ABS 026: Exploring Enantiomer Separation Using Structural Mass Spectrometry Methods

Rashi M. Gupta ¹ , Benjamin K. Blakley ¹ , Jody C. May¹ , John A. McLean ¹

¹ Department of Chemistry, Center for Innovative Technology, Institute for Integrative Biosystems Research and Education, Institute for Chemical Biology, Vanderbilt-Ingram Cancer Center

Van Wickle (2025) Volume 1, ABS 026

Introduction: The Thalidomide Crisis underscores the importance of developing rapid analytical methods which differentiate small molecule drug enantiomers. Previous work by the McLean Research Group demonstrated the differentiation of drug enantiomers by complexing them within a ternary copper complex of the form [(M)(L-His)(CuII) – H]+ using ion mobility-mass spectrometry (IM-MS). Here, we first explore amino acids beyond L-Histidine, such as L-Tyrosine, to expand the scope of our previous study. Additionally, we probe specific aspects of noncovalent complexes which might be leading to IM-based separation, leveraging structural mass spectrometry methods, including high resolution ion mobility (HRIM) and energy-resolved tandem mass spectrometry (ERMS). Ultimately, we hope that these results will deepen our understanding of the chemistry driving enantiomer separations using noncovalent complexation and IM-MS.

Methods: Various samples containing pure or racemic drug compounds were prepared with equimolar concentrations of a given chiral selector (CS), specifically various copper (II)-amino acid clusters. Amino acids under investigation include histidine, tyrosine, and beyond. Samples were directly infused (10 μL/min) into an ESI source (Jet Stream, Agilent Technologies) coupled to (1) a drift tube IM-MS (DTIMS; 6560, Agilent Technologies), or (2) a high resolution traveling wave IM-MS (TWSLIM; MOBILion Systems). IM separation was quantified using two-peak resolution (RP-P). For ERMS experiments, ions were fragmented using collision induced dissociation (CID). Stability for each complex was quantified using an EC50 value, defined as the voltage at which desired complex was fragmented to an abundance of 50% of its baseline abundance at 0V.

Results: Copper-amino acid complexes successfully enabled the gas-phase separation of chiral drugs, including metoprolol, thalidomide, and propranolol. ERMS data revealed that R-metoprolol formed more stable complexes (EC50 = 4.51 eV) than S-metoprolol (EC50 = 3.42 eV), with racemic samples intermediate at 3.58 eV. These values were consistent across three replicates. In contrast, ibuprofen—a drug not separated by IM-MS—exhibited a smaller ΔEC50 of 0.51 eV, supporting the proposed relationship between complex stability and separability. These findings support our hypothesis that gas-phase stability differences within diastereomeric complexes correlate with observed chiral separation in ion mobility experiments.

Discussion: Our data suggests a mechanistic link between complex stability and enantioselective separation in gas-phase ion mobility spectrometry. This correlation may provide a structural framework to rationalize why certain enantiomers resolve better than others and offers predictive insight for selector-analyte pairings. The inclusion of a negative control (ibuprofen) strengthened our hypothesis by showing that minimal separation aligns with minimal EC50 divergence. Moving forward, this framework will be extended to a broader panel of beta-blockers and tyrosine derivatives to explore structural features that govern enantioselectivity, with implications for developing generalizable chiral separation strategies in pharmaceutical analysis.