Measurement of phenylalanine and tyrosine in plasma by high-performance liquid chromatography using the inherent fluorescence of aromatic amino acids

Abstract

An isocratic high-performance liquid chromatography (HPLC) method is described using the natural fluorescence of phenylalanine and tyrosine compared with that of an internal standard N-methyl phenylalanine. Plasma precipitated with 6% perchloric acid was separated isocratically using a base-deactivated C18 column with 5% acetonitrile in water as the mobile phase. Fluorescent measurements at an excitation wavelength of 215 nm and emission 283 nm showed only three peaks for tyrosine, phenylalanine and the internal standard eluting within 9 min. Inter-batch coefficients of variation for phenylalanine were 2.9% and 1.8% at levels of 70 and 567 μmol/L, respectively, and 2.9% at a level of 63 μmol/L for tyrosine. The results for phenylalanine for this method showed a small mean positive bias (11 μmol/L) when compared with the target all-method means for UK National External Quality Assessment Scheme samples (n = 31). The results for tyrosine showed a small positive mean bias (10 μmol/L) when compared with an ion-exchange chromatographic method (n = 40). This method provides a quick and simple alternative to those using HPLC with pre- or post-column derivatization for monitoring patients with phenylketonuria. It is also less subject to interferences than HPLC methods using ultraviolet detection, particularly for the early eluting tyrosine peak.