Minimization of carbon addition during derivatization of monosaccharides for compound-specific delta13C analysis in environmental research

Little is known about the delta13C composition of monosaccharides representing the largest carbon reservoir in the biosphere. The main reason for this might be that monosaccharides have to be derivatized prior to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses and that a large isotopic correction is necessary for the carbon that has to be added to the original molecule during derivatization, resulting in large uncertainty of the calculated delta13C values of individual monosaccharides. The amount of added derivatization carbon is twice (alditol acetates) or even three times (trimethylsilyl (TMS) derivatives) as high as the amount of the original monosaccharide carbon. In addition, isotope fractionation occurs during acetylation. Therefore, the objectives of this study were (i) to minimize carbon addition during derivatization for GC/C/IRMS measurements of monosaccharides in soil and sediment samples and (ii) to quantify improvements in accuracy and precision of the final results. Minimization of carbon addition was accomplished by derivatization with methylboronic acid (MBA) and TMS thereafter (MBA method). Monosaccharides derivatized with the MBA method instead of TMS reduced the number of added carbon atoms from 2.2-2.7 to 0.3-0.8 per sugar carbon atom. Although the precision of GC/C/IRMS measurements with both methods is comparable (about 0.3 per thousand), delta13C values of an internal standard indicated that the newly developed MBA method is about 2 per thousand more accurate than the TMS method. delta13C comparison between soil samples that differed only slightly in their bulk carbon isotope signature showed that the MBA method is better in proving these small differences on a significant level. Total precision of the whole MBA method including all analytical and calculation steps is better by a factor of almost three than the TMS method.     

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.     

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.     

Optimisation of derivatisation procedures for the determination of delta13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.     

A single glucose derivative suitable for gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry

The incorporation of stable isotopes improves the assessment of glucose metabolism and, with some researchers using two tracers, (2)H-glucose assessed by gas chromatography/mass spectrometry (GC/MS) and (13)C-glucose by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), a common derivative for both is advantageous. The most commonly used derivatives for GC/MS are inappropriate for GC/C/IRMS as additional functional groups dilute the label. We therefore considered the suitability of six derivatives for both GC/MS and GC/C/IRMS. Glucose alkylboronates were prepared by adding the appropriate alkylboronic acid (butyl- or methylboronic acid) in pyridine to desiccated glucose. The derivatisation was completed by reacting this with either (a) acetic anhydride or trifluoroacetic anhydride (acetate derivatives) or (b) bis(trimethylsilyl)trifluoroacetamide BSTFA (TMS derivatives). All six derivatives were assessed using GC/MS and (13)C GC/C/IRMS.Neither TMS derivative exhibited any signal intensity in the molecular ion, although a M-15 ion showed good agreement between experimental and theoretical data and, whilst still low in intensity, could be suitable for isotope work. Similarly, none of the acetate derivatives showed any intensity at the molecular ion although three key fragmentation series were identified. The most attractive sequence, initiated by the loss of 1,2 cyclic boronate, resulted in the main fragment ion of interest, m/z 240, corresponding to the fluorinated methylboronate derivate. Minimal carbon and hydrogen atoms are added to this derivative making it an excellent choice for stable isotope work, while proving suitable for analysis by both GC/MS and GC/C/IRMS.     

Determination of the 13C/12C ratio of ethanol derived from fruit juices and maple syrup by isotope ratio mass spectrometry: collaborative study

A collaborative study of the carbon-13 isotope ratio mass spectrometry (13C-IRMS) method based on fermentation ethanol for detecting some sugar additions in fruit juices and maple syrup is reported. This method is complementary to the site-specific natural isotope fractionation by nuclear magnetic resonance (SNIF-NMR) method for detecting added beet sugar in the same products (AOAC Official Methods 995.17 and 2000.19), and uses the same initial steps to recover pure ethanol. The fruit juices or maple syrups are completely fermented with yeast, and the alcohol is distilled with a quantitative yield (>96%). The carbon-13 deviation (delta13C) of ethanol is then determined by IRMS. This parameter becomes less negative when exogenous sugar derived from plants exhibiting a C4 metabolism (e.g., corn or cane) is added to a juice obtained from plants exhibiting a C3 metabolism (most common fruits except pineapple) or to maple syrup. Conversely, the delta13C of ethanol becomes more negative when exogenous sugar derived from C3 plants (e.g., beet, wheat, rice) is added to pineapple products. Twelve laboratories analyzed 2 materials (orange juice and pure cane sugar) in blind duplicate and 4 sugar-adulterated materials (orange juice, maple syrup, pineapple juice, and apple juice) as Youden pairs. The precision of that method for measuring delta13C was similar to that of other methods applied to wine ethanol or extracted sugars in juices. The within-laboratory (Sr) values ranged from 0.06 to 0.16%o (r = 0.17 to 0.46 percent per thousand), and the among-laboratories (SR) values ranged from 0.17 to 0.26 percent per thousand (R = 0.49 to 0.73 percent per thousand). The Study Directors recommend that the method be adopted as First Action by AOAC INTERNATIONAL.     

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds.We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.     

Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.     

Development of a compound-specific isotope analysis method for acetone via 2,4-dinitrophenylhydrazine derivatization

A novel method has been developed for compound-specific isotope analysis for acetone via DNPH (2,4-dinitrophenylhydrazine) derivatization together with combined gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Acetone reagents were used to assess delta13C fractionation during the DNPH derivatization process. Reduplicate delta13C analyses were designed to evaluate the reproducibility of the derivatization, with an average error (1 standard deviation) of 0.17 +/- 0.05 per thousand, and average analytical error of 0.28 +/- 0.09 per thousand. The derivatization process introduces no isotopic fractionation for acetone (the average difference between the predicted and analytical delta13C values was 0.09 +/- 0.20 per thousand, within the precision limits of the GC/C/IRMS measurements), which permits computation of the delta13C values for the original underivatized acetone through a mass balance equation. Together with further studies of the carbon isotopic effect during the atmospheric acetone-sampling procedure, it will be possible to use DNPH derivatization for carbon isotope analysis of atmospheric acetone.     

Comparison of liquid chromatography-isotope ratio mass spectrometry (LC/IRMS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values.     

Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.     

Site-specific carbon isotope analysis of aromatic carboxylic acids by elemental analysis/pyrolysis/isotope ratio mass spectrometry

Site-specific carbon isotope composition of organic compounds can provide useful information on their origin and history in natural environments. Site-specific isotope analyses of small amounts of organic compounds (sub-nanomolar level), such as short-chain carboxylic acids and amino acid analogues, have been performed using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/pyrolysis/IRMS). These analyses were previously limited to volatile compounds. In this study, site-specific carbon isotope analysis has been developed for non-volatile aromatic carboxylic acids at sub-micromolar level by decarboxylation using a continuous flow elemental analysis (EA)/pyrolysis/IRMS technique. Benzoic acid, 2-naphthylacetic acid and 1-pyrenecarboxylic acid were pyrolyzed at 500-1000 degrees C by EA/pyrolysis/IRMS to produce CO2 for delta13C measurement of the carboxyl group. These three aromatic acids were most efficiently pyrolyzed at 750 degrees C. Conventional sealed-tube pyrolysis was also conducted for comparison. The delta13C values of CO2 generated by the continuous flow technique were within 1.0 per thousand of those performed by the conventional technique, indicating that the new continuous flow technique can accurately analyze the carbon isotopic composition of the carboxyl group in aromatic carboxylic acids. The new continuous flow technique is simple, rapid and uses small sample sizes, so this technique will be useful for characterizing the isotopic signature of carboxyl groups in organic compounds.     

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.     

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.     

Hydropyrolysis over a platinum catalyst as a preparative technique for the compound‐specific carbon isotope ratio measurement of C27 steroids

Compound‐specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is an important method for the determination of the ¹³C/¹²C ratios of biomolecules such as steroids, for a wide range of applications. However, steroids in their natural form exhibit poor chromatographic resolution, while derivatisation adds carbon thereby corrupting the stable isotopic composition. Hydropyrolysis with a sulphided molybdenum catalyst has been shown to defunctionalise the steroids, while leaving their carbon skeleton intact, allowing for the accurate measurement of carbon isotope ratios. The presence of double bonds in unsaturated steroids such as cholesterol resulted in significant rearrangement of the products, but replacing the original catalyst system with one of platinum results in higher conversions and far greater selectivity. The improved chromatographic performance of the products should allow GC/C/IRMS to be applied to more structurally complex steroid hormones and their metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric studies of the path of carbon in photosynthesis: positional isotopic analysis of (13)C-labelled C(4) to C(7) sugar phosphates

The (EIMS) electron ionization mass spectrometric fragmentation patterns of the methoxime- and ethoxime-trimethylsilyl (TMS) derivatives of C(4) to C(7) sugars involved as phosphates in the Calvin pathway of photosynthesis in plants were analysed by gas chromatography/EIMS using specifically labelled (13)C analogs. In general, most but not all of the major ions in the mass spectra arise from single carbon-carbon bond cleavages of the straight-chain derivatives. The results confirm that GC/MS of the alkoxime-TMS derivatives is a viable method for measuring (13)C incorporations at individual carbon atoms in each of the sugar phosphates during photosynthetic experiments with (13)CO(2).     

Selective conversion of plasma glucose into CO2 by Saccharomyces cerevisiae for the measurement of 13C abundance by isotope ratio mass spectrometry: proof of principle

To study carbohydrate digestion and glucose absorption, time-dependent (13)C enrichment in plasma glucose is measured after oral administration of naturally occurring (13)C-enriched carbohydrates. The isotope enrichment of the administered carbohydrate is low (APE <0.1%) and plasma (13)C glucose measurements are routinely determined with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) or liquid chromatography/combustion/isotope ratio mass spectrometry (LC/C/IRMS). In this study, plasma glucose was converted into CO(2) by an in-tube reaction with yeast permitting direct measurement of (13)CO(2) in the headspace. Saccharomyces cerevisiae incubated under anaerobic conditions was able to convert sufficient glucose into CO(2) to produce a consistent CO(2) peak in IRMS with little variation in peak area and precise delta(13)C(PDB) values for corn glucose: -11.40 +/- 0.16 per thousand, potato glucose: -25.17 +/- 0.13 per thousand, and plasma glucose: -26.29 +/- 0.05 per thousand. The measurement showed high linearity (R(2) = 0.999) and selectivity and was not affected by the glucose concentration in the tested range of 5-15 mM. Comparison with GC/C/IRMS showed a good correlation of enrichment data: R(2) > 0.98 for both sources of glucose and plasma samples. Commercially available, instant dried baker's yeast was qualitatively and quantitatively comparable with freshly prepared yeast: R(2) > 0.96, slope 1.03 and 1.08 for glucose solutions and plasma, respectively. Thus, yeast conversion of plasma glucose into CO(2) and (13)C measurement applying a breath (13)CO(2) analyzer is an inexpensive, simple and equally accurate alternative to the more expensive and laborious GC/C/IRMS and LC/C/IRMS measurements.     

Bulk and compound-specific isotopic characterisation of illicit heroin and cling film

Comparative analysis involves various but complementary methods and can be used for forensic intelligence purposes to group seizures of heroin into batches. Much forensic analysis now combines expertise in the traditional area of drugs investigation with a detailed understanding of supply, packaging, distribution, and drugs intelligence. It was the intention of this research to determine whether illicit heroin seizures and packaging material can be grouped according to isotopic compositions, and to explore factors that affect the isotopic compositions. In order to achieve these aims, 14 samples of seized heroin, thirteen provided by Avon and Somerset Constabulary (UK), were analysed by elemental analysis/isotope ratio mass spectrometry (EA/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) for carbon and hydrogen isotopes. These tests elucidated that a combination of the delta13C, delta15N, delta18O and delta2H results from EA/IRMS is able to distinguish between most samples of bulk heroin. We speculate that the delta13C values of the alkaloids, obtained by GC/C/IRMS, give indications of different geographical or temporal origins of some of the heroin samples. GC/C/IRMS of the cutting agent, caffeine, provides a means to link dilution events. Fifteen retail cling film samples and seven cling film samples from heroin seizures were analysed by EA/IRMS. A multivariate comparison of the carbon, hydrogen and oxygen isotope ratios was able to distinguish between most of the samples. This technique enabled the cling films from the heroin to be grouped according to seizure. Three solvents were tested on two samples of cling film of known composition. Methanol and chloroform were both found to extract material from PVC and from non-PVC cling films. Water-treated PVC was indistinguishable from the untreated PVC and thus water was found to be the most suitable solvent when washing cling film prior to IRMS analysis.     

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.     

A gas chromatography/combustion/isotope ratio mass spectrometry system for high-precision delta13C measurements of atmospheric methane extracted from ice core samples

Past atmospheric composition can be reconstructed by the analysis of air enclosures in polar ice cores which archive ancient air in decadal to centennial resolution. Due to the different carbon isotopic signatures of different methane sources high-precision measurements of delta13CH4 in ice cores provide clues about the global methane cycle in the past. We developed a highly automated (continuous-flow) gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) technique for ice core samples of approximately 200 g. The methane is melt-extracted using a purge-and-trap method, then separated from the main air constituents, combusted and measured as CO2 by a conventional isotope ratio mass spectrometer. One CO2 working standard, one CH4 and two air reference gases are used to identify potential sources of isotope fractionation within the entire sample preparation process and to enhance the stability, reproducibility and accuracy of the measurement. After correction for gravitational fractionation, pre-industrial air samples from Greenland ice (1831 +/- 40 years) show a delta13C(VPDB) of -49.54 +/- 0.13 per thousand and Antarctic samples (1530 +/- 25 years) show a delta13C(VPDB) of -48.00 +/- 0.12 per thousand in good agreement with published data.