Analysis of amino acid ¹³C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry

Abstract

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds. We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.

Single amino acid radiocarbon dating of Upper Paleolithic modern humans

“…The amino acid retention times were identified using standards, and spiking natural samples confirmed these retention times in archaeological samples. Detection by UV absorbance (32)(33)(34) and LC/MS analysis of selected amino acid fractions was used to confirm identification of peaks by mass. It is crucial that laboratory-derived carbonaceous material be kept as close to zero as possible.…”

An integrated stable isotope study of plants and animals from Kouphovouno, southern Greece: a new look at Neolithic farming

“…N values predominantly reflect the protein portion of the consumers' diets (Ambrose and Norr 1993;Chisholm et al 1982), with many amino acids being directly assimilated from the food source (Chikaraishi et al 2009;Corr et al 2005;McCullagh et al 2006). N values closer to the value of atmospheric nitrogen, which is 0‰ (Bernhard 2012;Delwiche et al 1979;Högberg 1997).…”

Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization

“…This process has been successfully applied to sugars 22 and amino acids, 21,23,24 and has been shown to be quantitative and without isotopic fractionation.…”

“…In LC/IRMS, the target molecule containing the heavy 13 C isotope is eluted after the molecule containing the light one ( 12 C). 23,24 The chromatographic isotope effect in LC seems to be related either to the nature of the compounds, to the stationary phases, or to both, and needs to be carefully investigated.…”