环境样品中苯丙氨酸 δ15N 值的高精度测量：一种结合高压液相色谱纯化和元素分析仪同位素比质谱的新方法。

High-precision measurement of phenylalanine δ15N values for environmental samples: a new approach coupling high-pressure liquid chromatography purification and elemental analyzer isotope ratio mass spectrometry.

机构信息

Ocean Sciences Department, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.

出版信息

Rapid Commun Mass Spectrom. 2013 Nov 15;27(21):2327-37. doi: 10.1002/rcm.6695.

DOI:10.1002/rcm.6695

PMID:24097388

Abstract

RATIONALE

Compound-specific isotope analysis of individual amino acids (CSI-AA) is a powerful new tool for tracing nitrogen (N) source and transformation in biogeochemical cycles. Specifically, the δ(15)N value of phenylalanine (δ(15)N(Phe)) represents an increasingly used proxy for source δ(15)N signatures, with particular promise for paleoceanographic applications. However, current derivatization/gas chromatography methods require expensive and relatively uncommon instrumentation, and have relatively low precision, making many potential applications impractical.

METHODS

A new offline approach has been developed for high-precision δ(15)N measurements of amino acids (δ(15)N(AA)), optimized for δ(15)N(Phe) values. Amino acids (AAs) are first purified via high-pressure liquid chromatography (HPLC), using a mixed-phase column and automated fraction collection. The δ(15)N values are determined via offline elemental analyzer-isotope ratio mass spectrometry (EA-IRMS).

RESULTS

The combined HPLC/EA-IRMS method separated most protein AAs with sufficient resolution to obtain accurate δ(15)N values, despite significant intra-peak isotopic fractionation. For δ(15)N(Phe) values, the precision was ±0.16‰ for standards, 4× better than gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS; ±0.64‰). We also compared a δ(15)N(Phe) paleo-record from a deep-sea bamboo coral from Monterey Bay, CA, USA, using our method versus GC/C/IRMS. The two methods produced equivalent δ(15)N(Phe) values within error; however, the δ(15)N(Phe) values from HPLC/EA-IRMS had approximately twice the precision of GC/C/IRMS (average stdev of 0.27‰ ± 0.14‰ vs 0.60‰ ± 0.20‰, respectively).

CONCLUSIONS

These results demonstrate that offline HPLC represents a viable alternative to traditional GC/C/IMRS for δ(15)N(AA) measurement. HPLC/EA-IRMS is more precise and widely available, and therefore useful in applications requiring increased precision for data interpretation (e.g. δ(15)N paleoproxies).

摘要

原理

对单个氨基酸的稳定同位素比值分析（CSI-AA）是一种追踪生物地球化学循环中氮（N）源和转化的强大新工具。具体来说，苯丙氨酸的δ（15）N 值（δ（15）N（Phe））代表了一种越来越常用的源δ（15）N 特征的代用指标，特别是在古海洋学应用方面具有很大的前景。然而，目前的衍生化/气相色谱法需要昂贵且相对罕见的仪器，并且精度相对较低，使得许多潜在的应用变得不切实际。

方法

我们开发了一种新的离线方法，用于高精度测定氨基酸的 δ（15）N 值（δ（15）N（AA）），并针对 δ（15）N（Phe）值进行了优化。首先通过高压液相色谱（HPLC），使用混合相柱和自动馏分收集来纯化氨基酸（AAs）。然后通过离线元素分析仪-同位素比质谱仪（EA-IRMS）来测定 δ（15）N 值。

结果

尽管存在明显的峰内同位素分馏，但是 HPLC/EA-IRMS 联合方法还是能够以足够的分辨率分离大多数蛋白质 AAs，从而获得准确的 δ（15）N 值。对于 δ（15）N（Phe）值，标准品的精度为±0.16‰，比气相色谱/燃烧/同位素比质谱（GC/C/IRMS；±0.64‰）高 4 倍。我们还比较了使用我们的方法和 GC/C/IRMS 从美国加利福尼亚州蒙特雷湾深海竹珊瑚获得的 δ（15）N（Phe）古记录。两种方法产生的 δ（15）N（Phe）值在误差范围内相等；但是，HPLC/EA-IRMS 的 δ（15）N（Phe）值的精度大约是 GC/C/IRMS 的两倍（平均标准差分别为 0.27‰±0.14‰和 0.60‰±0.20‰）。