Pharmaceutical impurity analysis by comprehensive two-dimensional temperature responsive × reversed phase liquid chromatography.

In this study, the possibilities of temperature responsive × reversed phase liquid chromatography (TRLC × RPLC) are assessed in terms of pharmaceutical impurity analysis. Due to the increased peak capacity per unit time they offer, two-dimensional LC approaches are gaining relevance for the analysis of complex drug formulations. Because the latter depicts a larger predisposition for the occurrence of an increased number of impurities, current 1D-HPLC approaches often prove insufficient. Since many LC × LC methods are limited by modulation, solvent compatibility, orthogonality, and sensitivity issues, the combination of TRLC × RPLC is explored in this work for pharmaceutical impurity analysis. As this combination of a purely aqueous separation with RPLC allows for systematic and optimization-free refocusing in the second dimension, it opens possibilities for generic LC × LC requiring minimal to no method development, in this way overcoming a major perceived contemporary hurdle of LC × LC. The approach is demonstrated with a representative mixture of 17 solutes comprising 11 corticosteroids and 6 progestogens. Orthogonality and peak capacities were assessed on three RP core-shell column selectivities (Poroshell EC-C18, phenyl-hexyl and PFP). Although the TRLC × EC-C18 combination offered somewhat better orthogonality, the combination with the PFP column proved the best for the separation at hand. Depending on the composition of the mixture, the use of full, shifted, or segmented gradients allowed facile optimization of the separation. The developed platform allowed detection of the impurities at the 0.05% level compared to a selected main compound, while also opening up possibilities for analysis of formulations comprising two active ingredients.