium acetate reagents were determined with a precision of better than 0.45‰. We found that the isotopic values of these reagents spanned the carbon isotopic range of acetic acid in biological and environmental samples. We also found that the isotope fractionation associated with the evaporation of acetic acid occurs solely on the methyl position, the carboxyl position being unaffected. CONCLUSIONS: These commercially available reagents will be used as RMs in the future for inter-laboratory calibration and/or inter-calibration with another intramolecular isotopic measurement technique, namely quantitative (13) C NMR. In cases where acetic acid is being used as a RM, its storage must be carefully controlled to prevent evaporation.

9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis.

Structural analysis of O-glycans from mucins and characterization of the interaction of these glycans with other biomolecules are essential for a full understanding of mucins. Various techniques have been developed for the structural and functional analysis of glycans. While 9-fluorenylmethyl chloroformate (Fmoc-Cl) is generally used to protect amino groups in peptide synthesis, it can also be used as a glycan-labeling reagent for structural analysis. Fmoc-labeled glycans are strongly fluorescent and can be analyzed with high sensitivity using liquid chromatography-fluorescence detection (LC-FD) analysis as well as being analyzed with high sensitivity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Fmoc-labeled glycans can be easily delabeled and converted to glycosylamine-form or free (hemiacetal or aldehyde)-form glycans that can be used to fabricate glycan arrays or synthesize glycosyl dendrimers. This derivatization allows for the isolation from biological samples of glycans that are difficult to synthesize chemically, as well as the fabrication of immobilized-glycan devices. The Fmoc labeling method promises to be a tool for accelerating O-glycan structural analysis and an understanding of molecular interactions. In this chapter, we introduce the Fmoc labeling method for analysis of O-glycans and fabrication of O-glycan arrays.

Liquid Chromatography and Capillary Electrophoresis Analysis of 2AA-Labeled O-Glycans.

To reveal O-glycan structures in mucins, it is necessary to release covalently bound O-glycans from the polypeptide backbone and derivatize to a form suitable for structural analysis. Various derivatization methods can now be applied in the analysis of O-glycans following the development of O-glycan release methods. Among the many derivatization methods available, we prefer to use fluorescent labeling with 2-aminobenzoic acid (anthranilic acid, 2AA). 2AA-labeled O-glycans can be detected with high sensitivity using liquid chromatography fluorescence detection (LC-FD) analysis because of the strong fluorescence. In addition, as 2AA has a carboxyl group that carries a negative charge, 2AA-labeled O-glycans can be analyzed with high sensitivity in negative ion mode mass spectrometry. Furthermore, because the negative charge of 2AA provides a driving force for electrophoresis, 2AA-labeled O-glycans can be analyzed using capillary electrophoresis (CE) and capillary affinity electrophoresis. High detection sensitivity and versatility are key advantages of the 2AA-labeling method. Here we present our preferred LC-FD and CE analytical methods for 2AA-labeled O-glycans.

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Accurate method for the determination of intramolecular 13C isotope composition of ethanol from aqueous solutions.

A new method, combining headspace solid phase microextraction (HS-SPME) with an online pyrolysis system coupled with isotope ratio mass spectrometry (IRMS), is developed for the determination of the intramolecular (13)C isotope composition of ethanol in aqueous solutions. The δ(13)C values of the pyrolytic fragments (CO, CH4, C2H4) are shown to be highly reproducible (sd <0.4‰). Furthermore, using 14 ethanol samples of known intramolecular isotope distribution, the CO and CH4 fragments are shown to arise solely from the methylene (CH2OH) and methyl (CH3) carbon atom positions of the original ethanol, respectively. Although the different steps (extraction and pyrolysis) fractionate between (12)C and (13)C, the isotopic fractionation is reproducible (sd <0.4‰), allowing correcting factors to be applied in order to back-calculate the original δ(13)CCH2OH and δ(13)CCH3 values of ethanol. The method thus allows the determination of the isotope composition of ethanol at the intramolecular and molecular levels, within a single run and a short experimental time (30 min), and with a very easy sample preparation. The method is then applied to alcoholic beverages to show its potential for authentication purposes.

Precision and long-term stability of clumped-isotope analysis of CO2 using a small-sector isotope ratio mass spectrometer.

RATIONALE: The ratio of the measured abundance of (13)C-(18)O bonding CO(2) to its stochastic abundance, prescribed by the δ(13)C and δ(18)O values from a carbonate mineral, is sensitive to its growth temperature. Recently, clumped-isotope thermometry, which uses this ratio, has been adopted as a new tool to elucidate paleotemperatures quantitatively. METHODS: Clumped isotopes in CO(2) were measured with a small-sector isotope ratio mass spectrometer. CO(2) samples digested from several kinds of calcium carbonates by phosphoric acid at 25 °C were purified using both cryogenic and gas-chromatographic separations, and their isotopic composition (δ(13)C, δ(18)O, Δ(47), Δ(48) and Δ(49) values) were then determined using a dual-inlet Delta XP mass spectrometer. RESULTS: The internal precisions of the single gas Δ(47) measurements were 0.005 and 0.02‰ (1 SE) for the optimum and the routine analytical conditions, respectively, which are comparable with those obtained using a MAT 253 mass spectrometer. The long-term variations in the Δ(47) values for the in-house working standard and the heated CO(2) gases since 2007 were close to the routine, single gas uncertainty while showing seasonal-like periodicities with a decreasing trend. Unlike the MAT 253, the Delta XP did not show any significant relationship between the Δ(47) and δ(47) values. CONCLUSIONS: The Delta XP gave results that were approximately as precise as those of the MAT 253 for clumped-isotope analysis. The temporal stability of the Delta XP seemed to be lower, although an advantage of the Delta XP was that no dependency of δ(47) on Δ(47) was found.