气相色谱-燃烧-同位素比值质谱法分析氨基酸氮稳定同位素并初步评估水生生物体营养级

Stable Nitrogen Isotope Analysis of Amino Acids by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry for High-Resolution Trophic Level Estimation

氨基酸稳定氮同位素(δ^(15)N)分析能准确有效地评估生物体的营养级以及氮在食物链中的流动。本研究优化了氨基酸氮同位素的分析方法:样品在酸性条件下水解后,释放出的蛋白质氨基酸经阳离子交换树脂纯化后,衍生为对应的N-新戊酰基,O-异丙醇(N-pivaloyl-isopropyl,NPP)酯,利用气相色谱-燃烧-同位素比值质谱仪(Gas chromatography-combustion-isotope ratio mass spectrometry,GC-C-IRMS)测定其δ^(15)N。经非极性气相色谱柱DB-5ms分离后,13种氨基酸NPP酯衍生物均可得到良好的基线分离。在样品量不低于20 ng N条件下,GC-C-IRMS方法的精密度优于1‰,测得的δ^(15)N值与EA-IRMS法测得的δ^(15)N值没有明显差异。阳离子树脂纯化前后各氨基酸δ^(15)N值差异低于1‰,表明没有产生明显的同位素分馏。采用本方法成功地估算了阿哈湖生态系统中常见水生生物的营养级,可作为研究氨基酸代谢以及生态系统特征的新方法。

The analysis of stable nitrogen isotopic composition （δ^15N） of individual amino acid was recognized as an effective method for estimating the trophic level of organisms and detecting the nitrogen flow in food webs. In this study, we evaluated a two-stage procedure of esterification followed by acylation, in which biological samples underwent hydrolysis in acid and the released individual amino acids were derivative into the corresponding N-pivaloyl-iso-propyl （NPP） esters for gas chromatography-combustion-isotope ratio mass spectrometric （GC-C-IRMS） analysis. A total of 13 kinds of individual amino acid derivatives were baseline separated on a nonpolar gas chromatography column （DB-Sms）. The amount of sample for each test was not less than 20 ng N on column. High correlations were observed between the δ^15N values respectively obtained by GC-C-IRMS and element analysis-isotope ration mass spectrometry （EA-IRMS）. Furthermore, the mean precision of this method was better than 1‰. Cation-exchange chromatograph was used to purify the samples, and the difference of the detection δ^15N values before and after purification by the resin was within 1‰. This method was applied to estimate the trophic level of various natural freshwater organisms from Aha Lake. The present study provided a new idea for the application of stable nitrogen isotope （ δ^15N） in the trophic level estimation of organisms and metabolism analysis of amino acid.