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.