Isolation of trace impurities and degradants from mixtures containing primarily an API, or an API plus excipients, is often necessary for structure elucidation purposes. Using chromatographic methods for this purpose can be a slow and painstaking work. The authors demonstrate that using supercritical fluid chromatography (SFC) offers distinct advantages in speed and in clean isolation of the desired peaks. These advantages are derived from the rapidity of method development, the efficiency of the preparative fractionation, and the ease with which commonly used reversed-phase high-performance liquid chromatography data may be correlated with SFC. Case studies are used to illustrate these efficiencies.
Chromatographic isolation of degradants and impurities, whether from stressed lots of pharmaceutical compounds or directly from the API, is often required when their structures are unknown and/or reference standards cannot be prepared by synthesis (1). Isolation of these (often trace) components can be a painstaking process for analytical laboratories that involve, even in the simplest scenarios, many repeated injections of material on a chromatographic column to accumulate the trace component chromatographic peak. Often the peak of interest is profiled using a stability-indicating method designed for optimum resolution of all possible impurities and degradants (2, 3). Adapting this method to preparative scale consumes huge quantities of solvent, has a long cycle time that slows accumulation of the trace component, and requires that large amounts of accumulated solvent be removed to recover a few milligrams of the peak in quantity sufficient for structural analyses using nuclear magnetic resonance (NMR) and multiple and sequential mass spectrometry (MSn ).
Using supercritical fluid chromatography (SFC) (4) in place of traditional reversed-phase and normal-phase high-performance liquid chromatography can greatly reduce the timelines for this process. Both the rapid method development cycle in SFC and the high efficiency of preparative SFC separations contribute to the reduced timelines (5). Solvent consumption is also reduced because SFC uses a mixed phase of solvent and recycled carbon dioxide (CO2). The accumulated fractions from SFC chromatography are highly concentrated compared with those collected with conventional liquid chromatography. In addition, the lability of compounds collected during the isolation process may be minimized in common SFC solvent systems and by the mild and fast evaporation conditions used for such highly concentrated fractions.SFC methods, however, are not commonly developed as stability-indicating methods. SFC is a normal-phase chromatography, and most stability-indicating methods use reversed-phase high-performance liquid chromatography (RP-HPLC). SFC methods must be developed a priori for isolation work. Using SFC as an isolation technique requires some investment in equipment, often in columns; however, the benefits to the pharmaceutical development process can be significant for a laboratory accustomed to RP-HPLC approaches.