Extraction of physostigmine from biologic fluids and analysis by liquid chromatography with electrochemical detection

A rapid and simple method is described for the extraction of physostigmine (Phy) and its hydrolysis product, eseroline, from plasma, whole blood, and cerebrospinal fluid (CSF) and their subsequent quantitation by high-performance liquid chromatography (HPLC) with dual electrode electrochemical detection. Phy and eseroline were extracted from biologic fluids with cyano-phase columns eluted with 0.1 M citrate buffer, pH 4 containing 20% acetonitrile. Phy recovery from citrate buffer and CSF was nearly 100%. Phy recovery from plasma was 82% when methanol was used to precipitate proteins and 62% when HClO4 was used to precipitate proteins. Phy recovery from whole blood was only 17%. These results are discussed in the context of attempting to measure Phy in fluids of patients receiving this drug in clinical trials for the treatment of Alzheimer's disease.