Fully automated reagent peak-free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column-switching system and fluorous scavenging derivatization

• Published in: Journal of separation science Vol. 36; no. 2; pp. 232 - 238
• Main Authors: Todoroki, Kenichiro, Hashimoto, Hiroki, Machida, Kazuyuki, Itoyama, Miki, Hayama, Tadashi, Yoshida, Hideyuki, Nohta, Hitoshi, Nakashima, Manabu, Yamaguchi, Masatoshi
• Format: Journal Article
• Language: English
• Published: Weinheim Blackwell Publishing Ltd 01-01-2013; Wiley; Wiley Subscription Services, Inc
• Subjects: Acids; Animals; Automated; Automation; Biological and medical sciences; Carboxylic acids; Carboxylic Acids - chemistry; Carboxylic Acids - urine; Chromatography; Chromatography, Liquid - instrumentation; Chromatography, Liquid - methods; Column switching; Diabetes; Diabetes mellitus; Diabetes Mellitus, Type 2 - urine; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Fluorescence; Fluorous scavenging derivatization; Humans; Liquid chromatography; Male; Mathematical analysis; Medical sciences; Mice; Scavenging; Urine; Endocrinopathy; Type 2 diabetes; Biological fluid; Chemical analysis; Column switching; Fluorogenic reagent; HPLC chromatography; Pyrene derivatives; Fluorescence; Derivatization; Carboxylic acid; Quantitative analysis; Urine; Validation; Automation; Fluorescence detector; Coupled method; Rodentia; Metabolic diseases; Carboxylic acids; Polar compound; Normal; Fluorous scavenging derivatization; Vertebrata; Reversed phase chromatography; Experimental disease; Trapping; Mammalia; Mouse; Fluorescent labelling; Animal
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.201200692

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‐1H‐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.