A novel fluorescent labeling reagent, 2-(9-acridone)-ethyl chloroformate, and its application to the analysis of free amino acids in honey samples by HPLC with fluorescence detection and identification with online ESI-MS.

In this study, a novel fluorescent labeling reagent 2-(9-acridone)-ethyl chloroformate (AEC-Cl) was designed, synthesized and applied for the determination of free amino acids by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The free amino acids were rapidly and efficiently labeled by AEC-Cl in the presence of basic catalyst (pH 9.0) within 5 min at room temperature (25 °C). The derivatives exhibited excellent stability and fluorescence properties, with maximum excitation and emission wavelengths at 268 nm and 438 nm, respectively. Derivatives of 22 kinds of natural amino acids were completely separated by gradient elution on a Hypersil ODS C18 column. Under the optimal conditions, the calibration curves exhibited excellent linear responses, with correlation coefficients of R2 > 0.9994. The detection and quantification limits were in the range of 0.61-2.67 μg kg-1 and 2.07-8.35 μg kg-1, respectively. Therefore, AEC-Cl was successfully applied for the detection of trace levels of free amino acids in honey samples. Graphical abstract A novel fluorescent labeling reagent was applied for the determination of free amino acids in honey by high-performance liquid chromatography with a fluorescence detector.