Protein amino acid analysis by an isotope ratio gas chromatography mass spectrometry computer technique

A method for quantitative analysis of protein amino acids by a gas chromatography mass spectrometry computer system is described. Amino acids were analysed as their N-trifluoroacetyl n-butyl ester derivatives. Isotope ratio determination was used as the quantitating technique via multiple internal standards. The exact composition of a deuterated amino acid mixture was determined against a standard amino acid calibration mixture and in turn the protein amino acid composition was determined against the deuterated amino acid mixture. The amount of protein taken for analysis was 100 micrograms and the procedure, excluding hydrolysis, could be performed with 2 1/2 hours. The introduction of the internal standards prior to protein hydrolysis provides a method with good precision (mean coefficient of variation less than 5%). The method, tested on insulin, gave results which agreed well with the known composition of the protein and with simultaneous analysis on ion exchangers.