氨基酸中氮的化合物特异性同位素分析(CSIA-AA,δ15NAA)在阐明食物链内的能量流和确定各种生物的营养位置方面越来越受欢迎。然而,对水解条件的影响缺乏研究,如HCl浓度和水解时间,生物群样品中的δ15NAA分析。在这项研究中,我们研究了两个HCl浓度(6M和12M)和四个水解时间(2小时,6h,12h,和24h)用于水解和衍生参考材料(金枪鱼)和小白鹭(n=4)的生物样品中的AA,夜鹭(n=4),sharpbelly(n=4)和藻类(n=1)使用正-新戊酰基-异丙基(NPIP)酯方法。Dowex阳离子交换树脂用于在衍生化之前纯化氨基酸。然后,我们使用气相色谱-燃烧-同位素比质谱(GC-C-IRMS)确定了δ15NAA值。结果表明,用不同HCl浓度或水解时间处理的样品之间的δ15NAA值没有显着差异(p>0.05)。特别是金枪鱼(12M)的δ15NGlx(范围:21.0-23.5‰)和δ15NPhe(范围:4.3-5.4‰)。根据2h时的δ15NAA计算的营养位置(TP)(小白鹭:2.9±0.1,夜鹭:2.8±0.1,尖胸:2.0±0.1和藻类:1.3±0.2)与24h时的营养位置(分别为3.1±0.1、2.8±0.1、2.2±0.1和1.1±0.1)一致,表明2小时的水解时间和6MHCl浓度是确定δ15NAA和估计TP的有效预处理条件。与目前使用的水解条件(24h,6米),建议的条件(2小时,6M)加速了δ15NAA测定,让它更快,更方便,更有效率。需要进一步研究以简化操作流程并降低时间成本,实现CSIA-AA的更有效应用。
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.