Electrospray has been applied to many applications due to its ability to multiply charge large analytes allowing them to be measured on an instrument with an m/z limit lower than the analyte's molecular weight. However, the same principle that provides the advantages of multiple charging can also be a disadvantage by leading to variable product ion spectra when different sites on the same molecule ionize. Large molecules can accommodate two or more charges leading to a single form of an analyte being detected in its multiply charged form. As molecular size decreases there is a decreased ability for a gas phase molecule to accommodate multiple charges. This leads to the possibility of two ionized forms of an analyte in gas phase arising from a single solution phase analyte. The different ionized forms are called protomers. Protomers arise based on an analyte's structure and will occur on any MS using electrospray. They provide a possible explanation for undesirable LC/MS method traits such as recoveries far in excess of 100%. Conditions such as pH and applied voltage can effect the likelihood of one or the other site on a small molecule to preferentially ionize over another. This can cause variability in an assay when a given parameter is not properly controlled. Since the different forms of the ionized analyte are exactly the same elemental composition high resolution alone cannot distinguish between the different forms. However, since the site of ionization effects gas phase conformation and interaction with the drift gas in an ion mobility cell, these species can often be individually determined through the combined use of ion mobility with high resolution MS. Q/IMS/TOF, therefore, allows direct investigation of protomers so that directed method development can be applied to improve the reproducibility as well as accuracy and precision of methods targeting these challenging analytes. The ability to obtain accurate mass measured product ion spectra for the separate protomeric forms of an analyte provides additional coverage of fragments investigated during structural elucidation of unknowns. The additional detail obtained from analyzing protomers improves specificity and more thoroughly supports or disproves proposed structures. Data will be presented demonstrating IMS on a series of protomers encountered in the analysis of fluoroquinolones. The results of investigation of parameters that effect the distribution of protomers will be presented.

Ion mobility for the investigation of electrospray variability and structural elucidation: Applied protomer analysis

249th National Meeting (2015)

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