Influence of Solvent Additive Composition on Chromatographic Separation and Sodium Adduct Formation of Peptides in HPLC–ESI MS

Abstract

Mobile phase additives have a strong influence on sensitivity and spectral quality when HPLC is coupled to mass spectrometry. They may cause ion suppression, as frequently observed with trifluoroacetic acid, or may lead to increased adduct formation, complicating data analysis and interfering with automated LC–MS–MS analysis. When reversed-phase HPLC is performed under neutral conditions, ammonium acetate is the additive of choice because of its volatility. Because of the limited solubility of ammonium acetate in commonly employed organic solvents like acetonitrile usually a small amount of water is added for dissolution, and in addition, acetic acid can be added to obtain a buffered system. We compared six different acetonitrile/ammonium acetate blends, differing in their water and acetic acid content and evaluated their performance for HPLC–MS analysis of model peptides (bradykinins) under neutral conditions. The results demonstrate that mobile phase preparation strongly influences chromatographic separation and adduct formation in electrospray ionization MS, as shown for a series of bradykinin peptides. By varying the solvent composition only slightly to influence the pH curves during gradient elution, three nearly coeluting peptides could be baseline separated. At the same time, the formation of sodiated instead of protonated peptides was found to vary by up to a factor of four, depending on the preparation of the solvent blends.