Multidimensional Separations in Complex Biological Matrices Utilizing Chromatography, Ion Mobility, and Mass Spectrometry
Davis, Don Earl
Mass spectrometry (MS) has been a rapidly growing analytical technique, especially when analyzing complex biological matrices in metabolomics, clinical applications, anti-doping applications, and routine testing analyses. With new MS platforms being developed every year, the resolution of separation by mass analyzers has increased, increasing utility tremendously. Even though more and more fundamental research is being developed for improving the efficiency of mass analyzers, several challenges remain when analyzing complex biological samples. More specifically, when analyzing isomeric species such as structural isomers (constitutional isomers) or stereoisomers. This issue holds true for metabolomics, clinical, anti-doping, and routine testing applications where laboratories are typically doing this type of work in regulated environments get audited. Their current analytical techniques can not compensate for issues as specific as isomers. Therefore, to address this issue, ion mobility techniques in conjunction with liquid chromatography (LC) and MS provide a reasonable solution in separating isomers without adding additional analysis time. Ion mobility spectrometry (IMS) is a gas-phase separation technique that distinguishes ions based on their size, shape, and charge state. The IMS size and shape measurement takes the form of an ion collision cross section (CCS), a coarse-grained area measurement (reported in square angstroms, Å2) encompassing the ion size as well as its interaction with the neutral gas. IMS separates ions based on differences in gas phase electrophoretic mobility. Gas-phase IMS analysis is rapid, typically occurs on a time scale of less than 100 ms per spectrum. In contrast, condensed phase LC-MS is on the time scale of minutes. Therefore, IMS can be included in workflows without compromising analytical throughput, providing an additional separation dimension and an associated molecular descriptor (CCS) to support analyte detection and identification and minimize false positive/negative results. IMS experiments provide a dimension of separation and LC that can be coupled to mass analysis to potentially distinguish isomeric interferences and provide further confidence in analyte identification in complex biological samples. Furthermore, IMS can identify analytes not reported in relevant samples through a combination of targeted and untargeted analysis because CCS values derived from IMS show utility in improving analyte identification accuracy.