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Fully automated reagent peak‐free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column‐switching system and fluorous scavenging derivatization
Author(s) -
Todoroki Kenichiro,
Hashimoto Hiroki,
Machida Kazuyuki,
Itoyama Miki,
Hayama Tadashi,
Yoshida Hideyuki,
Nohta Hitoshi,
Nakashima Manabu,
Yamaguchi Masatoshi
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201200692
Subject(s) - reagent , chemistry , derivatization , chromatography , carboxylic acid , elution , fluorescence , detection limit , carbodiimide , reversed phase chromatography , high performance liquid chromatography , organic chemistry , physics , quantum mechanics
In this study, we combined a column‐switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak‐free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1‐pyrenemethylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and 1‐hydroxy‐1 H ‐benzotriazole. Residual excess of the unreacted reagent was tagged with 2‐(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column‐switching system placed in front of an analytical reversed‐phase column. The signal of the fluorous‐tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type‐II model mice and their control.