High performance liquid chromatography/electrospray ionization ion-trap mass spectrometry analysis of chitooligosaccharides by pre-column derivatization with 3-amino-9-ethylcarbazole

A pre-column derivatization method was established for chitooligosaccharides (COS) analysis, employing 3-amino-9-ethylcarbazole (AEC) as a derivatization reagent. The enamine was generated by the reaction of the reducing end of COS and the primary amine of AEC, and then reduced to secondary amines by NaBH3 CN. Under the optimized high performance liquid chromatography (HPLC) and electrospray ionization ion-trap mass spectrometry (ESI-MS) conditions, the HPLC separation was carried out on an RP C18 column (250 mm x 4.6 mm, 5 microm) with a gradient elution using acetonitrile and ammonium acetate as mobile phase. The derivative was successfully analyzed by HPLC, ESI-MS and on line HPLC-ESI-MS in positive-ion mode. The established method is simple, high sensitive and reproducible, which has great potential application to component analysis, quality control and structure activity relationship analysis of COS.