ANALYSIS OF DRUG PLASMA-PROTEIN INTERACTIONS BY MEANS OF ASYMMETRICALFLOW-FIELD FLOW FRACTIONATION

Purpose, The applicability of Asymmetrical Flow Field-Flow Fractionati on (Asymmetrical Flow FFF) as an alternative tool to examine the distr ibution of a lipophilic drug (N-Benzoyl-staurosporine) within human pl asma protein fractions was investigated with respect to high separatio n speed and loss of material on surfaces due to adsorption. Methods. F ield-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicoc hemical properties. This method was used to separate human plasma in i ts protein fractions. The drug distribution in the fractions was inves tigated by monitoring the fractionated eluate for drug content by fluo rescence spectroscopy. Results. Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha(2)-macroglo bulin and low density lipoprotein (LDL) fractions in less than ten min utes. Calibration of the system and identification of the individual f ractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composi tion on the absolute recovery of N-Benzoyl-staurosporine and fluoresce in-isothiocyanate-albumin (FITC-albumin) was found to be quite signifi cant. Both factors were-optimized during the course of the investigati ons. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. Conclusions. If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorptio n at the separation membrane, Asymmetrical Flow FFF shows high recover ies and fast separation of human plasma proteins, and can be a reliabl e tool to characterize drug / plasma protein interactions. For analyti cal purposes it has the potential to rival established technologies li ke ultracentrifugation in terms of ease-of-use, precision, and separat ion time.