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

Abstract

Rationale: The 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 (δ13CCARBOXYL values). This automation makes PSIA measurements easier and routine.

Methods: An 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.

Results: The methodology described produced δ13CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ13CCARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ13CCARBOXYL values of a number of internationally available amino acid reference materials.

Conclusions: The 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.