High-performance liquid chromatographic method for determination of amino acids by precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

This work presents an high-performance liquid chromatography method for the determination of amino acids after precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) which can readily react with both primary and secondary amines. The precolumn derivatization conditions, including the CNBF concentration, reaction pH, temperature and reaction time were investigated for method optimization. In pH 9.0 borate buffer, the reaction of amino acids with CNBF was carried out at 60 °C for 30 min, the optimized concentration of CNBF was 70 mmol L−1 and the molar ratio of amino acids to CNBF was 1:5.25. The chromatographic separation of 19 amino acids derivatives was performed on a Kromasil ODS C18 column (250 mm × 4.6 mm, 5 μm) with good reproducibility, and ultraviolet detection was applied at 260 nm. The mobile phase was a mixture of phase A (acetonitrile) and phase B (acetate buffer, acetonitrile, triethylamine; 82.8:17:0.2, pH 4.9), and the flow rate was 0.4 mL min−1. The separation of all the labeled amino acids was achieved within 45 min at room temperature by gradient elution mode. The method linearity, calculated for each amino acid, had a correlation coefficient higher than 0.9979, in concentrations ranging from 9.60 to 3330.00 μmol L−1. The detection limits of amino acids were 2.40–6.50 μmol L−1, at a signal-to-noise ratio of 3. The proposed method was applied for the determination of amino acids in beer with recoveries of 97.0–103.9% and relative standard deviations of 2.62–4.22%, respectively. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.