Simultaneous high-performance liquid chromatography analysis of 18 amino acids in special formula milk powder: development and validation

A high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of 18 amino acids in special formula milk powder. This method was based on pre-column derivatization with phenyl isothiocyanate, separation on a C₁₈ column, ultraviolet detection, and quantification using external standards. Acid hydrolysis using 6 mol/L HCl under autoclaving at 120 °C for 2 h was optimized and compared with conventional oven hydrolysis at 110 °C for 24 h. The results revealed that all 18 amino acids yielded linear correlation coefficients exceeding 0.999. The limits of detection and quantification ranged from 0.07 to 0.32 μg/mL and from 0.23 to 1.07 μg/mL, respectively. The precision, repeatability, and stability were 0.06%–2.44%, 0.32%–2.22%, and 0.19%–2.77%, respectively, and the spiked recoveries ranged between 85.92% and 107.55%. The developed method allowed the analytical run time for all amino acids to be reduced to 35 min. Another advantage was that the proposed method enabled quantification of tryptophan, asparagine, and glutamine, which are not detected by the standard method defined in GB 5009.124–2016 National food safety standards–determination of amino acids in food. Overall, the obtained results demonstrate that our method allowed the simultaneous quantification of 18 amino acids in special formula milk powder with improved precision, sensitivity, and efficiency after optimization of the hydrolysis and detection conditions. This method is expected to prove suitable for the quality control of foods for special medical purposes.