Enhancing isomer specificity in mass spectrometry by combining silver ion adduction and ion mobility

• The combination of theoretical and experimental results show that silver ion adduction introduces conformational changes in steroids • The conformational changes that are isomer specific and enables mobility separation of steroid isomers even at low resolution • The combination of ion mobility and tandem mass spectrometry can annotate steroids with isomeric specificity Identification and characterization of steroids from complex mixtures with isomeric precision is key to studying endocrine-related metabolism and disorders. Whereas the golden standard chromatography, including liquid chromatography and gas chromatography, can be coupled with mass spectrometry to separate steroids prior to ionization, this separation is time-consuming. Contrarily, direct infusion techniques can offer increased throughput; however, these are often hampered by limited structural specificity. Thus, it is important to develop new analytical tools for direct infusion mass spectrometry that will provide isomer specificity. Herein, we show that direct infusion with electrospray ionization in combination with silver adduction and cyclic ion mobility mass spectrometry enables the mobility separation and detectability of steroid isomers. Specifically, silver ion adduction of steroids increases instrumental response up to 14 times and enables almost baseline mobility separation of closely related structural steroid isomers even at low IMS resolution. By combining experimental and theoretical data, we show that the silver interacts with the steroid isomer at single or multi-sites, which introduces conformational changes that enable mobility separation. Moreover, we show that the combination of cyclic ion mobility spectrometry and silver adduct fragmentation in tandem mass spectrometry provides an additional dimension of annotation for steroid isomers. Thus, the simple introduction of silver ions into the electrospray solvent provides a great gain in the analytical discernment of steroid isomers. For the first time, we show that the use of silver adduction introduces a conformational change in steroids that allows for them to be separated with low-resolution ion mobility spectrometry without any prior derivatization, chromatographic separation, or instrumental modification. This is a new and important tool for analyzing steroid isomer specificity that can unravel their importance in biological systems.