Automated study of ligand-receptor binding using solid-phase microextraction.
An automated ligand-receptor binding study was performed for the first time using solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry. A new multi-fibre SPME system, which relies on multi-well plate technology and allows parallel preparation of up to 96 samples was used in order to obtain all data points of the binding curve in a single experiment. The binding of diazepam to human serum albumin was used as the model system in order to evaluate the performance of automated SPME. The time required to establish equilibrium was 30 min; this was verified experimentally by constructing extraction time profiles in the presence and absence of receptor molecules. Fibre constant calibration was used to remove inter-fibre variability from the binding data. Using a simple one-site binding model, a binding constant of 9.1 x 10(5)+/-3 x 10(5)l/mol was obtained. This result is in excellent agreement with values for equilibrium dialysis and manual SPME procedures reported in the literature. The proposed method can be further extended to study plasma-protein binding or drug binding to whole blood. In comparison to other methods, SPME is simpler, faster and fully automated, can be combined with any analytical detection method, and can be used to directly study complex samples.
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.
Dajana
D
Vuckovic
Janusz
J
Pawliszyn
D002853:Chromatography, Liquid / Q000379:methods*; D003975:Diazepam / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D052617:Solid Phase Microextraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013678:Technology, Pharmaceutical / Q000379:methods; D013997:Time Factors
D008024:Ligands; D012709:Serum Albumin; D003975:Diazepam