Direct quantitative analysis of aromatic amino acids in human plasma by four-way calibration using intrinsic fluorescence: Exploration of third-order advantages

A novel intrinsic fluorescence method for the direct determination of l-phenylalanine, l-tyrosine, and l-tryptophan in human plasma is presented. By using fluorescence excitation–emission–pH–sample data array in combination with four-way calibration method based on the quadrilinear component model, the proposed approach successfully achieved quantitative analysis of the aromatic amino acids in human plasma, even in the presence of an unknown, uncalibrated serious interferent. It needs little preparation, uses the “mathematical separation” instead of “analytical separation”, what makes it fast and environmentally friendly. Satisfactory results have been achieved for calibration set, validation set, and prediction set. The ranges for phenylalanine, tyrosine, and tryptophan are 2.0×103–20.0×103, 50.0–500.0, and 20.0–200.0 ng mL−1 respectively. Average spike recoveries (mean±standard deviation) are 93.3±7.7%, 104.3±6.6%, and 99.5±9.0% respectively. The real concentrations in human plasma are 10.2±0.3, 6.6±0.1, and 5.3±0.1 μg mL−1 respectively, which are consistent with the results obtained by LC–MS/MS method and reference values. In addition, we explored the third-order advantages through the real four-way array; it has shown that higher resolving power is one of the main advantages of higher-order tensor calibration method. These results demonstrated that the proposed method is sensitive, accurate, and efficient for direct quantitative analysis of aromatic amino acids in human plasma.