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.
Yoshida
Hideyuki
H
Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Yamada
Akifumi
A
Todoroki
Kenichiro
K
Imakyure
Osamu
O
Nohta
Hitoshi
H
Yamaguchi
Masatoshi
M
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