Evaluation and comparison of various separation techniques for the analysis of closely-related compounds of pharmaceutical interest

The aim of the present work was to compare various separation techniques for the fast analysis of closely-related compounds, including structurally-related compounds, positional isomers, diastereoisomers, Z/E isomers. Three analytical techniques were evaluated, namely ultra high performance liquid chromatography (UHPLC), ultra high performance supercritical fluid chromatography (UHPSFC), both with sub-2 μm particles, and capillary electrophoresis (CE) using non-aqueous solvents. To fairly compare the three analytical techniques, only two starting conditions for further method development were considered. All the selected mobile phases or background electrolyte were MS-compatible. As expected, CE often provided excellent results for the analysis of basic compounds but it was difficult to find out conditions that could be widely applied. On the other hand, UHPLC and UHPSFC were more generic and the performance was better than CE for the analysis of neutral and acidic compounds. In all cases, the analysis time was systematically lower than 3 min. In conclusion, UHPLC was the most versatile strategy for the analysis of closely-related compounds and should be tested in a first instance. UHPSFC and CE approaches offered some drastic changes in selectivity and should be considered a second choice to reach alternative selectivity as they also allow high throughput separations.