Gradient elution of organic acids on a beta-cyclodextrin column in the polar organic mode and its application to drug discovery.

A high performance liquid chromatographic method was developed that separated organic acids using the polar organic mode. The separation was obtained using a β-cyclodextrin stationary phase with a mobile phase that was composed of acetonitrile/methanol/triethylamine (TEA)/acetic acid. The compounds were eluted under gradient conditions and the elution order depended on the number, type and position of the hydrogen bonding functional groups present in the molecule. Adjusting the acid to base ratio resulted in the biggest change in selectivity. In addition, increasing the methanol concentration decreased the retention times of the analytes, which had little effect on the selectivity. Using a certain set of conditions one could separate a large number of organic acids, which allowed these acids to be detected by UV and mass spectrometry. These conditions were used to evaluate the purity of potential pharmaceutical drug candidates that showed activity towards a kinase target vascular endothieal growth factor (Vegf). Each compound contained a carboxylic acid group that was critical to the activity. The method was able to give purity estimates of these samples, which were difficult to determine by other HPLC methods.