A new approach to δ15N compound‐specific amino acid trophic position measurements: preparative high pressure liquid chromatography technique for purifying underivatized amino acids for stable isotope analysis

Abstract

Compound specific isotope analysis of amino acids (CSI‐AA) has emerged as an important new method for investigating trophic dynamics in both aquatic and terrestrial systems. Multiple studies have shown that δ15N values of glutamic acid (Glu) and phenylalanine (Phe) can be coupled to provide precise estimates of trophic position (TP), while simultaneously decoupling baseline δ15N values from the effects of trophic transfer. However, the current standard gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) approach is limited by high expense, limited availability, and relatively low precision. We present a new method for making TP estimates in biological samples by CSI‐AA (TPCSIA), based on a high‐pressure liquid chromatography (HPLC) purification of underivatized amino acids, followed by offline elemental analysis‐isotope ratio mass spectrometry (EA‐IRMS). We compare results from our new HPLC/EA‐IRMS method versus GC‐C‐IRMS in both standard and natural materials. Nitrogen isotopic values of purified Glu and Phe standards were identical within error for both methods. In five widely different marine organisms, the δ15N values of Glu and Phe were also indistinguishable within error between the two approaches; however, the δ15N values produced by the HPLC/EA‐IRMS approach had higher average precision (average SD = 0.3 ± 0.2 ‰) than the GC‐C‐IRMS measurements (average SD = 0.45 ± 0.15 ‰). The resulting TPCSIA estimates were statistically indistinguishable (t < 1.2, df = 6, P > 0.3) between the two methods for all organisms examined. Our HPLC/EA‐IRMS method may therefore allow significant expansion of TPCSIA applications, requiring only commonly available instrumentation to produce high precision TPCSIA values.