The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.
Chromatography, Liquid Ligands