A simple and sensitive method of acetylcholine identification and assay. Bioassay combined with minicolumn gel filtration or high-performance liquid chromatography.

ABSTRACT

A modified technique of acetylcholine assay on the guinea pig ileum has been combined with either minivolume gel filtration or high-performance liquid chromatography separation of the samples. In addition, labeled acetylcholine (14C-ACh) was eluted with unlabeled acetylcholine or with the samples expected to contain acetylcholine, and their elution profiles were compared by bioassay plus radioassay of eluate fractions. When the elution of both biological activity and label occurred in the same eluates, it was concluded that the substance assayed on guinea pig ileum was acetylcholine. The method was sensitive to 0.5 ng (1.8 pmol) of acetylcholine and its reproducibility was within 5%. It thus represents a substantial improvement in chemical specificity over the previous bioassay method described by Paton and Vizi (1969).