Reagent peak-free liquid chromatography-fluorescence analysis of carboxylic acids using a fluorous scavenging-derivatization method.
We developed a fluorous scavenging-derivatization method for reagent peak-free liquid chromatography (LC)-fluorescence analysis of carboxylic acids. In this method, carboxylic acids were fluorescently derivatized with 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and could be selectively removed by microfluorous solid-phase extraction before LC analysis. With use of this method, eight fluorescent derivatives of linear aliphatic carboxylic acids (C(1)-C(8)) can be separated within 30 min by reversed-phase LC with gradient elution. In the chromatogram obtained, the fluorous-tagged unreacted reagent peak is greatly decreased after microfluorous solid-phase extraction and does not interfere with the quantification of each acid. With use of microfluorous solid-phase extraction with 80% (v/v) aqueous methanol elution, over 99.9% of the unreacted fluorescent reagent was removed. The detection limits (signal-to-noise ratio of 3) for the carboxylic acids examined are 2.3-8.0 fmol per 10-microL injection. We also applied this method successfully to the analysis of highly polar carboxylic acids such as alpha-keto acids and tricarboxylic acid cycle metabolites.
Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Kenichiro
K
Todoroki
Hiroki
H
Hashimoto
Tomohiko
T
Mikawa
Miki
M
Itoyama
Tadashi
T
Hayama
Eijiro
E
Kojima
Hideyuki
H
Yoshida
Hitoshi
H
Nohta
Masatoshi
M
Yamaguchi
D002264:Carboxylic Acids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008744:Methylamines / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry
C510196:1-pyrenemethylamine; D002264:Carboxylic Acids; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008744:Methylamines; D011721:Pyrenes