Ion-pair reversed-phase liquid chromatography-electrospray mass spectrometry for the analysis of underivatized small peptides.

The single run analysis of 23 small peptides (principally glycyl and lysyl dipeptides) is performed by ion-pair reversed-phase liquid chromatography coupled with evaporative light scattering detection or electrospray (tandem) mass spectrometry. Several perfluorinated carboxylic acid homologues are evaluated with an octadecyl silica stationary phase (Supelcosil ABZ+ Plus). Among the perfluorocarboxylic acids tested the nonafluoropentanoic acid and the tridecafluoroheptanoic acid gave the best results. Special attention was paid to the separation of isomer/isobar dipeptides (e.g., Gly–Ile, Gly–Leu, Leu–Gly, as well as Gly–Gln, Gly–Lys, etc.) which is usually necessary in spite of the high specificity of mass spectrometry. Before LC–MS analysis, ion-spray fragmentation as well as optimization of MS parameters of the analysed peptides was investigated. The optimum collision energy of glysyl peptides, Ala–Gln, Asp–Asp and Asp–Asp–Asp (13–18 eV) was different from that of the lysyl peptides, Tyr–Glu and oxidised glutathione (25–32 eV). Limits of detection varied from 0.1 to 1.2 mg l−1 for simple MS and 0.05 to 25 mg l−1 for tandem MS.