Practical considerations in the determination of compound-specific amino acid δ15N values in animal and plant tissues by gas chromatography-combustion-isotope ratio mass spectrometry, following derivatisation to their N-acetylisopropyl esters

Rationale: Stable nitrogen isotope (δ(15)N) 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 δ(15)N value remain little understood. Determination of individual amino acid (AA) δ(15)N 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 δ(15)N values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Amino acid standards of known δ(15)N 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 δ(15)N 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‰ of their δ(15)N values measured separately by elemental analyser (EA)-IRMS, with a precision of better than 0.8‰. The δ(15)N values of AAs after Dowex ion-exchange chromatography were within ±0.9‰ of their values prior to ion-exchange chromatography. The AA δ(15)N values of bone collagen hydrolysed with and without starch were within ±0.8‰.

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 δ(15)N values by GC-C-IRMS.