A generic fast solid-phase extraction high-performance liquid chromatography/mass spectrometry method for high-throughput drug discovery.

High-performance liquid chromatography/mass spectrometry (HPLC/MS) is increasingly perceived to be an essential tool in drug discovery at many key steps, like drug screening, lead identification, ADME profiling, and drug metabolism and pharmacology studies. High-throughput screenings in the early phase for metabolic stability, protein binding, permeability (ADME) and bioavailability are widely used to weed out compounds that do not exhibit the necessary characteristics. For such high-throughput LC/MS studies, a generic LC/MS method that can be used for a variety of compounds is desired. In this study, we used a small set of compounds with a wide range of properties to guide method development, and achieved a sample throughput of 1.7 min/sample. Here, we present a generic fast method that achieves good peak separation and peak shape on conventional HPLC systems using a column-switching mechanism for on-line solid-phase extraction (SPE)-HPLC/MS analysis. The method has a linear response range from 1 to 500 nM for the tested compounds. When a larger set of 658 randomly picked small molecules were analyzed using this method, 612 were observed with good signal intensity and HPLC peak shapes. This generic fast SPE-LC/MS method has been used to screen more than 1.5 million compounds repetitively against over 200 protein targets for hit confirmation and semi-quantitation of binding constants from biological assays. Over 7000 different compounds for a variety of protein-binding assays have been studied using this method for quantitative analysis as well.