B-197 Separation of Bile Acid Muricholic Acid Enantiomers by C18 Reversed-phase HPLC as a Function of Component Polarity Values of the Mobile Phase
Authors: Khair M , Anderson D J 
Published: September 2023

Abstract

Objective
Optimize the reversed phase HPLC technique for the separation of alpha-muricholic acid and beta-muricholic bile acid enantiomers, by investigating the effects of the mobile phase’s three polarity components [hydrogen bond donor acidity (α), hydrogen bond donor basicity (β) and dipolarity/polarizability (π)] on the separation of the enantiomers.

Background
The importance of drug enantiomers in clinical pharmacology is well recognized, where often only one of the enantiomer pair effectively binds to the target protein site to exert its therapeutic effect. Same for some endogenous compounds, only one of the enantiomer pair molecules binds to an enzyme or receptor to exert its physiological action, while the other molecule is inactive. It is essential for the analytical method to separately determine the two compounds of the pair. Usual HPLC techniques for separating enantiomers employ specialized chiral columns. In this work, steroid isomers are separated on a C18 column, with the separation controlled by mobile phase (MP) component polarity adjusted with different proportions of binary organic modifiers added to the MP.

Methods
The bile acid enantiomers alpha-muricholic acid and beta-muricholic acid were separated on a C18 column (150 mm × 2.1 mm, 3.5 μm, 40 °C) using two different organic modifiers in the MP at a flow rate of 0.2 mL/min, with ESI-LC/MS detection. The isocratic MP consisted of 0.1% formic acid in 100% water for MP A and 0.1% formic acid and five or more different proportions (separate runs) of the two organic modifiers for MP B to adjust MP polarity. MP component polarities was determined from: 1) the literature values for the total polarity (P) for each of the pure solvents in the MP; 2) published values of the fraction of the total polarity for the polarity components (α + β + π = 1) for each solvent; and 3) the fraction proportion of each solvent in the MP. The experimentally determined selectivity factor is then plotted vs the polarity component value of the MP.

Results
When the selectivity factor vs the values of the MP polarity components was plotted for six different organic modifier pair combinations on the same plot, the summed dipolarity/polarizability (and basicity polarity (β) components of the MP showed reasonable correlation (R2 = 0.72), displaying mostly a linear superimposed trend for all the organic modifier pairs, while the acidity component (α, R2 = 0.13) and total polarity (P) (R2 = 0.002) of the MP did not. Thus, the π + β summed MP polarities is a significant determiner of selectivity, irrespective of the organic modifier pair used. The bile acid enantiomers were baseline separated, with a selectivity factor of 1.3 being the highest value obtained.

Conclusion
For the laboratory determination of steroid enantiomers, our results suggest that calculated component polarity values of the MP can be used to direct laboratorians for achieving desired separation of steroid enantiomers (as opposed to a trial-and-error approach for achieving the separation). This study shows that the separation of these bile acid enantiomers significantly depends on the summed π and β polarities of the MP, with poor correlation for total polarity (P) and α.