The effect of dispersing a solvent into charged droplets, when applying a high electric potential to an effluent capillary, was first described by Zeleny in 1917 (1). It was investigated in more detail in 1955 by Drozin (2) and used in pioneering work for an electrospray interface by Dole et al (3,4). This was continued in 1984 by Yamashita and Fenn (5,6), which finally resulted in a description of an LC–MS interface in 1985 (7). Today electrospray ionization (ESI) is the most widely used ionization technique in LC–MS. This is especially true in protein mass spectrometry, where series of multiply charged ions are observed and used to determine the molecular mass (8,9). Furthermore, after tryptic digestion, the resulting peptides can be measured with LC–ESI-MS, to obtain amino acid sequence information after a second MS/MS-step (10–14).

When characterizing peptides using LC–ESI-MS, often multiply charged ions are observed (usually doubly charged [M+2H]²⁺ ions in the instance of tryptic peptides). When the charge state is identified (15), the (monoisotopic) molecular mass can be calculated and the ion can be isolated to permit MS/MS for amino acid sequence information. With the growing demand for such data in bioscience (proteomics), the ability to do this in automated mode has been discussed (16–18). For this purpose, it is necessary to identify without doubt the correct doubly or multiply charged ions as precursors for MS/MS; in the ideal instance all relevant ions should occur at high abundance. However, this is not always the situation, because sometimes protonation is not the only ionization process.

Since the early days of electrospray, theories of ion formation have been discussed. The hypothesis of Dole (3), based upon the assumption that the observed ions are produced by simply desolvating the charged droplets, is called the "charge residue model". Roellgen et al (19). postulated a "soft desolvation" of ions by solvent evaporation from small charged droplets by either electrohydrodynamic or mechanical instabilities (or both). These theories led to the present model of a "coulomb explosion", where the charge stays with the analyte when a charged droplet becomes smaller and eventually "explodes", losing all neutral solvent molecules (20). To produce charged droplets in a first step it is necessary to have ions present in the effluent, which undergoes the electrospray process. To drive the ionization in the direction of the protonated molecules, it is common to add formic or acetic acid to the solvent, usually in concentrations of 0.1 to 0.5%. The protons are transferred to the analyte during the process of ionization in the electrospray interface. If the solvent contains other cations, these may compete with the protons to form adducts with the analyte. Wang and Cole (21) investigated the influence of ionic strength on the positive-ion electrospray mass spectra of myoglobin and reported a 10-fold decrease in signal intensity and almost no change of charge-state distribution, when increasing the salt concentration. This effect can be used in a positive way for some applications (22,23) and is especially helpful in MS of glycosides. However, it is normally undesirable in protein and peptide analysis because here the desired result is the formation of the doubly charged molecule ion at high abundance, which has to be identified and isolated for subsequent MS–MS analysis.

The unwanted formation of metal ion adducts is often caused by salt impurities from sample preparation or from the solvents (eluents). The aim of this work was to study the influence of the ubiquitous sodium and potassium ions, present in trace amounts in the solvent, on the electrospray mass spectra of peptides, using human gastrin as a model peptide (24,25). Water and methanol were chosen because of their protic behavior and their ability to leach Na⁺ and K⁺ from surfaces (i.e., from glass bottles used for solvent storage.)