Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection

• Phenylalanine and tyrosine ratio is important for neurotransmitter biosynthesis.
• An HPLC method for serum phenylalanine and tyrosine concentrations is described.
• Both amino acids are measured utilizing their natural fluorescence.
• Rapid and sensitive measurement of phenylalanine and tyrosine is achieved.
• Method can be adapted easily for kynurenine and tryptophan measurements.

ObjectivesAn HPLC method was developed to quantify serum concentrations of phenylalanine and tyrosine simultaneously using fluorescence detection without derivatization.MethodsSerum protein is precipitated with trichloroacetic acid, 0.015 mM dihydrogen-phosphate solution is used for separation on reversed-phase C18 material, and acetonitrile is avoided. Both amino acids are monitored utilizing their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths.ResultsOne analytical run is completed within 7 min. Lower detection limit for Phe and Tyr is 0.3 μM. Comparison of the new method with a classical HPLC method for total amino acids and using UV-absorption detection reveals a highly significant relationship for Phe and Tyr.ConclusionThe new HPLC method allows rapid and very sensitive measurement of phenylalanine and tyrosine concentrations.