Optimization and validation of a bioanalytical HPLC–UV technique for simultaneous determination of underivatized phenylalanine and tyrosine in the blood for phenylketonuria diagnosis and monitoring

This study aimed to develop a fast, accurate, and precise high-performance liquid chromatography with UV detection method for simultaneous analysis of underivatized phenylalanine (Phe) and tyrosine (Tyr) in biological samples. Separation of the analytes was accomplished using a Discovery HS F5-3 column, which offered better retention and peak symmetry for the tested analytes. Chromatographic conditions were optimized using central composite experimental design, and three factors were investigated: the concentration of ammonium acetate (A), the acetonitrile proportion in the mobile phase (B) and the column oven temperature (C). The approach was verified using β-expectation tolerance intervals for total error measurement that did not exceed 15%. Optimal settings were A = 50 mm, B = 24% and C = 28°C. The method applicability was determined using human plasma from 75 volunteers. The limits of detection and quantification of the technique were satisfactory at 9 and 29 μm for Phe and 4 and 13 μm for Tyr. The mean analytical bias in spiking levels was acceptable, ranging from -1.649 to +1.659% for both substances, with RSD <5% in all instances. The suggested approach was successfully used to analyze Phe and Tyr in human blood samples and calculate the Phe/Tyr ratio.