Rapid Chiral Analysis of Drugs with Multiple Stereocenters via High-Resolution Ion Mobility

The increasing structural complexity of small-molecule therapeutics requires sensitive analytical techniques that facilitate rapid chiral analysis of drugs with multiple chiral centers. Here, we demonstrate ultrafast chiral separations for compounds possessing 3 chiral centers (8 stereoisomers, 4 enantiomer pairs) using gas-phase ion mobility spectrometry-mass spectrometry (IM-MS). Separations are achieved using spontaneous noncovalent copper-tyrosine complexation.This complexation strategy converts all stereoisomers into diastereomers that are structurally distinguishable by IM-MS. Notably, this strategy is over 3 orders of magnitude faster than traditional liquid-phase chiral chromatography (ms vs min, respectively). Using IM-MS, we achieved direct baseline separation of enantiomer pairs and progress toward differentiation of all 8 stereoisomers. Results suggest that higher-order complexes (i.e., copper-tyrosine-bound drug dimers) enhance chiral differentiation at the expense of increased spectral complexity. Exchanging the chirality of the amino acid for complexation resulted in an inversion of separation elution ordering. Peak fitting analysis suggests that an IM resolving power of several thousand is necessary to resolve all stereoisomers for quantitative purposes, which is approximately an order of magnitude higher than the current state-of-the-art. Collectively, these findings demonstrate the speed and versatility of gas-phase ion mobility spectrometry for complex chiral analysis.