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.     

Error assessment of nitrogen and oxygen isotope ratios of nitrate as determined via the bacterial denitrification method

Currently, bacterial denitrification is becoming the accepted method for δ¹⁵N‐ and δ¹⁸O‐NO determination. However, proper correction methods with international references (USGS32, USGS34 and USGS35) are needed. As a consequence, it is important to realize that the corrected isotope values are derived from a combination of several other measurements with associated uncertainties. Therefore, it is necessary to consider the propagated uncertainty on the final isotope value. This study demonstrates how to correctly estimate the uncertainty on corrected δ¹⁵N‐ and δ¹⁸O‐NO values using a first‐order Taylor series approximation. The bacterial denitrification method errors from 33 batches of 561 surface water samples varied from 0.2 to 2.1‰ for δ¹⁵N‐NO and from 0.7 to 2.3‰ for δ¹⁸O‐NO, which is slightly wider than the machine error, which varied from 0.2 to 0.6‰ for δ¹⁵N‐N₂O and from 0.4 to 1.0‰ for δ¹⁸O‐N₂O. The overall uncertainties, which are composed of the machine error and the method error, for the 33 batches ranged from 0.3 to 2.2‰ for δ¹⁵N‐NO and from 0.8 to 2.5‰ for δ¹⁸O‐NO. In addition, the mean corrected δ¹⁵N and δ¹⁸O values of 132 KNO₃‐IWS (internal working standard) measurements were computed as 8.4 ± 1.0‰ and 25.1 ± 2.0‰, which is a slight underestimation for δ¹⁵N and overestimation for δ¹⁸O compared with the accepted values (δ¹⁵N = 9.9 ± 0.3‰ and δ¹⁸O = 24.0 ± 0.3‰). The overall uncertainty of the bacterial denitrification method allows the use of this method for source identification of NO. Copyright © 2010 John Wiley & Sons, Ltd.     

Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association

We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.     

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.     

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.     

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins

In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. The present study takes the approach of extracting the protein components of plant tissues before hydrolysis and using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not require derivatisation, for measurement of the isotopic enrichment of the amino acids. The protocol developed offers a methodology for consistent measurement of the δ(13)C values of amino acids, allowing isotopic differences between the individual amino acids from different plant tissues to be identified. In particular, there are highly significant differences between leaf and seed protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰), aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended application in archaeology, the technique will be of value in the fields of plant sciences, nutrition and environmental food-web studies.     

δ15N of soil N and plants in a N‐saturated,subtropical forest of southern China

We investigated the δ¹⁵N profile of N (extractable NH, NO, and organic N (EON)) in the soil of a N‐saturated subtropical forest. The order of δ¹⁵N in the soil was EON > NH > NO. Although the δ¹⁵N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5‰. The difference in δ¹⁵N between bulk soil N and EON (Δ¹⁵N_bulk‐EON) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high δ¹⁵N of EON, as in the case of soil microbial biomass δ¹⁵N. A thorough δ¹⁵N survey of available N (NH, NO, and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the δ¹⁵N of the available N forms did not fully overlap with the δ¹⁵N of plants. This mismatch in δ¹⁵N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N‐saturated forest. Copyright © 2010 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.     

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.     

Geographical traceability of wheat and its products using multielement light stable isotopes coupled with chemometrics

The present study was aimed to investigate the variation of stable isotopic ratios of carbon, nitrogen, hydrogen, and oxygen in wheat kernel along with different processed fractions from three geographical origins across 5 years using isotope ratio mass spectrometry (IRMS). Multiway ANOVA revealed significant differences among region, harvest year, processing, and their interactions for all isotopes. The region contributed the major variability in the δ¹³C ‰, δ²H ‰, δ¹⁵N ‰, and δ¹⁸O‰ values of wheat. Variation of δ¹³C ‰, δ¹⁵N ‰, and δ¹⁸O ‰ between wheat whole kernel and its products (break, reduction, noodles, and cooked noodles) were ?0.7‰, and no significant difference was observed, suggesting the reliability of these isotope fingerprints in geographical traceability of wheat‐processed fractions and foods. A significant influence of wheat processing was observed for δ²H values. By applying linear discriminant analysis (LDA) to the whole dataset, the generated model correctly classified over 91% of the samples according to the geographical origin. The application of these parameters will assist in the development of an analytical control procedure that can be utilized to control the mislabeling regarding geographical origin of wheat kernel and its products.     

Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

We report the first high‐precision characterization of molecular and intramolecular δ¹⁵N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid ¹⁵ N or ¹³ C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ¹⁵N and δ¹³C values characterized. Mean diet δ¹⁵N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ¹⁵N = +4.1 ± 0.7‰ and ?4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ¹⁵N were +1.4 ± 0.4, –2.1 ± 0.9, –7.2 ± 0.3, and ?10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ¹⁵N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ¹⁵N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ¹⁵N_ASP‐PUR, δ¹⁵N_ASP‐PYR, δ¹⁵N_GLN, and δ¹⁵N_GLY +11.9 ± 2.3‰, +7.0 ± 2.0‰, –9.1 ± 2.4‰, and ?31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ¹⁵N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ¹⁵N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ¹⁵N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ¹⁵N values vary over more than 40‰ and are not influenced by dietary protein level or age. 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.     

Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Individual free amino acid δ¹⁵N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ¹⁵N values. High correlations were observed between the δ¹⁵N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ¹⁵N values of plant leave samples following incubation in ¹⁵N-nitrate at different time points were determined. A typical foliar free amino acid ¹⁵N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ¹⁵N values. With the known amino acid metabolism pathways and δ¹⁵N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.

     

Adaptation of continuous‐flow cavity ring‐down spectroscopy for batch analysis of δ13C of CO2 and comparison with isotope ratio mass spectrometry

Measurements of δ¹³C in CO₂ have traditionally relied on samples stored in sealed vessels and subsequently analyzed using magnetic sector isotope ratio mass spectrometry (IRMS), an accurate but expensive and high‐maintenance analytical method. Recent developments in optical spectroscopy have yielded instruments that can measure δ¹³CO₂ in continuous streams of air with precision and accuracy approaching those of IRMS, but at a fraction of the cost. However, continuous sampling is unsuited for certain applications, creating a need for conversion of these instruments for batch operation. Here, we present a flask (syringe) adaptor that allows the collection and storage of small aliquots (20–30 mL air) for injection into the cavity ring‐down spectroscopy (CRDS) instrument. We demonstrate that the adaptor's precision is similar to that of traditional IRMS (standard deviation of 0.3‰ for 385 ppm CO₂ standard gas). In addition, the concentration precision (±0.3% of sample concentration) was higher for CRDS than for IRMS (±7% of sample concentration). Using the adaptor in conjunction with CRDS, we sampled soil chambers and found that soil‐respired δ¹³C varied between two different locations in a piñon‐juniper woodland. In a second experiment, we found no significant discrimination between the respiration of a small beetle (~5 mm) and its diet. Our work shows that the CRDS system is flexible enough to be used for the analysis of batch samples as well as for continuous sampling. This flexibility broadens the range of applications for which CRDS has the potential to replace magnetic sector IRMS. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography/mass spectrometry stable isotope analysis of dissolved organic carbon in stream and soil waters

A commercial interface coupling liquid chromatography (LC) to a continuous‐flow isotope ratio mass spectrometry (CF‐IRMS) instrument was used to determine the δ¹³C of dissolved organic carbon (DOC) in natural waters. Stream and soil waters from a farmland plot in a hedgerow landscape were studied. Based on wet chemical oxidation of dissolved organics the LC/IRMS interface allows the on‐line injection of small volumes of water samples, an oxidation reaction to produce CO₂ and gas transfer to the isotope ratio mass spectrometer. In flow injection analysis (FIA) mode, bulk DOC δ¹³C analysis was performed on aqueous samples of up to 100 μL in volume in the range of DOC concentration in fresh waters (1–10 mg C.L^–1). Mapping the DOC δ¹³C spatial distribution at the plot scale was made possible by this fairly quick method (10 min for triplicate analyses) with little sample manipulation. The relative contributions of different plot sectors to the DOC pool in the stream draining the plot were tentatively inferred on the basis of δ¹³C differences between the hydrophilic and hydrophobic components. Copyright © 2011 John Wiley & Sons, Ltd.     

The role of liquid chromatography and flow injection analyses coupled to isotope ratio mass spectrometry for studying human in vivo glucose metabolism

Under most physiological conditions, glucose, or carbohydrate (CHO), homeostasis is tightly regulated. In order to mechanistically appraise the origin of circulating glucose (e.g. via either gluconeogenesis, glycogenolysis or oral glucose intake), and its regulation and oxidation, the use of stable isotope tracers is now a well-accepted analytical technique. Methodologically, liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) can replace gas chromatography coupled to combustion-isotope ratio mass spectrometry (GC/C/IRMS) for carrying out compound-specific (13)C isotopic analysis. The LC/IRMS approach is well suited for studying glucose metabolism, since the plasma glucose concentration is relatively high and the glucose can readily undergo chromatography in an aqueous mobile phase. Herewith, we report two main methodological approaches in a single instrument: (1) the ability to measure the isotopic enrichment of plasma glucose to assess the efficacy of CHO-based treatment (cocoa-enriched) during cycling exercise with healthy subjects, and (2) the capacity to carry out bulk isotopic analysis of labeled solutions, which is generally performed with an elemental analyzer coupled to IRMS. For plasma samples measured by LC/IRMS the data show a isotopic precision SD(δ(13)C) and SD(APE) of 0.7 ‰ and 0.001, respectively, with δ(13)C and APE values of -25.48 ‰ and 0.06, respectively, being generated before and after tracer administration. For bulk isotopic measurements, the data show that the presence of organic compounds in the blank slightly affects the δ(13)C values. Despite some analytical limitations, we clearly demonstrate the usefulness of the LC/IRMS especially when (13)C-glucose is required during whole-body human nutritional studies.     

Visible light induced free radical promoted cationic polymerization using thioxanthone derivatives

The free radical promoted cationic polymerization cyclohexene oxide (CHO), was achieved by visible light irradiation (λ_inc = 430–490 nm) of methylene chloride solutions containing thioxanthone‐fluorene carboxylic acid (TX‐FLCOOH) or thioxanthone‐carbazole (TX‐C) and cationic salts, such as diphenyliodonium hexafluorophosphate (Ph₂I⁺PF) or silver hexafluorophosphate (Ag⁺PF) in the presence of hydrogen donors. A feasible initiation mechanism involves the photogeneration of ketyl radicals by hydrogen abstraction in the first step. Subsequent oxidation of ketyl radicals by the oxidizing salts yields Bronsted acids capable of initiating the polymerization of CHO. In agreement with the proposed mechanism, the polymerization was completely inhibited by 2,2,6,6‐tetramethylpiperidinyl‐1‐oxy and di‐2,6‐di‐tert‐butylpyridine as radical and acid scavengers, respectively. Additionally polymerization efficiency was directly related to the reduction potential of the cationic salts, that is, Ag⁺PF (E = +0.8 V) was found to be more efficient than Ph₂I⁺PF (E = ?0.2 V). In addition to CHO, vinyl monomers such as isobutyl vinyl ether and N‐vinyl carbazole, and a bisepoxide such as 3,4‐epoxycyclohexyl‐3′,4′‐epoxycyclohexene carboxylate, were polymerized in the presence of TX‐FLCOOH or TX‐C and iodonium salt with high efficiency. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

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.     

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.     

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.