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

Our previous experiments on ESI-MS analysis of reaction mixture solutions containing HEPES (4-(-2- hydroxyethyl)-1-piperazineethanesulfonic acid), 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, regarding HEPES monomer ions formation, was observed. This enhanced formation of HEPES multimer ions is much higher than the one 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 with information from mass spectra. The results obtained led us to conclude that the formation of HEPES ions is related with 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 ions abundances, which were found to be consistent with those observed for analyte monomer ions abundances, were related with type of analyte, i.e. to their acid/base nature. Strikingly, the variations observed between HEPES monomer and multimer ions abundances, enable to discriminate among the different influence of analyte species on HEPES species formation. The results obtained also enabled to provide an explanation for the observation that HEPES species do not suppress significantly analyte species ion signals, when high concentrated HEPES solutions with analyte are electrosprayed. According to our results, the association behaviour between HEPES species seems to be preserved in the gas phase during electrospray ionization. This observation may provide some information that may be useful regarding the behaviours involved in the gas phase ion formation process from charged droplets during electrospray ionization or, at least, to differentiate among behaviours.