Determination of heptylphysostigmine in plasma by high-performance liquid chromatography with electrochemical detection

Heptylphysostigmine is a new and very promising cholinergic drug for the treatment of Alzheimer disease. A method has been developed for its determination in plasma with a detection limit of 50 pg/ml. The drug was extracted in n-hexane by a simple one-step procedure, after buffering with sodium bicarbonate. Samples were analysed on a 25 cm x 4.6 mm I.D. silica column (5 microns particle size) using a mixture of acetonitrile, methanol and ammonium nitrate as mobile phase. Since this molecule is quite unstable in plasma, pyridostigmine bromide was added to samples to limit the decomposition. Physostigmine was employed as internal standard. The molecule was electrochemically detected by oxidizing potential (+0.75 V). The method was applied to the analysis of blood samples taken from one healthy volunteer administered this drug. In the same subject the inhibition rate of acetylcholinesterase in plasma and red cells was also measured.