HPLC measurement of phenylalanine by direct injection of plasma onto an internal-surface reversed-phase silica support

Abstract

Direct-injection analysis for phenylalanine in plasma by isocratic 'high-performance' liquid chromatography (HPLC) is described. For chromatographic separation I use an internal-surface reversed-phase silica support. The method involves ultraviolet detection, requires as little as 10 μL of plasma (routinely 20 μL), and supplies results in <7.5 min from injection. The intra-batch coefficient of variation for phenylalanine in human plasma samples (197-1104 μmol/L) ranged between 3.0% and 6.0% and the range of linearity was 10-1250 μmol/L for a 20-μL sample injection loop. The upper limit can readily be extended by using a smaller volume loop. Phenylalanine results from direct plasma injection (y) correlated well with those of a protein precipitation/HPLC technique (x): y = 0.931x + 46.4 (r = 0.951, n = 41, range 78-1344 μmol/L).