Liquid chromatographic determination of acetylcholine based on pre-column alkaline cleavage reaction and post-column tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection.

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre-column alkaline cleavage of ACh to form trimethylamine (TMA) and the post-column tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180 degrees C in the presence of lithium hydroxide, and the produced TMA was separated on a cation-exchange/reversed-phase dual-functional column using a mixture of 0.2 M potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2'-bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal-to-noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 microL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations.