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.