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.     

Hydropyrolysis as a preparative method for the compound-specific carbon isotope analysis of fatty acids

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry is an effective and risk-free means of investigating fatty acid metabolism. Straightforward analysis, however, leads to poor chromatographic resolution, while derivatization adds carbon thereby corrupting the starting stable isotopic composition. Hydropyrolysis is a new approach which defunctionalizes fatty acids to yield the corresponding n-alkanes thus retaining the carbon skeleton intact and improving chromatography, allowing the faithful measurement of carbon isotope ratios.     

Use of a temperature‐programmable injector coupled to gas chromatography–combustion–isotope ratio mass spectrometry for compound‐specific carbon isotopic analysis of polycyclic aromatic hydrocarbons

Compound‐specific isotopic analysis (CSIA) can provide information about the origin of analysed compounds; for instance, polycyclic aromatic hydrocarbons (PAHs) in aerosols. This could be a valuable tool in source apportionment of particulate matter (PM) air pollution. Because gas chromatography–combustion–isotope ratio mass spectrometry (GC‐C‐IRMS) analysis requires an amount of at least 10 ng of an individual PAH, a high concentration of PAHs in the injected extract is needed. When the concentration is low a large volume injector creates the possibility of introducing a satisfactory amount of individual PAHs. In this study a temperature‐programmable injector was coupled to GC‐C‐IRMS and injection parameters (solvent level, transfer column flow, transfers time) were optimised using six solid aromatic compounds (anthracene, fluoranthene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene) dissolved in n‐pentane and EPA 610 reference mixture. CSIA results for solid PAHs were compared with results obtained for the single components analysed by elemental analysis–isotope ratio mass spectrometry. The injection method was validated for two sample injection volumes, 50 and 100 µL. This method was also compared with commonly used splitless injection. To be included in the study, measurements had to have an uncertainty lower than 0.5‰ for and a minimum peak height of 200 mV. The lower concentration limits at which these criteria were fulfilled for PAHs were 30 mg/L for 1 µL in splitless injection and 0.3 and 0.2 mg/L for 50 and 100 µL, respectively, in large volume injection. Copyright © 2009 John Wiley & Sons, Ltd.     

Membrane permeation continuous‐flow isotope ratio mass spectrometry for on‐line carbon isotope ratio determination

Gaseous membrane permeation (MP) technologies have been combined with continuous‐flow isotope ratio mass spectrometry for on‐line δ¹³C measurements. The experimental setup of membrane permeation‐gas chromatography/combustion/isotope ratio mass spectrometry (MP‐GC/C/IRMS) quantitatively traps gas streams in membrane permeation experiments under steady‐state conditions and performs on‐line gas transfer into a GC/C/IRMS system. A commercial polydimethylsiloxane (PDMS) membrane sheet was used for the experiments. Laboratory tests using CO₂ demonstrate that the whole process does not fractionate the C isotopes of CO₂. Moreover, the δ¹³C values of CO₂ permeated on‐line give the same isotopic results as off‐line static dual‐inlet IRMS δ¹³C measurements. Formaldehyde generated from aqueous formaldehyde solutions has also been used as the feed gas for permeation experiments and on‐line δ¹³C determination. The feed‐formaldehyde δ¹³C value was pre‐determined by sampling the headspace of the thermostated aqueous formaldehyde solution. Comparison of the results obtained by headspace with those from direct aqueous formaldehyde injection confirms that the headspace sampling does not generate isotopic fractionation, but the permeated formaldehyde analyzed on‐line yields a ¹³C enrichment relative to the feed δ¹³C value, the isotopic fractionation being 1.0026 ± 0.0003. The δ¹³C values have been normalized using an adapted two‐point isotopic calibration for δ¹³C values ranging from ?42 to ?10‰. The MP‐GC/C/IRMS system allows the δ¹³C determination of formaldehyde without chemical derivatization or additional analytical imprecision. Copyright © 2009 John Wiley & Sons, Ltd.     

Advantages of compound‐specific stable isotope measurements over bulk measurements in studies on plant uptake of intact amino acids

Increasing interest in the ability of plants to take up amino acids has given rise to questions on the accuracy of the commonly used bulk method to measure and calculate amino acid uptake. This method uses bulk measurements of ¹³C and ¹⁵N enrichment in plant tissues after application of dual‐labelled amino acids but some authors have recommended the use of compound‐specific stable isotope (CSI) analysis of the plants' amino acids instead. However, there has never been a direct evaluation of both methods. We conducted a field study applying dual‐labelled (¹³C, ¹⁵N) amino acids (glycine, valine, tyrosine and lysine) to soil of a Plantago lanceolata monoculture. Root and shoot samples were collected 24 h after label application and the isotope composition of the plant tissues was investigated using bulk and CSI measurements. Enrichment of ¹³C in the case of CSI measurements was limited to the applied amino acids, showing that no additional ¹³C had been incorporated into the plants' amino acid pool via the uptake of tracer‐derived C‐fragments. Compared with this rather conservative indicator of amino acid uptake, the ¹³C enrichment of bulk measurements was 8, 5, 1.6 and 6 times higher for fine roots, storage roots, shoot and the whole plant, respectively. These findings show that the additional uptake of tracer‐derived C‐fragments will result in a considerable overestimation of amino acid uptake in the case of bulk measurements. We therefore highly recommend the use of CSI measurements for future amino acid uptake studies due to their higher accuracy. Copyright © 2009 John Wiley & Sons, Ltd.     

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

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

New protocol for compound‐specific radiocarbon analysis of archaeological bones

Rationale

For radiocarbon results to be accurate, samples must be free of contaminating carbon. Sample pre‐treatment using a high‐performance liquid chromatography (HPLC) approach has been developed at the Oxford Radiocarbon Accelerator Unit (ORAU) as an alternative to conventional methods for dating heavily contaminated bones. This approach isolates hydroxyproline from bone collagen, enabling a purified bone‐specific fraction to then be radiocarbon dated by accelerator mass spectrometry (AMS).

Methods

Using semi‐preparative chromatography and non‐carbon‐based eluents, this technique enables the separation of underivatised amino acids liberated by hydrolysis of extracted bone collagen. A particular focus has been the isolation of hydroxyproline for single‐compound AMS dating since this amino acid is one of the main contributors to the total amount of carbon in mammalian collagen. Our previous approach, involving a carbon‐free aqueous mobile phase, required a two‐step separation using two different chromatographic columns.

Results

This paper reports significant improvements that have been recently made to the method to enable faster semi‐preparative separation of hydroxyproline from bone collagen, making the method more suitable for routine radiocarbon dating of contaminated and/or poorly preserved bone samples by AMS. All steps of the procedure, from the collagen extraction to the correction of the AMS data, are described.

Conclusions

The modifications to the hardware and to the method itself have reduced significantly the time required for the preparation of each sample. This makes it easier for other radiocarbon facilities to implement and use this approach as a routine method for preparing contaminated bone samples.

     

Isolation and desalting with cation‐exchange chromatography for compound‐specific nitrogen isotope analysis of amino acids: application to biogeochemical samples

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound‐specific nitrogen isotopic compositions of amino acids. We employed cation‐exchange chromatography of protein and non‐protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation‐exchange chromatography. We applied the procedure to various environmental samples including ‘hard’ (calcareous, siliceous, rock and sediment samples) and ‘soft’ materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation‐exchange chromatography is a pre‐treatment procedure which should be widely useful for the determination of compound‐specific nitrogen isotopic compositions of amino acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of nitrogen‐15 isotope fractionation in tropine: evaluation of extraction protocols for isotope ratio measurement by isotope ratio mass spectrometry

N‐Demethylation of tropine is an important step in the degradation of this compound and related metabolites. With the purpose of understanding the reaction mechanism(s) involved, it is desirable to measure the ¹⁵N kinetic isotope effects (KIEs), which can be accessed through the ¹⁵N isotope shift (Δδ¹⁵N) during the reaction. To measure the isotope fractionation in ¹⁵N during tropine degradation necessitates the extraction of the residual substrate from dilute aqueous solution without introducing artefactual isotope fractionation. Three protocols have been compared for the extraction and measurement of the ¹⁵N/¹⁴N ratio of tropine from aqueous medium, involving liquid‐liquid phase partitioning or silica‐C18 solid‐phase extraction. Quantification was by gas chromatography (GC) on the recovered organic phase and δ¹⁵N values were obtained by isotope ratio measurement mass spectrometry (irm‐MS). Although all the protocols used can provide satisfactory data and both irm‐EA‐MS and irm‐GC‐MS can be used to obtain the δ¹⁵N values, the most convenient method is liquid‐liquid extraction from a reduced aqueous volume combined with irm‐GC‐MS. The protocols are applied to the measurement of ¹⁵N isotope shifts during growth of a Pseudomonas strain that uses tropane alkaloids as sole source of carbon and nitrogen. The accuracy of the determination of the ¹⁵N/¹⁴N ratio is sufficient to be used for the determination of ¹⁵N‐KIEs. Copyright © 2009 John Wiley & Sons, Ltd.