Stability of acetylcholine chloride solution in autonomic testing

Acetylcholine (ACh) is the neurotransmitter used as an agent to evoke a sudomotor axon reflex response in autonomic testing. Adequate stimulus of postganglionic axons requires ACh solutions to be stable, but its stability in the clinical laboratory is uncertain. We evaluated the stability of standard (0.55 M) ACh solutions stored at temperatures of - 20°C, 4°C, 25°C, and 50°C for 10 time points between 0 and 84 days. ACh and choline (Ch) were measured by reverse-phase HPLC with electrochemical detection using an Acetylcholine/Choline Assay Kit. Linear regressions of ACh and Ch standards were used to calculate the levels in the stored samples. The inherent levels of Ch were used as the internal standard. Regression analyses were used to examine the effects of length of storage and temperature. The samples of ACh stored at - 20°C and 4°C showed an extremely small breakdown over the 84-day period and had no evidence to show the regression lines differed. ACh solution stored at 25°C was stable for about 28 days, after such time, modest breakdown occurs. At a temperature of 50°C, ACh showed a rapid breakdown after 1 day. We conclude ACh solution should not be stored at room temperature for more than 28 days and should not be exposed to higher temperatures to assure an adequate axon stimulus.