Determination of the δ2 H values of high molecular weight lipids by high-temperature gas chromatography coupled to isotope ratio mass spectrometry.

RATIONALE: The hydrogen isotopic composition of lipids (δ2 Hlipid ) is widely used in food science and as a proxy for past hydrological conditions. Determining the δ2 H values of large, well-preserved triacylglycerides and other microbial lipids, such as glycerol dialkyl glycerol tetraether (GDGT) lipids, is thus of widespread interest but has so far not been possible due to their low volatility which prohibits analysis by traditional gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS). METHODS: We determined the δ2 H values of large, polar molecules and applied high-temperature gas chromatography (HTGC) methods on a modified GC/P/IRMS system. The system used a high-temperature 7-m GC column, and a glass Y-splitter for low thermal mass. Methods were validated using authentic standards of large, functionalised molecules (triacylglycerides, TGs), purified standards of GDGTs. The results were compared with δ2 H values determined by high-temperature elemental analyser/pyrolysis/isotope ratio mass spectrometry (HTEA/P/IRMS), and subsequently applied to the analysis of GDGTs in a sample from a methane seep and a Welsh peat. RESULTS: The δ2 H values of TGs agreed within error between HTGC/P/IRMS and HTEA/IRMS, with HTGC/P/IRMS showing larger errors. Archaeal lipid GDGTs with up to three cyclisations could be analysed: the δ2 H values were not significantly different between methods with standard deviations of 5 to 6 ‰. When environmental samples were analysed, the δ2 H values of isoGDGTs were 50 ‰ more negative than those of terrestrial brGDGTs. CONCLUSIONS: Our results indicate that the HTGC/P/IRMS method developed here is appropriate to determine the δ2 H values of TGs, GDGTs with up to two cyclisations, and potentially other high molecular weight compounds. The methodology will widen the current analytical window for biomarker and food light stable isotope analyses. Moreover, our initial measurements suggest that bacterial and archaeal GDGT δ2 H values can record environmental and ecological conditions.

Isotope effects associated with the preparation and methylation of fatty acids by boron trifluoride in methanol for compound-specific stable hydrogen isotope analysis via gas chromatography/thermal conversion/isotope ratio mass spectrometry.

RATIONALE: Compound-specific stable hydrogen isotope analysis of fatty acids is being used increasingly as a means of deriving information from a diverse range of materials of archaeological, geological and environmental interest. Preparative steps required prior to isotope ratio mass spectrometry (IRMS) analysis have the potential to alter determined δD values and hence must be accounted for if accurate δD values for target compounds are to be obtained. METHODS: Myristic, palmitic, stearic, arachidic and behenic saturated fatty acids were derivatised to their respective fatty acid methyl esters (FAMEs), using 14% (w/v) boron trifluoride in methanol then analysed by gas chromatography/thermal conversion/IRMS (GC/TC/IRMS). FAMEs generated from fatty acid sodium salts of unknown δD values were then used to test a correction factor determined for this method of derivatisation. RESULTS: Derivatisation was found to alter the hydrogen isotopic composition of FAMEs although this effect was reproducible and can be accounted for. The difference between the mean corrected and mean bulk δD values was always less than 6.7 ‰. Extraction of saturated fatty acids and acyl lipids from samples, subsequent hydrolysis, then separation on a solid-phase extraction cartridge, was found to alter the determined δD values by less than one standard deviation. CONCLUSIONS: Overall, it has been shown that for natural abundance hydrogen isotope determinations, the isolation and derivatisation of extracted fatty acids alters the determined δD values only by a numerical increment comparable with the experimental error. This supports the use of the described analytical protocol as an effective means of determining fatty acid δD values by GC/TC/IRMS.

Identification of a disinterred grave by molecular and stable isotope analysis.

Confirmation of a potential disinterred grave was sought by GC and GC/MS analyses of lipid extracts of whole soils and white particulate matter. Fatty acid profiles and concentrations determined for three of the soils correlated with the deposition of a large amount of exogenous organic matter, most likely adipocere and/or decomposed body fat. Determination of C16:0 and C18:0 fatty acid delta13C values by GC/C/IRMS revealed the input to be isotopically distinct from common British domesticated animals, plotting closely to values determined for adipose fat obtained from of a murder victim. By considering the difference between delta13C values (delta13C18:0-16:0) a potential isotopic proxy for identifying the source of adipocere (human) and adipose tissue was proposed.

Four millennia of dairy surplus and deposition revealed through compound-specific stable isotope analysis and radiocarbon dating of Irish bog butters.

Bog butters are large white or yellow waxy deposits regularly discovered within the peat bogs of Ireland and Scotland. They represent an extraordinary survival of prehistoric and later agricultural products, comprising the largest deposits of fat found anywhere in nature. Often found in wooden containers or wrapped in animal bladders, they are considered to have been buried intentionally by past farming communities. While previous analysis has determined that Irish bog butters derive from animal fat, their precise characterisation could not be achieved due to diagenetic compositional alterations during burial. Via compound-specific stable isotope analysis, we provide the first conclusive evidence of a dairy fat origin for the Irish bog butter tradition, which differs from bog butter traditions observed elsewhere. Our research also reveals a remarkably long-lived tradition of deposition and possible curation spanning at least 3500 years, from the Early Bronze Age (c. 1700 BC) to the 17th century AD. This is conclusively established via an extensive suite of both bulk and compound-specific radiocarbon dates.

Development of N-acetyl methyl ester derivatives for the determination of delta13C values of amino acids using gas chromatography-combustion- isotope ratio mass spectrometry.

A novel derivatization procedure, N-acetyl methyl (NACME) esterification, was developed to improve the accuracy and precision of amino acid delta13C value determination using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS). Standard mixtures of 15 protein amino acids were converted to NACME and N-acetyl-isopropyl (NAIP) esters; the latter established derivative was employed for comparison purposes. Both procedures yielded baseline-resolved peaks for all 15 amino acids when GC columns coated with polar stationary phases were employed. For NACME esters, the methylation conditions governed reaction yields, with highest yields observed when a 1 h, 70 degrees C methylation procedure (anhydrous MeOH/acetyl chloride, 25:4, v/v) was performed. The mean derivatization yields expressed relative to an underivatized coinjected standard (n-nonadecane) for both NACME and NAIP esters were identical. Likewise, the mean kinetic isotope effects (KIEs) were not significantly different (KIE(NACME) = 1.036; KIE(NAIP) = 1.038) and were shown in both cases to be reproducible. The mean reproducibility obtained from 15 replicates (3 x batches of 5) of both derivatives was strong (mean STDV(NACME) = 0.3 per thousand and STDV(NAIP) = 0.4 per thousand). The isotopic robustness of both derivatization procedures was observed over a concentration range of 52,500 microg of amino acid. NACME esters displayed low errors (+/-0.6 per thousand for phenylalanine to +/-1.1 per thousand for serine) due to the higher sample-to-derivative carbon ratio of this derivative. Finally, the integrity of the new NACME procedure was confirmed through analysis of diet and bone collagen amino acids of rats reared on C3 or C4 diets, which indicated the high degree of both accuracy and precision of the delta13C values obtained for individual amino acids.

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.

Characterisation of "bog butter" using a combination of molecular and isotopic techniques.

The chemical analyses of "bog butters" recovered from peat bogs of Scotland were performed with the aim of determining their origins. Detailed compositional information was obtained from "bog butter" lipids using high temperature gas chromatography (HTGC) and GC-mass spectrometry (GC-MS). The results indicate the degree to which "bog butters" have undergone diagenetic alterations during burial to form an adipocere like substance, consisting predominantly of hexadecanoic (palmitic) and octadecanoic (stearic) acids. GC-combustion-isotope ratio MS (GC-C-IRMS) was used to determine delta13C values for the dominant fatty acids present, revealing for the first time that "bog butters" were derived from both ruminant dairy fats and adipose fats. The results are compared and contrasted with modern reference fats and adipoceres produced in vitro.

Direct dating of archaeological pottery by compound-specific 14C analysis of preserved lipids.

A methodology is described demonstrating the utility of the compound-specific 14C technique as a direct means of dating archaeological pottery. The method uses automated preparative capillary gas chromatography employing wide-bore capillary columns to isolate individual compounds from lipid extracts of archaeological potsherds in high purity (>95%) and amounts (>200 microg) sufficient for radiocarbon dating using accelerator mass spectrometry (AMS). A protocol was developed and tested on n-alkanes and n-carboxylic acids possessing a broad range of 14C ages. Analytical blanks and controls allowed background 14C measurements to be assessed and potential sources of errors to be detected, i.e., contamination with modern or dead 14C, isotopic fraction effects, etc. A "Russian doll" method was developed to transfer isolated target compounds onto tin powder/capsules prior to combustion and AMS analyses. The major advantage of the compound-specific technique is that 14C dates obtained for individual compounds can be directly linked to the commodities processed in the vessels during their use, e.g., animal fats. The compound-specific 14C dating protocol was validated on a suite of ancient pottery whose predicted ages spanned a 5000-year date range. Initial results indicate that meaningful correlations can be obtained between the predicted date of pottery and that of the preserved lipids. These findings constitute an important step forward to the direct dating of archaeological pottery.

Chemistry of archaeological animal fats.

Animal fats are preserved at archaeological sites in association with unglazed pottery, human and animal remains, and other deposits or hoards. High-temperature gas chromatography (HT-GC) and combined HT-GC/mass spectrometry (HT-GC/MS) has confirmed the presence of animal fats in lipid extracts of artifacts. Degradation products and pathways have been discerned through the analyses of archaeological finds and the products of laboratory and field-based decay experiments. The origins of preserved fats have been determined through detailed compositional analysis of their component fatty acids by GC, by GC/MS of dimethyl disulfide derivatives of monoenoic components, and by GC-combustion-isotope ratio-MS (GC-C-IRMS), to derive diagenetically robust delta(13)C values. Regiospecific analysis of intact triacylglycerols by high-performance liquid chromatography/MS (HPLC/MS), with atmospheric pressure chemical ionization, provides a further criterion for establishing the origin of fats. Preparative GC has been employed to isolate individual fatty acids from archaeological pottery in sufficient amounts for (14)C dating.