A METHOD FOR MEASURING NANOGRAM QUANTITIES OF ACETYLCHOLINE BY PYROLYSIS-GAS CHROMATOGRAPHY: THE DEMONSTRATION OF ACETYLCHOLINE IN EFFLUENTS FROM THE RAT PHRENIC NERVE-DIAPHRAGM PREPARATION

¹ Department of Pharmacology, Mount Sinai School of Medicine, New York, New York

A specific, sensitive, simple and rapid method for analyzing acetylcholine is described. Acetylcholine is precipitated as the enneaiodide in the presence of tetramethylammonium iodide as a co-precipitant and, for quantification, with the addition of a known amount of butyrylcholine as an internal standard. After sublimation of excess iodine, the precipitate is dissolved, concentrated in a pyrolyzing chamber connected to a gas chromatograph and flash-heated. Pyrolysis causes N-demethylation to yield dimethylaminoethyl acetate from acetylcholine and dimethylaminoethyl butyrate from butyryicholine. The relative ratios of the peak areas are linearly related to the amounts of acetylcholine. The method permits measurements of acetylcholine in quantities as low as 2 ng (i.e., 13.7 pmol) in 5 ml of Tyrode's solution and 5 ng in 300 ml of Tyrode's solution. The phrenic nerve-diaphragm preparation of the rat released material which is chromatographically indistinguishable from acetylcholine and which is destroyed by electric eel acetyicholinesterase. Electrical stimulation caused increased release of of acetylcholine from the phrenic nerve-diaphragm. No other differences between the effluents from the stimulated and unstimulated preparation were seen.