Efficient acid hydrolysis for compound-specific δ15N analysis of amino acids for determining trophic positions.

ABSTRACT

Compound-specific isotope analysis of nitrogen in amino acids (CSIA-AA, δ15NAA) has gained increasing popularity for elucidating energy flow within food chains and determining the trophic positions of various organisms. However, there is a lack of research on the impact of hydrolysis conditions, such as HCl concentration and hydrolysis time, on δ15NAA analysis in biota samples. In this study, we investigated two HCl concentrations (6 M and 12 M) and four hydrolysis times (2 h, 6 h, 12 h, and 24 h) for hydrolyzing and derivatizing AAs in reference materials (Tuna) and biological samples of little egret (n = 4), night heron (n = 4), sharpbelly (n = 4) and Algae (n = 1) using the n-pivaloyl-iso-propyl (NPIP) ester approach. A Dowex cation exchange resin was used to purify amino acids before derivatization. We then determined δ15NAA values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The results revealed no significant differences (p > 0.05) in δ15NAA values among samples treated with different HCl concentrations or hydrolysis times, particularly for δ15NGlx (range 21.0-23.5‰) and δ15NPhe (range 4.3-5.4‰) in Tuna (12 M). Trophic positions (TPs) calculated based on δ15NAA at 2 h (little egret 2.9 ± 0.1, night heron 2.8 ± 0.1, sharpbelly 2.0 ± 0.1 and Algae 1.3 ± 0.2) were consistent with those at 24 h (3.1 ± 0.1, 2.8 ± 0.1, 2.2 ± 0.1 and 1.1 ± 0.1, respectively), suggesting that a 2-h hydrolysis time and a 6 M HCl concentration are efficient pretreatment conditions for determining δ15NAA and estimating TP. Compared to the currently used hydrolysis conditions (24 h, 6 M), the proposed conditions (2 h, 6 M) accelerated the δ15NAA assay, making it faster, more convenient, and more efficient. Further research is needed to simplify the operational processes and reduce the time costs, enabling more efficient applications of CSIA-AA.