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Publication - Dr Timothy Knowles

    Practical considerations in the determination of compound-specific amino acid delta N-15 values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetylisopropyl esters

    Citation

    Styring, AK, Kuhl, A, Knowles, TDJ, Fraser, RA, Bogaard, A & Evershed, RP, 2012, ‘Practical considerations in the determination of compound-specific amino acid delta N-15 values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetylisopropyl esters’. Rapid Communications in Mass Spectrometry, vol 26., pp. 2328-2334

    Abstract

    RATIONALE Stable nitrogen isotope (d15N) values of bone collagen are routinely used to inform interpretations of diet and trophic positions within contemporary and ancient ecosystems, yet the underlying physiological and biochemical factors which contribute to the bulk collagen d15N value remain little understood. Determination of individual amino acid (AA) d15N values in animal and plant proteins can help to elucidate the cycling of nitrogen and inform predictions of palaeodiet and ecology. METHODS In this study we present a methodology for the measurement of amino acid d15N values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Amino acid standards of known d15N values were derivatised to their N-acetylisopropyl (NAIP) esters and purified through Dowex ion-exchange resin to determine any isotopic fractionation associated with derivatisation and ion-exchange chromatography. The effect of starch on AA d15N values was also determined by hydrolysing bone collagen with and without the presence of starch. RESULTS The amino acids derivatised to their NAIP esters give values within +/-0.8 parts per thousand of their d15N values measured separately by elemental analyser (EA)-IRMS, with a precision of better than 0.8 parts per thousand. The d15N values of AAs after Dowex ion-exchange chromatography were within +/-0.9 parts per thousand of their values prior to ion-exchange chromatography. The AA d15N values of bone collagen hydrolysed with and without starch were within +/-0.8 parts per thousand. CONCLUSIONS Hydrolysis of lipid-extracted plant material followed by purification of AAs using Dowex ion-exchange resin and derivatisation to their NAIP esters is a suitable protocol for the accurate determination of individual plant and animal AA d15N values by GC-C-IRMS. Copyright (c) 2012 John Wiley & Sons, Ltd.

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