[HPLC for the diagnosis and monitoring of phenylketonuria and ketoaciduria].

A simple, rapid, and sensitive HPLC is proposed to test six amino acids in plasma/serum. Deproteinization was carried out with acetonitrile; derivation was made with the orthophthalic aldehyde 2-mercaptoethanol. Separation was accompanied on a Chromolith (Merck), 4.6 mm in size, with monolytic reverse-phase silica gel in the isocratic mode with ultraviolet detection at 230 nm. The eluent contained a 50% mixture of methanol-acetonitrile-isopropanol (90:5:5, v/v/v) and 50% 0.01 M phosphate buffer (pH 6.7); flow rate 1000 ul/min; pressure 42 bars. Complete separation lasted at least 10 min. The detection limit was about 1 ng for phenylalanine, leucine, and isoleucine and less than 0.5 ng for tryptophan, valine, and methionine at a signal/noise ratio of 3.0. The simplicity, reproducibility, and sufficient sensitivity of the technique along with the feasibility of its application on standard chromatographic equipment (an isocratic pump and a ultraviolet detector) make it suitable for routine clinical application.