Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence

• Main Authors: Christian G. Kendall, Amanda M. Stockton, Stephen Leicht, Heather McCaig, Shirley Chung, Valerie Scott, Fang Zhong, Ying Lin
• Format: Article
• Language: English
• Published: Hindawi Limited 2015-01-01
• Series: Journal of Analytical Methods in Chemistry
• Online Access: http://dx.doi.org/10.1155/2015/368362
• ISSN: 2090-8865; 2090-8873

Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5–17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.