Poterat and Hamburge respectively,and attracted much attention erectile mant MPinto the evant for rmant-GMP.Active detected by nown PDE inhibitors hase com theophyll ine (23 22 isdetecledve absorbance peak7 int oceiNeancoherpoeeTee some ations de for on-line sys ms 50 he -up The considerabl a major cost factor when oper ation line co iniectio the MS-Based Detection Systems CAPILLARY ELECTROPHORESIS-BASED ASSAYS Mass spectrometry is an attractive to Capillary electrophoresis is a well-established technique for high resolutio eparatio r does s the binding of sm As a ther as shift in electrophe ctic to ch often cau ng of has beer mixtures suc streptavidin/biotin and anti An'AC Ma the b Cetek Corpor the the -MS (M wa alidated a for 621 .It is based on the ase of the MS th of a o of a und The in resp n The ction o iswell suited for th can be re g natural product crude extracts,combinatorial libraries and 908 Current Organic Chemistry, 2006, Vol. 10, No. 8 Potterat and Hamburger phosphodiesterase (PDE) inhibitors [60]. This enzyme family hydrolyses cGMP and cAMP to GMP and AMP, respectively, and has attracted much attention recently since selective inhibitors have a therapeutic potential in diseases such as hypertension, asthma, heart failure and erectile dysfunction. The test system was based on the conversion of the fluorescent reporter substrate mant-cGMP into the less fluorescent mant-GMP. Active compounds were detected by means of an increase in fluorescence. The assay was validated by spiking extracts with the known PDE inhibitors papaverine (22) and theophylline (23). In spite of their intrinsic selectivity, fluorescence-based assays suffer from background interferences which may decrease their sensitivity and lead to the assignment of false positives. Therefore, attempts have been made to switch to detection methods with increased selectivity, such as fluorescence resonance energy transfer detection (FRET). The suitability of FRET for on-flow systems has been demonstrated using the protease subtilisin as a model enzyme [61]. A further selectivity increase could be achieved with time-resolved FRET, which entirely suppresses the interferences from short lived fluorescence. A flow injection kinase assay using TR-FRET has been recently reported. The assay measures the phosphorylation of poly(GT)-biotin by the receptor tyrosine kinase EGFR [62]. While FRET fluorescence has been applied so far only to flow injection assays, work aiming at coupling this method to liquid chromatography is ongoing. MS-Based Detection Systems Mass spectrometry is an attractive alternative to fluorescence detection in on-flow assays. The principle consists of continuously monitoring a MS reporter molecule at its specific m/z. A major advantage over a fluorescence based measurement is that it does not require the synthesis and purification of labeled ligands [56]. As a further asset, this format is insensitive to natively fluorescent compounds which often cause problems in the screening of natural product extracts. The feasibility of MS based detection was demonstrated using streptavidin/biotin and anti￾digoxigenin/digoxin competitition model assays. Subsequently, a substrate conversion based assay involving the detection of the reaction products by ESI-MS was described [63]. The assay was validated with the cysteine protease cathepsin B. It is based on the decrease of the MS signal corresponding to the reaction product in response to the presence of an inhibitor in the effluent. The reduction of the product signal can be readily detected using the single ion trace locked on the product mass. Operating the MS in the scan mode provides structural information about the compounds responsible for the inhibition. Besides the research work carried out at the University of Amsterdam, there have been a few attempts to establish on￾line detection systems for biologically relevant properties. Of particular interest is an on-line HPLC procedure for the detection of radical scavenging compounds in plant extracts. The method measures the reduction of the DPPH radical and can be run with mobile phase compositions ranging from 10 to 90% organic solvent in water or buffer. Reduction of the radical is detected as a negative absorbance peak at 517 nm [64]. Full integration of the bioassay into the HPLC flow, providing both chemical and biological information in a single run, can be viewed as the ultimate achievement in interfacing bioactivity and chromatography. However, there are some limitations for on-line systems: Separation and bioassay conditions must be compatible, or rendered to be so, with the aid of make-up buffers. The considerable consumption of immunochemicals, receptors or enzymes may represent a major cost factor when operating such on￾line assays. The resolution is typically lower than in an off￾line configuration, since band broadening is induced by the post-column reaction. A further drawback in comparison to the off-line HPLC-based activity profiling is that on-line assays require substantial development and optimization for each target to be screened. CAPILLARY ELECTROPHORESIS-BASED ASSAYS Capillary electrophoresis is a well-established technique for high resolution separation of compounds based on their charge-to-mass ratio and shape. Affinity capillary electrophoresis (ACE) detects the binding of small molecules to a labeled macromolecule (enzyme, receptor, nucleic acids) through a shift in electrophoretic mobility due to changes in surface charges or target conformation. The ACE technology has been recently optimized for the detection of bioactive compounds in complex mixtures such as natural product extracts [65]. An ACE screening platform has been commercialized by the biotech company Cetek Corporation (Marlborough, MA), which claims to have established validated assays for more than 150 pharmaceutical targets (CE Assay™). The basic principle is the detection of a shift in the capillary electrophoretic mobility of a fluorescently￾labeled target in response to the binding of an active compound. The technology is well suited for the screening of natural product crude extracts, combinatorial libraries and N O O O O papaverine (22) O O OH HO OH OH kaempferol (21) N N N H N O O theophylline (23)