Method Optimisation in Hydrophilic-Interaction Liquid Chromatography by Design of Experiments Combined with Quantitative Structure-Retention Relationships

Accurate prediction of the separation conditions for a set of target analytes with no retention data available is fundamental for routine analytical assays but remains a very challenging task. In this paper, a quality by design (QbD) optimisation workflow capable of discovering the optimal chromatographic conditions for separation of new compounds in hydrophilic-interaction liquid chromatography (HILIC) is introduced. This workflow features the application of quantitative structure - retention relationship (QSRR) methodology in conjunction with design of experiments (DoE) principles and was used to carry out a two-level full factorial DoE optimisation for a mixture of pharmaceutical analytes on zwitterionic, amide, amine, and bare silica HILIC stationary phases, with mobile phases containing varying acetonitrile content, mobile phase pH, and salt concentration. A dual-filtering approach that considers both retention time (t(R)) and structural similarity was used to identify the optimal set of analytes to train the QSRR in order to maximise prediction accuracy. Highly predictive retention models (average R-2 of 0.98) were obtained and statistical analysis of the prediction performance of the QSRR models demonstrated their ability to predict the retention times of new compounds based solely on their molecular structures, with root-mean-square errors of prediction in the range 7.6-11.0 %. Further, the obtained retention data for pharmaceutical test compounds were used to compute their separation selectivity, which was used as input into a DoE optimiser in order to select the optimal separation conditions. Experimental separations performed under the chosen optimal working conditions showed good agreement with the theoretical predictions. To the best of our knowledge, this is the first study of a QbD optimisation workflow assisted with dual-filtering-based retention modelling to facilitate the method development process in HILIC.