The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application.

In spite of its physiological importance acetylcholine has been determinable chemically only after isolation by time-consuming procedures. Of necessity workers in this field have resorted to pharmacological bioassays whose specificity seems open to doubt. The finding that choline is readily acetylated by the action of a coenzyme-linked enzyme system in which adenosine triphosphate serves as an energy source (1) lends added interest to the development of adequate analytical procedures for acetylcholine. This communication concerns certain properties of the interaction of scetylcholine with hydroxylamine, and the means of using this reaction, RCOOR’ + HzNOH ---f RCONHOH + R’OH, for the quantitative determination of acetylcholine. Feigl, Anger, and Frehden (2) have described the use of hydroxylamine as a spot test reagent (cf. also (3)). Lipmann and Tuttle (4) have based a specific quantitative method for the acyl phosphates on their ability to react with hydroxylamine in water at pH 6. Hill (5, 6) has used the reaction of est,ers with hydroxylamine at alkaline pH in anhydrous solvents to determine long chain fatty acid esters (cf. also (7)). The present method, which is designed for use with short chain 0-acyl derivatives, is based on the finding that hydroxylamine at an alkaline pH in water rapidly converts acetylcholine stoichiometrically to hydroxamic acid throughout a wide range of ester concentration. The specificity of the reaction as between esters and products of t.heir hydrolysis and its rapidity and technical simplicity have afforded an analytical method which is adaptable to widely different conditions. Hydroxylamine has been employed a,s a trapping reagent for acyl anhydrides in mixed enzyme systems by Lipmann and Tuttle (8) and others (9-11). Chantrenne (12) has shown that hydroxylamine at pH 7.1 also traps carboxylic acid esters. Experiments reported below evaluate the