Application of 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)–ethoxy] Ethyl Chloroformate as a Pre-column Derivatization Reagent for Determination of Amino Acids from Hydrolyzed Cicada by High Performance Liquid Chromatography with Fluorescence Detection and Mass Sp

Based on the use of 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC-Cl) as a sensitive pre-column derivatization reagent, a simple method for evaluating the chemical compositions of the hydrolyzed protein from the cicada nymphs has been developed. Studies on derivatization conditions indicated that the labeling reaction proceeded rapidly and smoothly in the presence of a base catalyst (pH 9.0) in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at λex of 300 nm and an emission maximum at λem of 395 nm. The separation of amino acid derivatives was carried out on a reversed-phase Hypersil BDS-C18 (200 mm × 4.6 mm, 5 μm) column in combination with a gradient elution. The complete baseline resolution for 20 amino acid derivatives was achieved within 65 minutes. Identification of amino acid derivatives was carried out by the on-line ion trap/mass spectrometry with electrospray ion source in positive-ion mode. Excellent linear response was observed with coefficients > 0.9992. Fluorescent detection limits calculated from 25 fmol injection (at a signal-to-noise ratio of 3) were 2.60–24.3 fmol. The established method for the determination of amino acids from the hydrolyzed cicada sample was satisfactory.