Liquid-chromatographic measurement of phenylalanine and tyrosine in serum

With phenylalanine ammonia-lyase (EC 4.3.1.5) we converted phenylalanine (Phe) and tyrosine (Tyr) to transcinnamic acid and p-coumaric acid, respectively. These were separated by "high-performance" liquid chromatography and detected at 280 nm. We measured the Phe and Tyr content of human serum by adding 100 mU of the enzyme to a 20-microL serum aliquot, mixing for 2 h at 24 degrees C, then stopping the reaction with 1 mL of cold methanol. Precipitated proteins were removed by centrifugation and the separated clear supernates were stored at -20 degrees C. For chromatographic separation, detection, and quantification, we used a system equipped with a C-18 reversed-phase column, a variable-wavelength spectrophotometer, a printer-plotter, and a microcomputer. The mobile phase was a mixture of dilute aqueous (50 g/L) acetic acid and CH3CN (80/20, by vol). CVs for specimens containing 100 mg of Phe or Tyr per liter varied from 5 to 10%. Analytical recoveries were near 100%.