Phenylalanine: Application of a Simple HPLC Technique to its Measurement in Dried Blood Spots

Recent recommendations regarding the management of phenylketonuria suggest that to attain maximum achievable intelligence quotients plasma phenylalanine levels need to be more strictly controlled. Twice weekly monitoring and a target range of 120-300 1-LII10l/L have been advocated.' To achieve the necessary turnround time and meet the analytical requirements of such a policy laboratories need simple, rapid methods for phenylalanine estimation which are both accurate and precise particularly at the lower end of the suggested range. High performance liquid chromatography (HPLC) can be accurate and precise but most methods require derivatization, gradient separation or fluorimetric detection.s-' A method based on isocratic separation and ultraviolet (UV) detection of the aromatic amino acids by HPLC without derivatization has been used for the measurement of phenylalanine in plasma deproteinized with perchloric acid (PCA).s This report describes the extension of the method to the estimation of phenylalanine in extracts of dried blood spots on paper which are considered to be a safe and convenient method of collecting blood at home and are also used for neonatal screening. HPLC was performed at 30°C on Waters equipment (Millipore (UK) Limited, Watford WD1 8YW, UK) using a 501 pump, 712 automated injector and 455 UV detector at 206 nm. Data acquisition and calculation of results based on peak height was performed by Baseline 810 software on a NEC Advanced Personal Computer. The octadecylsilane analytical column was as used by Atherton and Green.s Stock phenylalanine standard (5 mmol/L), stock internal standard (c-methylphenylalanine, 5 mmol/L) and working internal standard (500 ILmol/L) were