Behaviour of 4-(−2-Hydroxyethyl)-1-Piperazineethanesulphonic Acid under Electrospray Ionisation Mass Spectrometry Conditions

Abstract

Our previous experiments on electrospray ionisation mass spectrometry (ESI-MS) analysis of reaction mixture solutions containing 4-(−2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), a commonly used buffer, indicated that HEPES species did not significantly suppress analyte species, even in reaction mixture solutions with significant amounts of HEPES. With the purpose of investigating the behaviour of HEPES under ESI-MS conditions, HEPES aqueous solutions and HEPES aqueous solutions containing analyte with high- and low polarity and with different acid/base chemistry, were therefore investigated. For electrosprayed aqueous solutions of HEPES with concentrations above 10⁻⁵ M, an enhanced formation of HEPES multimer ions, showing HEPES monomer ion formation, was observed. This enhanced formation of HEPES multimer ions is much higher than those observed for other polar compounds, such as acetyl–arginine, acetyl–lysine and histidine. Information from solution behaviour such as HEPES concentration, solution pH and instrumental factors, namely the capillary temperature, was related to information from mass spectra. The results obtained led us to conclude that the formation of HEPES ions is related to the initial solution composition. The influence of analyte species on HEPES species formation, for electrosprayed HEPES solutions with analyte, was also investigated. The variations observed for HEPES monomer and multimer ion abundances, which were found to be consistent with those observed for analyte monomer ion abundances, were related to the type of analyte, i.e. to their acid/base nature. Strikingly, the variations observed between HEPES monomer and multimer ion abundances enable the discrimination of different influences of analyte species on HEPES species formation. The results obtained also provided an explanation for the observation that HEPES species do not significantly suppress analyte species ion signals when highly-concentrated HEPES solutions with analyte are electrosprayed. According to our results, the associated behaviour between HEPES species seems to be preserved in the gas phase during electrospray ionisation. This observation may provide some information that may be useful regarding the behaviour involved in the gas-phase ion formation process from charged droplets during electrospray ionization or, at least, to differentiate between behaviours.