Capillary zone electrophoresis determination of phenylalanine in serum: a rapid, inexpensive and simple method for the diagnosis of phenylketonuria.

Abstract

A simple, rapid, and quantitative capillary zone electrophoresis method for phenylalanine analysis in serum has been developed, with the aim of providing an analytical tool, as an alternative to liquid and gas chromatography, for the routine laboratory diagnosis of phenylketonuria. Electrophoresis was carried out in a 65 cm long, 50 microns wide bare silica capillary, using 0.025 M borate (adjusted to pH 10 with 1 M NaOH) at a potential of 20 kV, with in-column UV detection at 214 nm. Under these conditions, the three aromatic amino acids (tryptophan, phenylalanine and tyrosine) migrated according to the pKs of the respective amine (and hydroxyl) groups. The efficiency of separation was about 150,000 plates/column for phenylalanine. Diprophylline was adopted as internal standard. The injection of ethanol-deproteinized normal control serum gave rise to only a few major peaks not interfering with phenylalanine; phenylalanine in serum at normal concentrations appeared in a clean region of the electropherogram as a symmetrical peak with a migration time of about 11 min. The sensitivity was > or = 3 micrograms/mL, with s/n ratio = 3. The linearity, in the range of 5-175 micrograms/mL, was described by the equation y = 1.407-0.583 x, r2 = 0.9998. Accuracy and precision were satisfactory, with intra-day and inter-day coefficients of variation lower than 4% and 7%, respectively. The injection of sera from five phenylketonuria patients gave electropherograms clearly showing huge peaks of phenylalanine, thus allowing an easy laboratory diagnosis of phenylketonuria.