Approaches to Singleton Achiral Purification of Difficult Samples for Discovery Research Support in the Pharmaceutical Industry

Discovery purification support is a challenging task. Purification scientists often are confronted with large sample loads and short turnaround time. The ability to develop analytical separation methods rapidly and the subsequent ability to scale up separations to preparative columns are critical for meeting sample turnaround timelines. Difficult samples require more time for analytical method development and more effort in handling these compounds on preparative scale. Difficult samples are defined as follows: minimal retention on the regular reversed-phase columns due to high polarity, minimal separation on the reversed-phase columns due to structurally similar impurities such as diastereomers and regioisomers, requirement for large sample amount for numerous injections, and degradation in aqueous solution.

The second approach involved using hydrophilic endcapping columns to retain highly polar compounds. These columns retain polar compounds more efficiently due to a unique hydrophilic endcapping. The combination of a moderately covered hydrophobic group and an additional polar group gives selectivity when compared to a traditional reversed-phase column — yet it allows for more retention of polar compounds. These columns can operate in a highly aqueous solution such as 100% water. Under this condition, some polar compounds can be retained on the column for two or three voids. Under some situations, this retention time should be sufficient for isolating desired components from its crude form.

The third approach pertained to supercritical fluid chromatography (SFC) for achiral purifications. SFC has been proven as a very efficient tool for separation of various compounds (7,8). It is used widely in the pharmaceutical industry to support discovery research. The low viscosity and high diffusivity of supercritical fluid allows the process to work at a higher linear velocity when pass through the column. SFC separations are normally fast and use less organic solvent compared with HPLC. Therefore, SFC is suitable for large-scale separations from grams to kilograms. Gram-scale samples can be time consuming to purify using reversed-phase HPLC–mass spectrometry (MS) columns. SFC not only speeds up the purification process, but also avoids sample degradation in aqueous solution. This is because SFC operates in normal-phase mode. A variety of columns can be used for SFC achiral purifications, including reversed-phase C-18 columns, normal-phase silica gel columns, and pyridine and bonded-phase columns (cyano, diol, and amino). SFC also is used very commonly for chiral separation. Chiral stationary phases also can be used to isolate the structurally similar impurities such as diastereomers and regioisomers.

Difficult samples obtained from Pfizer discovery research presented a challenging task. However, the use of HILIC, hydrophilic endcapping reversed-phase columns, and normal-phase SFC technologies offset these challenges. The following article is a discussion of the analytical method developments, the preparative experimental conditions, and the final purification results obtained from each of the three experiments. The purity and yield of each experiment also are discussed.