Two-dimensional isotopic shifts for steroid isomer delineation with high-resolution cyclic ion mobility separations

Recently, the use of mass distribution-based isotopic shifts in high-resolution ion mobility spectrometry-mass spectrometry-based separations have enabled isomer delineation by measuring the relative arrival times of their heavy and light isotopologues. However, all previous efforts to induce such shifts have focused solely on the introduction of one type of isotopic substitution for a given molecule or isomer set. Herein, for the first time, we present a two-dimensional isotopic labeling strategy where two unique derivatizations are performed on various steroid isomer molecules to induce two distinct isotopic shifts and thus simultaneously measure them in a single ion mobility separations experiment. Derivatization strategies were chosen to target two specific functional groups in these steroids (i.e., hydroxyl and carbonyl), and heavy-labeled versions of the derivatizing reagents were used to induce isotopic shifts at each of these positions. We found that isotopic shifts were orthogonal to one another, diagnostic for certain steroid isomers, and that the simultaneous analysis of two different isotopic shifts was necessary for complete characterization of each steroid isomer set. We envision this multidimensional isotopic shift strategy as a new method for delineating amongst isomeric molecules, especially those with several different functional groups causing their isomerism.