Determination of Amino Acids from Hydrolyzed Bovine Serum Albumin by High Performance Liquid Chromatography and Identification with Electrospray Ionization Mass Spectrometry

A precolumn derivatization method for the determination of 20 amino acids using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as precolumn derivatization reagent was developed. Amino acid derivatives were successfully separated on a reversed-phase Hypersil BDS C18 (4.6 mm×200 mm, 5 μm) column in conjunction with a gradient elution. Studies on derivatization conditions indicated that amino acids could rapidly and mildly react with BCEOC reagent at room temperature in the presence of sodium borate catalyst in acetonitrile solvent to give the corresponding sensitive fluorescent derivatives. Maximal derivatization yields close to 100% were observed with a 4-fold molar reagent excess. The fluorescence excitation and emission wavelengths were 333 nm and 390 nm, respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) in a positive-ion mode. Excellent linear responses were obtained with the coefficients over 0.9990. Detection limits (at a signal-to-noise of 3:1) were 6.3–117.6 fmol for the labeled amino acids.