Sensitive determination of G-protein-coupled receptor binding ligands by solid phase extraction-electrospray...

High affinity Histamine H2-receptor binding ligands were assayed by automated solid phase extraction (SPE) coupled via electrospray ionization with a Quadrupole-Time-of-Flight mass spectrometer (Q-ToF-MS). The mass spectrometric behavior of these analytes was tested in aqueous solutions with several (nine) volatile salts, in different pH, and with various methanol contents. Out of the high amount of available ligands, three fluorescent-labeled molecules (5706, 5707, and 5708) were studied in detail. The limits of detection (LODs) for all three compounds obtained in mass spectrometric detection was 1 fmol (absolute) in continuous flow and FIA (flow injection analysis) measurements. The results obtained with FIA-fluorescence detection gave LODs a factor 10-100 times higher. A systematic investigation of sample solving conditions, loading flow conditions, and elution flow conditions made the automated SPE-MS coupling efficient. Ideally, the ligands were dissolved in MeOH-25 mM phosphate buffer (30:70 v/v; pH 11), the SPE loading flow comprised MeOH-25 mM phosphate buffer (30:70 v/v; pH 11) and the SPE elution flow contained MeOH-100 mM ammonium formate solution (90:10 v/v; pH 3). Using this method on a C18-modified silica cartridge (C18, 5 microm, 100 A, 300 microm i.d. x 5 mm, LC Packings) assures high recovery and achieved LODs for all three compounds of 5 fmol (absolute). As an absolute amount of ligands specifically bound on H2-receptors in biochemical experiments is, as will be published elsewhere, between 10 and 100 fmol, the SPE-MS method for the basic compounds can be directly applied for these Histamine H2-receptors.