Separation and identification of positively charged and neutral nucleoside adducts by capillary electrochromatography-microelectrospray mass spectrometry

Capillary electrochromatography (CEC) is shown to be capable of separating mixtures containing both positively charged and neutral styrene oxide–adenosine adducts. In a study of the mechanism of deamination of positively charged 1-(2-hydroxy-1-phenylethyl) adenosine using ¹⁸O-labeled water, possible contamination of the chromatographically purified deamination product, 1-(2-hydroxy-1-phenylethyl) inosine, with the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine was observed. Because the deamination product and the presumed contamination have the same molecular weights and similar structures, CEC-microelectrospray mass spectrometry (CEC-μESI/MS) was used to confirm the presence and identity of the suspected impurity. A trace amount of the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine, which could not be observed by either HPLC-UV or CEC-UV, was detected by CEC-μESI/MS. This discriminatory ability of CEC-μESI/MS is attributed to the fact that positive ion mode ESI-MS is a more sensitive detector for a positively charged compound than a UV detector, and that the combination of electroosmotic and electrophoretic flows and hydrophobic interactions with the stationary phase contributes to the separation of the positively charged compound. As a result, the positively charged compound was observed to elute much earlier and with much sharper peaks than the neutral compounds for which electroosmotic flow is the only “pumping” force for the solvent.