Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry

The analysis of the 20 underivatized protein amino acids by liquid chromatography ionspray tandem mass spectrometry is investigated in positive ion mode. First, by direct infusion, amino acid fragmentation was investigated based on the product-ion mass spectrum of the parent compound in three different collision energies (10, 20, 30 eV). Then, the relative abundance of fragment ions was studied as a function of the collision energy in order to select the product ion with the highest abundance and to obtain the maximum sensitivity for each amino acid, by using the optimum collision energy. Depending on the amino acid, the loss of H2O or NH3 or CH2O2 was selected as the product ion from the molecular ion [M+H]+ in selective reaction monitoring mode. 15 eV was chosen as a mean value of collision energy to obtain satisfactory sensitivity for the simultaneous determination of the 20 protein amino acids. In spite of the specificity of mass spectrometry, and in order to obtain maximum sensitivity, several pairs of amino acids had to be separated. The separation of these amino acids pairs was achieved in less than 20 min by using a porous graphitic carbon column and nonafluoropentanoic acid as ion-pairing reagent. Detection limits depending on the amino acid varied from 500 fmol to 40 pmol (using a 10 μl loop).