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The measurement of the nitrogen isotope ratio of taurine (2-aminoethanesulphonic acid) in biological samples has a large number of potential applications. Taurine is a small water-soluble molecule which is notoriously difficult to analyze due to its polarity and functionality. A method is described which allows the determination of the natural abundance delta N-15 values of taurine and structural analogues, such as 3-amino-1-propanesulphonic acid (APSA), by isotope ratio mass spectrometry interfaced to gas chromatography (GC-irm-MS). The one-step protocol exploits the simultaneous derivatization of both functionalities of these aminosulphonic acids by reaction with triethylortho-acetate (TEOA). Conditions have been established which ensure quantitative reaction thus avoiding any nitrogen isotope fractionation during derivatization and workup. The differences in the delta N-15 values of derivatized and non-derivatized taurine and APSA all fall within the working range of 0.4 parts per thousand (-0.02 to 0.39 parts per thousand). When applied to four sources of taurine with various delta N-15 values, the method achieved excellent reproducibility and accuracy. The optimized method enables the determination of the natural abundance delta N-15 values of taurine over the concentration range 1.5-7.84 mu mol.mL(-1) in samples of biological origin. Copyright (C) 2010 John Wiley & Sons, Ltd.

Multi-element stable isotope ratios have been assessed as a means to distinguish between fermented cocoa beans from different geographical and varietal origins. Isotope ratios and percentage composition for C and N were measured in different tissues (cotyledons, shells) and extracts (pure theobromine, defatted cocoa solids, protein, lipids) obtained from fermented cocoa bean samples. Sixty-one samples from 24 different geographical origins covering all four continental areas producing cocoa were analyzed. Treatment of the data with unsupervised (Principal Component Analysis) and supervised (Partial Least Squares Discriminant Analysis) multiparametric statistical methods allowed the cocoa beans from different origins to be distinguished. The most discriminant variables identified as responsible for geographical and varietal differences were the δ15N and δ13C values of cocoa beans and some extracts and tissues. It can be shown that the isotope ratios are correlated with the altitude and precipitation conditions found in the different cocoa-growing regions.

International audience; In the article (Romek et al. 2013) we reported the values of δ15N (‰) and δ13C (‰) obtained bySince exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.

International audience; RATIONALE In isotope tracer experiments used in nutritional studies, it is frequently desirable both to determine the 15N/14N ratios of target compounds and to quantify these compounds. This report shows how this can be achieved in a single chromatographic run for protein amino acids using an isotope ratio mass spectrometer. METHODS Protein hydrolysis by acidic digestion was used to release amino acids, which were then derivatized as their N-pivaloyl-O-isopropyl esters. Suitable conditions for sample preparation were established for both hair and milk proteins. The N-pivaloyl-O-isopropyl esters of amino acids were separated by gas chromatography (GC) on a 60m ZB-WAX column linked via a combustion interface to an isotope ratio mass spectrometer. The 15N/14N ratios were obtained from the m/z 28, 29 and 30 peak intensities and the quantities from the Area All (Vs) integrated peak areas. RESULTS It is shown from a five-point calibration curve that both parameters can be measured reliably within the range of 1.0 to 2.0mg/mL for the major amino acids derived from the hydrolysis of human maternal milk or hair samples. The method was validated in terms of inter-day and inter-user repeatability for both parameters for 14 amino acids. The amino acid percentage composition showed a good correlation with literature values. The method was applied to determine the variability in a population of lactating mothers and their infants. CONCLUSIONS It has been established that 15N values can be simultaneously determined for a complex mixture of amino acids at variable concentrations. It is shown that the percentage composition obtained correlates well with that obtained by calculation from the protein composition or from literature values. This procedure should provide a significant saving in analysis time, especially in those cases where the GC run-time is necessarily long. It allows the satisfactory determination of the variation within a sample population. Copyright (c) 2013 John Wiley & Sons, Ltd.

Two-dimensional nuclear magnetic resonance (2D NMR) forms a powerful tool for the quantitative analysis of complex mixtures such as samples of metabolic relevance. However, its use for quantitative purposes is far from being trivial, not only because of the associated experiment time, but also due to its subsequent high sensitivity to hardware instabilities affecting its precision. In this paper, an alternative approach is considered to measure absolute metabolite concentrations in complex mixtures with a high precision in a reasonable time. It is based on a ``multi-scan single shot'' (M3S) strategy, which is derived from the ultrafast 2D NMR methodology. First, the analytical performance of this methodology is compared to the one of conventional 2D NMR. 2D correlation spectroscopy (COSY) spectra are obtained in 10 min on model metabolic mixtures, with a precision in the 1-4% range (versus 5-18% for the conventional approach). The M3S approach also shows a better linearity than its conventional counterpart. It ensures that accurate quantitative results can be obtained provided that a calibration procedure is carried out. The M3S COSY approach is then applied to measure the absolute metabolite concentration in three breast cancer cell line extracts, relying on a standard addition protocol. M3S COSY spectra of such extracts are recorded in 20 min and give access to the absolute concentration of 14 major metabolites, showing significant differences between cell lines.