First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

Method optimization for drug impurity profiling in supercritical fluid chromatography: Application to a pharmaceutical mixture.

A supercritical chromatographic method for the separation of a drug and its impurities has been developed and optimized applying an experimental design approach and chromatogram simulations. Stationary phase screening was followed by optimization of the modifier and injection solvent composition. A design-of-experiment (DoE) approach was then used to optimize column temperature, back-pressure and the gradient slope simultaneously. Regression models for the retention times and peak widths of all mixture components were built. The factor levels for different grid points were then used to predict the retention times and peak widths of the mixture components using the regression models and the best separation for the worst separated peak pair in the experimental domain was identified. A plot of the minimal resolutions was used to help identifying the factor levels leading to the highest resolution between consecutive peaks. The effects of the DoE factors were visualized in a way that is familiar to the analytical chemist, i.e. by simulating the resulting chromatogram. The mixture of an active ingredient and seven impurities was separated in less than eight minutes. The approach discussed in this paper demonstrates how SFC methods can be developed and optimized efficiently using simple concepts and tools.

Investigation of the effect of column temperature and back-pressure in achiral supercritical fluid chromatography within the context of drug impurity profiling.

Twenty commercially available stationary phases were characterized in supercritical fluid chromatography (SFC) using a diverse set of pharmaceutical compounds. Six dissimilar phases were selected, and a benzodiazepine and a trimethoprim impurity mixture were screened on these phases. Two stationary phases were then selected for each mixture to study the effect of temperature and back-pressure on retention, separation and chromatographic efficiency using a response surface design approach. The maximal feasible domain for each phase was examined and column performance was monitored for stability during the duration of the study. Chromatographic responses of the individual mixture components, such as retention time, peak width and apparent plate count, were modelled as a function of temperature and back-pressure. The use of high temperatures led to improved separations and higher efficiencies while high back-pressures resulted in reduced retention. For the two mixtures, the response surface plots of the resolution of the worst-separated peak pair over the experimental domain allowed the identification of the temperature and back-pressure leading to the maximal resolution for the worst-separated peak pair. For the mixtures investigated, the use of high temperatures led to improved separations and high efficiencies, while high back-pressures resulted in reduced retention. These factors are fine-tuning parameters in SFC, and similarly to the modifier composition, they lead to local, rather than global selectivity differences.

Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle based approach in achiral SFC.

Defining a method development methodology for achiral drug impurity profiling in SFC requires a number of steps. Initially, diverse stationary phases are characterized and a small number of orthogonal or dissimilar phases are selected for further method development. In this paper, we focus on a next step which is the investigation of the modifier composition on chromatographic selectivity. A solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH), propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in terms of elution order, retention and mixture resolution. However, varying only the modifier composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore the modifier composition is an optimization parameter, with the stationary phase being the factor determining most the selectivity of a given mixture in achiral SFC.