HPLC combined with electrochemical detection and using bioractors is the most accepted method to measure ACh. For more details visit the web-site on the company Antec (The Netherlands).
Even Simple TLC with pure Acetylcholine can be of great help, if not, run GC with ECD as detector and of course pure acetylcholine too, If you could get HPTLC it would be great.
I would even make it simpler for you (of course if you have the primary standard of acetyl choline) to use GC with any column (porapaq Q or R) and even TCD detector.