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

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 (δ¹³ C_CARBOXYL values). This automation makes PSIA measurements easier and routine.

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.

The methodology described produced δ¹³ C_CARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ¹³ C_CARBOXYL values) between amino acids over a 1-year period. First estimates are presented for the δ¹³ C_CARBOXYL values of a number of internationally available amino acid reference materials.

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.