A Validated Method for the Simultaneous Measurement of Tryptophan, Kynurenine, Phenylalanine, and Tyrosine by High-Performance Liquid Chromatography–Ultraviolet/Fluorescence Detection in Human Plasma and Serum

Aromatic amino acids are precursors of neurotransmitters
and immunomodulatory
molecules, and their catabolism is dysregulated in various disorders
associated with inflammation. This dysregulation often correlates
with disease stage, symptom severity, comorbidities, quality of life,
and cognitive performance, making its measurement valuable for research,
diagnostics, and personalized monitoring. We developed a rapid, reliable,
and cost-effective HPLC method for the simultaneous quantification
of phenylalanine, tyrosine, tryptophan, and kynurenine in human serum
and plasma. After protein precipitation, analytes were separated on
a reversed-phase C18 column under isocratic conditions. Detection
was performed based on intrinsic fluorescence for tryptophan, phenylalanine,
and tyrosine and on UV absorption for kynurenine and the internal
standard nitrotyrosine. The method showed linearity (R2 > 0.99) over 0.31–20 μM for kynurenine, 1.56–200
μM for phenylalanine, 0.08–200 μM for tryptophan,
and 0.78–200 μM tyrosine. Limits of detection were 0.01
μM for tryptophan, 0.08 μM for tyrosine and kynurenine,
and 0.39 μM for phenylalanine. Precision and accuracy were within
15%, and recovery rates ranged from 98 to 100%. Samples remained stable
after processing and after three freeze–thaw cycles. Interlaboratory
testing confirmed the reproducibility of the results. This validated
method enables sensitive, accurate, and simultaneous quantification
of key aromatic amino acids, providing a practical alternative to
LC–MS/MS for routine diagnostics and biomarker studies.