Increased Sample Throughput For Ketoconazole Analysis By Automated 96-Well Sample Preparation And Multiplexed HPLC

By K. Szczap, C. Loop, D. Gray, H. Freiser, R. Shoup, and A. Witkowski

In order to increase the sample throughput of a manual LC-FL assay for ketoconazole, the method was automated and a multiplexed HPLC system was employed.

The original ketoconazole method involved manual protein precipitation, followed by HPLC-FL analysis with a run time of approximately 15 minutes. The sample preparation was converted to the 96 well format and automated using the Tomtec Quadra96. In addition, a multiplexed HPLC system was configured to cut the HPLC run times in half. The LC configuration enabled alternate sample injections to travel down one of two HPLC columns. The injections were staggered such that the eluting peaks could be diverted to a single FL detector.

The resulting assay was fully validated and readily met ±15/15/15% acceptance criteria. Based on the assay's performance statistics, the automated method was found to be robust, even though two separate analytical columns were used for the same batch. Additional data using high-flow HPLC on a monolithic rod column also looks very promising.