Automated online liquid chromatographic/mass spectrometric metabolic study for prodrug stability

In vitro metabolic stability studies are performed routinely in drug discovery to determine the rate of metabolism as well as the metabolic fate of compounds. These studies are labor intensive, involving incubation of the compound with a biological matrix, sampling at various time points, stopping the reaction, and sample preparation for analysis. All of these steps involve manual pipetting in the conventional method. An automated method for in vitro metabolism studies is reported here. The method reduces the time and manual labor required and has other advantages, such as better reproducibility and unattended operation. This method utilizes an autosampler custom configured with cooling and incubation capabilities. The autosampler is programmed to directly inject incubation samples at set time points onto an online extraction column. The extracted sample then enters an analytical column for separation and ultimately the mass spectrometer for detection. The injection has the dual function of stopping the reaction and starting the analysis on the LC–MS. This method was used for the metabolic stability study of a prodrug in plasma and liver S9 fractions of five different species. The stability data from the automated method were similar to those obtained using the conventional method. The potential for this method to increase throughput of metabolic stability studies in drug discovery is demonstrated.