Amine analysis using AlexaFluor 488 succinimidyl ester and capillary electrophoresis with laser-induced fluorescence

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. © 2015 christian g. kendall et al.