Identifying and Quantifying Relative Concentrations of Epimers in Mixtures via Cyclic Ion Mobility Mass Spectrometry: Dexamethasone and Betamethasone as a Case Study

Epimers can show different biological activities and different pharmacological behaviors; therefore, their separation and analysis are crucial in the drug development process. Due to their similar chemical and physical properties, separation of epimers is challenging. This study demonstrates the application of cyclic ion mobility–mass spectrometry to separate, identify, and quantify dexamethasone and betamethasone in a binary mixture. Cyclic IMS separation of the isolated protonated dimer resulted in three peaks: dexamethasone homodimer, betamethasone homodimer, and their heterodimer. Besides providing improved separation over the protonated monomer, the presence of a heterodimer peak provides additional confirmation of an isomeric mixture. We identified the dexamethasone and betamethasone homodimer peaks by infusing pure solutions of each epimer and measuring each pure homodimer's arrival time. The measured peak areas indicated that the heterodimer is formed at twice the rate of each homodimer and that dexamethasone and betamethasone contribute equally to the heterodimer signal. Using this observation, we could accurately calculate the relative concentrations of each epimer by adding half of the heterodimer peak area to each homodimer peak area. These findings enable the identification and quantification of dexamethasone and betamethasone based on the arrival time distributions of their protonated dimers. This is the first demonstration of accurate relative quantification of epimers by separating charged dimers in the gas phase.