Sensitive detection of selected cholinergic channel activators derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole.

A high-performance liquid chromatographic method with fluorescence detection was developed for a series of cholinergic channel activators using 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescent labeling reagent. This method includes three separate steps: extraction of parent compound and internal standard with organic solvent from plasma, reaction of parent and internal standard in NBD-F precolumn to yield a fluorescent product and extraction of the resultant fluorophores with organic solvents. The extraction and reaction procedures were optimized for this series of structurally new compounds. The method showed high sensitivity, selectivity and reproducibility and proved useful for the determination of ng ml-1 plasma levels of selected cholinergic channel activators.