Determination of phenylalanine and tyrosine in plasma and dried blood samples using HPLC with fluorescence detection

The determination of phenylalanine and tyrosine is presently the most reliable direct approach to the diagnosis of phenylketonuria. An HPLC method for the simultaneous measurement of phenylalanine and tyrosine in samples of dried blood spots and plasma has been developed and evaluated. We have used an inherent fluorescence of both phenylalanine and tyrosine. For the separation, a reverse-phase column LiChroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of ethanol and deionized water (5:95, v/v) was used as a mobile phase. Analytical performance of this method is satisfactory for both phenylalanine and tyrosine: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma and blood samples were between 92.0 and 102.9%. The limit of detection was 10.0 and 5.0 μmol/L, respectively. The preliminary reference ranges of phenylalanine and tyrosine in a group of newborns are 69.3 ± 13.1 and 42.7 ± 12.9 μmol/L, in a group of blood donors are 68.4 ± 9.9 and 52.1 ± 10.9 μmol/L. The presented method is inexpensive and suitable for diagnosis of phenylketonuria.