Hydropyrolysis of steroids: a preparative step for compound-specific carbon isotope ratio analysis

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry is an important method for the detection of steroid abuse in athletes. However, steroids in their natural form exhibit poor chromatographic resolution, while derivatization adds carbon thereby corrupting the starting stable isotopic composition. Hydropyrolysis is a new approach, which defunctionalizes steroids but leaves their carbon skeleton intact. The process improves chromatography, allowing the faithful measurement of carbon isotope ratios and enabling a more effective apportionment for the source of steroids and their metabolites.     

Stable carbon isotope ratios of methyl-branched fatty acids are different to those of straight-chain fatty acids in dairy products

Methyl-branched fatty acids (MBFAs) are the dominant form of fatty acid found in many bacteria. They are also found at low levels in a range of foodstuffs, where their presence has been linked to bacterial sources. In this study we evaluated the potential of compound-specific isotope analysis to obtain insights into the stable carbon isotope ratios (δ¹³C values in ‰) of individual MBFAs and to compare them to the stable carbon isotope ratios of straight-chain fatty acids in food. Due to their low abundance in foodstuffs, the MBFAs were enriched prior to gas chromatography coupled to isotope ratio mass spectrometric (GC–IRMS) analysis. After transesterification, urea complexation was used to suppress the 16:0 and 18:0 methyl esters that were dominant in the samples. Following that, silver-ion high performance liquid chromatography was used to separate the saturated from the unsaturated fatty acids. The resulting solutions of saturated fatty acids obtained from suet, goat’s milk, butter, and human milk were studied by GC–IRMS. The δ¹³C values of fatty acids with 12–17 carbons ranged from −25.4‰ to −37.6‰. In all samples, MBFAs were most depleted in carbon-13, followed by the odd-chain fatty acids 15:0 and 17:0. 14:0 and 16:0 contained the highest proportions of carbon-13. The results from this study illustrate that MBFAs have distinctive δ¹³C values and must originate from other sources and/or from very different substrates. These measurements support the initial hypothesis that δ¹³C values can be used to attribute MBFAs to particular sources.     

Calling all archaeologists: guidelines for terminology,methodology, data handling,and reporting when undertaking and reviewing stable isotope applications in archaeology

Stable isotope analysis has been utilized in archaeology since the 1970s, yet standardized protocols for terminology, sampling, pretreatment evaluation, calibration, quality assurance and control, data presentation, and graphical or statistical treatment still remain lacking in archaeological applications. Here, we present recommendations and requirements for each of these in the archaeological context of: bulk stable carbon and nitrogen isotope analysis of organics; bulk stable carbon and oxygen isotope analysis of carbonates; single compound stable carbon and nitrogen isotope analysis on amino acids in collagen and keratin; and single compound stable carbon and hydrogen isotope analysis on fatty acids. The protocols are based on recommendations from the Commission on Isotopic Abundances and Atomic Weights of the International Union of Pure and Applied Chemistry (IUPAC) as well as an expanding geochemical and archaeological science experimental literature. We hope that this will provide a useful future reference for authors and reviewers engaging with the growing number of stable isotope applications and datasets in archaeology.     

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

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

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.     

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.     

Increasing the sensitivity of delta13C and delta15N abundance measurements by a high sensitivity elemental analyzer connected to an isotope ratio mass spectrometer

A common elemental analyzer system connected to a temperature-controlled gas chromatography (GC) column and coupled to an isotope ratio mass spectrometer was improved to decrease the determination limit for a simultaneous stable isotope ratio measurement of nitrogen and carbon dioxide. The additional use of a special ashtray system to collect the combustion residuals permitted more time-efficient work. These modifications to the elemental analyzer allowed precise measurements to be made down to 1.5 microg nitrogen and 10 microg carbon for stable isotope analysis. Low system background values and an acceptable signal-to-noise ratio have made an additional blank correction for these low sample measurements unnecessary. We provide a precision of this stable isotope analysis for lowest amounts of 1.2-2 microg nitrogen with a standard deviation of +/-0.496 per thousand (n = 27) and for 8.2-15 microg carbon with a standard deviation of +/-0.257 per thousand (n = 31) across different sample runs under stipulated conditions. This application can be established in an automatic mode without cryofocusing procedures.     

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.     

Determination of stable carbon isotopes of organic acids and carbonaceous aerosols in the atmosphere

A wet oxidation method for the compound-specific determination of stable carbon isotopes (delta(13)C) of organic acids in the gas and aerosol phase, as well as of water-soluble organic carbon (WSOC), is presented. Sampling of the organic acids was done using a wet effluent diffusion denuder/aerosol collector (WEDD/AC) coupled to an ion chromatography (IC) system. The method allows for compound-specific stable carbon isotope analysis by collecting different fractions of organic acids at the end of the IC system using a fraction collector. delta(13)C analyses of organic acids were conducted by oxidizing the organic acids with sodium persulfate at a temperature of 100 degrees C and determining the delta(13)C value of the resulting carbon dioxide (CO(2)) with an isotope ratio mass spectrometer. In addition, analysis of delta(13)C of the WSOC was performed for particulate carbon collected on aerosol filters. The WSOC was extracted from the filters using ultrapure water (MQ water), and the dissolved organic carbon was oxidized to CO(2) using the oxidation method. The wet oxidation method has an accuracy of 0.5 per thousand with a precision of +/-0.4 per thousand and provides a quantitative result for organic carbon with a detection limit of 150 ng of carbon.     

A method for carbon stable isotope analysis of methyl halides and chlorofluorocarbons at pptv concentrations

A pre-concentration system has been validated for use with a gas chromatography/mass spectrometry/isotope ratio mass spectrometer (GC/MS/IRMS) to determine ambient air (13)C/(12)C ratios for methyl halides (MeCl and MeBr) and chlorofluorocarbons (CFCs). The isotopic composition of specific compounds can provide useful information on their atmospheric budgets and biogeochemistry that cannot be ascertained from abundance measurements alone. Although pre-concentration systems have been previously used with a GC/MS/IRMS for atmospheric trace gas analysis, this is the first study also to report system validation tests. Validation results indicate that the pre-concentration system and subsequent separation technologies do not significantly alter the stable isotopic ratios of the target methyl halides, CFC-12 (CCl(2)F(2)) and CFC-113 (C(2)Cl(3)F(3)). Significant, but consistent, isotopic shifts of -27.5 per thousand to -25.6 per thousand do occur within the system for CFC-11 (CCl(3)F), although the shift is correctible. The method presented has the capacity to separate these target halocarbons from more than 50 other compounds in ambient air samples. Separation allows for the determination of stable carbon isotope ratios of five of these six target trace atmospheric constituents within ambient air for large volume samples (相似文献   

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.     

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.     

液相色谱-同位素比质谱技术发展及应用研究进展

液相色谱-同位素比质谱（LC-IRMS）是一种特征化合物同位素分析技术,该技术利用LC IsoLink接口设备实现液相色谱与同位素比质谱的联用,通过检测目标物质的稳定碳同位素比（δ¹³C）,实现样品的产地来源与品质真实性鉴定。该文总结了IRMS与LC-IRMS技术的概况,以及过去20年LC-IRMS的发展历程;归纳整理了LC-IRMS在食品安全、生态与环境、生命科学及考古学等领域的应用情况;评述了LC-IRMS面临的技术局限、挑战及其未来的发展趋势。     

常见炸药的稳定同位素比值分析方法研究进展

炸药的深度比对与溯源对于爆炸案事件的侦破具有重大意义,以不同地域来源的原料或不同生产工艺生产的炸药,其组成元素的稳定同位素比值具有差异,因而稳定同位素比值可作为炸药深度比对与溯源的重要指标。稳定同位素比值质谱法(IRMS)作为一种高精度的稳定同位素比值测量手段,已逐渐发展成熟,与元素分析仪、气相色谱仪、液相色谱仪等仪器联用,在食品安全、环境保护、法庭科学等领域应用广泛。IRMS在炸药比对与溯源上亦发挥了重要作用,自1975年IRMS被应用于区分不同国家生产的三硝基甲苯(TNT)以来,IRMS已成功用于多种炸药的分析。但目前尚未见有文献系统地总结常见炸药的稳定同位素比值分析研究进展。该文介绍了稳定同位素比值分析的相关原理、仪器组成及特点,分别总结了硝酸铵、黑火药、TNT、太恩、黑索金等常见炸药的稳定同位素比值分析方法,汇总了文献报道的不同国家生产的硝酸铵、黑火药、TNT等炸药的稳定同位素比值。文章就不同炸药的稳定同位素比值差异、炸药生产、存储过程中相关因素对同位素比值的影响,爆炸前后稳定同位素比值的变化情况等内容进行了分析。本文还指出了目前炸药的稳定同位素比值分析研究中存在的问题,对可能的解决办法进行了讨论,对未来的发展方向提出了建议。     

Reconstructing bulk isotope ratios from compound‐specific isotope ratios

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

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

Quantitative analysis using gas chromatography/combustion/isotope ratio mass spectrometry and standard addition of intrinsically labelled standards (SAIL)--application to isoflavones in foods

We have investigated a novel application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) for the quantitative analysis of the isoflavones in food matrices. Previous methods have been hampered by the lack of analytical standards to introduce early enough in the extraction procedure to allow compensations for losses at all stages of the procedure. In this work we have produced standard materials that can be added at the initial extraction, by intrinsically labelling soya plants by growing them in an atmosphere enriched in the stable isotope of carbon in CO(2). On analysis these plants were shown to contain phytoestrogens at a high (up to 20%) level of enrichment. The dried plant material has been used to estimate the isoflavone concentrations of a set of spiked flours. For daidzein the methodology was shown to produce results comparable to those achieved by GC/MS techniques. The method was less successful for genistein, possibly due to the greater fragility of this compound under the conditions required for the analysis.     

Measurement of oleic acid in human plasma by isotope dilution mass spectrometry

Summary A method for the quantitative determination of oleic acid in human plasma by isotope-dilution mass spectrometric technique is described. For the measurement of the fatty acid concentration (1-¹³C) oleic acid is added to the plasma sample. The fatty acids are extracted with n-hexane. Portions of the extract are esterified by the boron trifluoridemethanol method or stable isotope methylation. The methyl ester derivatives of the fatty acids are separated and detected by GC-MS with the mass spectrometer set to m/z 296 and 297. For the measurement of oleic acid after stable isotope methylation the m/z 299 and m/z 300 are monitored. The amounts of oleic acid in the plasma are calculated from the isotope ratios measured by selected ion monitoring. The recovery of the methylation step and the precision and accuracy of the GC-MS method are presented and discussed.