Comparison of solid phase microextraction versus spectroscopic techniques for binding studies of carbamazepine.

The binding of carbamazepine to human serum albumin was studied in vitro using solid-phase microextraction (SPME) with liquid chromatography-ultraviolet detection (LC-UV), as well as spectroscopic fluorescence and nuclear magnetic resonance ((1)H NMR) techniques. We were able to recognize one high affinity binding site with both fluorescence and SPME methods. Additionally, SPME experiment showed the existence of one lower affinity binding site for carbamazepine at the range of concentrations studied with fluorescence. The analysis of Hill's plot indicated positive cooperativity between drugs located in these two binding sites. Two low affinity-binding sites have been found with SPME-LC-UV analysis performed in parallel to (1)H NMR study, which does not show any complex formation. In conclusion, the results of the studies with carbamazepine as a model drug showed the advantages of simultaneous use of solid phase microextraction and spectroscopic methods in protein binding studies and indicated complementary information, which can be obtained with the use of SPME. Furthermore, we show that SPME in combination with liquid chromatography-mass spectrometry permitted direct in vitro determination of plasma-protein binding and direct in vivo evaluation of inter-animal variability in free concentrations of carbamazepine at physiologically relevant concentrations, the type of experiments typically inaccessible by spectroscopic techniques due to poor sensitivity and different mode of implementation.