Development of a negligible depletion-solid phase microextraction method to determine the free concentration of chlorpromazine in aqueous samples containing albumin

► The free concentration of compounds is influenced by the addition of proteins. ► A nd-SPME – HPLC–UV method was developed in this study. ► The developed method was used to measure the free concentration of chlorpromazine. ► The method was optimized for coating thickness and exposure time. ► Our findings can be used to correct in vitro data for protein binding. The addition of proteins to in vitro systems influences the free concentration of the test compound in the medium. The objective of this study was to set up a negligible depletion-solid phase microextraction method, coupled to high-performance liquid chromatography (nd-SPME – HPLC) to measure the free concentration of chlorpromazine (CPZ) in medium containing albumin. The nd-SPME method was optimized for coating thickness (polyacrylate coating) and exposure time, and potential effects from the addition of bovine serum albumin (BSA) were studied. It was shown that the addition of albumin did not cause fouling or influenced the uptake kinetics of CPZ into the fiber. At a realistic in vivo albumin concentration of 40 g/L of albumin, 94% of CPZ was protein bound. This is in line with findings in vivo, reporting a protein binding for CPZ of 92–99%. The nd-SPME – HPLC method described in this study can be used to measure the free concentration of chlorpromazine in medium containing proteins. These findings can be used to correct in vitro data for protein binding of chlorpromazine and this information is essential for the extrapolation to in vivo data.