Liquid chromatographic determination of acetylcholine based on pre-column alkaline cleavage reaction and post-column tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection.
A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre-column alkaline cleavage of ACh to form trimethylamine (TMA) and the post-column tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180 degrees C in the presence of lithium hydroxide, and the produced TMA was separated on a cation-exchange/reversed-phase dual-functional column using a mixture of 0.2 M potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2'-bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal-to-noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 microL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations.
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Hideyuki
H
Yoshida
Akifumi
A
Yamada
Kenichiro
K
Todoroki
Osamu
O
Imakyure
Hitoshi
H
Nohta
Masatoshi
M
Yamaguchi
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000737:chemistry; D000109:Acetylcholine / Q000032:analysis*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D057230:Limit of Detection; D049449:Luminescence; D009942:Organometallic Compounds / Q000737:chemistry*; D015203:Reproducibility of Results
D009942:Organometallic Compounds; C058901:tris(2,2'-bipyridine)ruthenium III; D015082:2,2'-Dipyridyl; D000109:Acetylcholine