Position-specific 13 C/12 C analysis of amino acid carboxyl groups - automated flow-injection analysis based on reaction with ninhydrin.

Abstract

RationaleThe fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off‐line preparation to reveal this information. An automated position‐specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ 13CCARBOXYL values). This automation makes PSIA measurements easier and routine.MethodsAn existing high‐performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post‐column derivatisation unit between the HPLC system and the interface. The post‐column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin.ResultsThe methodology described produced δ 13CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ 13CCARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ 13CCARBOXYL values of a number of internationally available amino acid reference materials.ConclusionsThe PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole‐molecule level. When combined with on‐line chromatographic separation or off‐line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position‐specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.