Comprehensive two dimensional liquid chromatography as analytical strategy for pharmaceutical analysis
Résumé
Comprehensive on-line two-dimensional liquid chromatography (LCxLC) is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceuticals. A comparative study of different sets of chromatographic conditions including stationary phase, pH additive and organic modifier was carried out with two real pharmaceutical samples in order to find out the best analytical conditions for implementation of one or several generic on-line LCxLC separations. Our choice was based on the evaluation of both degree of orthogonality and practical sample peak capacity under linear gradient conditions. The potential of 190 combinations of chromatographic systems was compared. A set of 3 RPLCxRPLC configurations was found to be very attractive for both samples and in good agreement with the findings of a previous study carried out with 17 model compounds, thereby supporting the idea of using generic LCxLC conditions in the pharmaceutical area. The three selected 2D-systems were implemented for the on-line RPLCxRPLC-UV/MS analysis of two pharmaceutical samples. It was shown, for each sample, that these 2D-systems were able to generate an effective peak capacity close to 1000 in less than 50min. For each sample, baseline separation was obtained for every known compound and furthermore a large number of unknown impurities could also be separated and identified. Finally, in the proposed conditions, the total number of compounds detected was significantly improved from one RPLC separation to one RPLCxRPLC separation. Only a small additional gain was observed by performing a second RPLCxRPLC separation or even a third one.