Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.

The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (R) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. R values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental R values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.