High-throughput screening (HTS) technologies have become one of the most important tools in modern drug discovery to accelerate the development of novel lead compounds [1]. HTS technologies have been developed and implemented that are able to test tens of thousands of compounds or more per day for their activity in various assay types, ranging from receptor binding and enzyme inhibition to whole-cell assays. While HTS techniques are highly efficient in the screening of pure compound samples, the screening of complex mixtures is more demanding, involving a close coordination between chemical analysis, sample fractionation and biological screening.

Complex mixtures in drug discovery are samples originating from natural products, reaction mixtures from solution-phase combinatorial chemistry and in vitro or in vivo metabolic profiling. In all cases, non-active sample constituents at widely different concentration ranges are present next to an unknown number of pharmacologically active compounds. Identification requires fractionation, mostly performed by off-line liquid chromatography (LC), in combination with fraction collection. Fractionation can be performed prior to or after primary screening of, for example, natural product extracts [2, 3] and or combinatorial chemistry libraries [4, 5], but always requires a follow-up screening step. The whole process of screening and fractionation must be repeated until the bioactive compound against the molecular target is isolated. It is obvious that this process can be very laborious and time-consuming.

In recent years, analytical screening technologies were described that facilitate the determination and identification of bioactive compounds in complex mixtures. Both high-performance liquid chromatography (HPLC) [6-8] and capillary electrophoresis (CE) [9-11] were employed to separate the compound mixture during or prior to the biological screening. In contrast to the microtiter plate format dominating HTS assays, analytical screening assays are carried out in continuous-flow systems to be compatible with the separation technique em ployed. Bother receptor ligand binding and enzyme inhibition assays were compatible with the continuous-flow assay formats.

Both modern microtiter plate and continuous-flow biochemical assays are based on fluorescence detection principles as the most common readout principle. These assay types require fluorescent labels to generate a readout signal reflecting the affinity of the compound(s) tested for the biomolecular target. In more advanced systems, mass spectrometry (MS) was used in parallel to simultaneously measure MS and MS-MS spectra of biologically active compounds. Methods using LC-UV/MS [12-14] and LC-fluorescence/MS [15, 16] allowed the simultaneous detection of bioactivity and characterization of the bioactive molecules in a single analysis. While fluorescence based biochemical assays typically are characterized by a high detection sensitivity and robustness, the need to prepare a fluorescent label or substrate that retains a significant receptor or enzyme affinity often hampers the speed of assay development. Also, in complex samples, the presence of natively fluorescent compounds with excitation/emission spectra, that overlap the spectra of the fluorescent label, may complicate data interpretation.

In the present review, we focus on the use of MS for the detection of both chemical and biochemical characteristics of bioactive compounds present in complex mixtures. The biochemical assays on which these methodologies are based rely on the direct or indirect detection of binding interactions by MS.

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