Aim: The purpose of the study was to find methods suitable for measuring the free concentrations of testosterone and phenytoin. Materials & methods: Sample solutions of the compounds in buffer and human albumin were processed using liquid-liquid extraction, microextraction and ultrafiltration and analyzed by LC-MS/MS. Results: Liquid-liquid extraction with dibutyl phthalate provided complete extraction from buffer solutions and partial extraction from albumin samples. Spintip C18 devices provided exhaustive extraction from buffer and albumin samples. Spintip C8 devices offered complete extraction from buffer and approximately 50% recovery from albumin samples. Centrifree ultrafiltration devices showed high recovery of free concentrations from all the samples, while Amicon and Nanosep devices provided partial recovery. Conclusion: Spintip C8 and Centrifree devices proved useful for measuring free concentrations.
Keywords: albumin; free concentration; liquid–liquid extraction; phenytoin; plasma protein binding; solid-phase microextraction; testosterone; ultrafiltration.