April - June 2015, Volume 11, Issue 2

Ladasiu Ciolacu FC, Ardelean A, Turcus V, Mândrutiu I, Belengeanu AD, Bechet D, Frentescu L, Mihali CV, Benga G

A Simple, Sensitive and Highly Accurate Procedure for Plasma Phenylalanine Determination by HPLC

Acta Endo (Buc) 2015, 11 (2): 143-146
doi: 10.4183/aeb.2015.143

Phenylketonuria (PKU) is the most frequent inherited amino acid metabolic disorder, and it may also be treated by dietary means. The determination of phenylalanine (Phe) levels in the blood plasma is important not only in early diagnostic, but also in monitoring the treatment of PKU. Purpose. The aim of our work was to develop a simple, sensitive and highly accurate procedure to determine the plasma concentration of Phe. Procedure. The measurement of plasma Phe concentration involves two steps: a) separation of plasma (from the blood taken on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex-50X8 and evaporation of the eluate in vacuum at 40˚C), and b) determination of Phe concentration in the solution of amino acids by HPLC. This analysis was performed using a Dionex Ultimate 3000 instrument equipped with a Ultimate 3000 diode array detector (DAD). The values of Phe concentration in the plasma of several patients were calculated using a calibration curve made with standards of Phe (dilutions of a stock solution of 50 mg/ dL). The measurements in duplicate (plasma Phe) or a greater number of samples from the same concentration of standards of Phe showed extremely small sample to sample differences. Concentrations as low as 0.2 mg/dL could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and highly accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentration of Phe in patients with PKU.

Keywords: phenylalanine, phenylketonuria, plasma, HPLC.

Correspondence: Gheorghe Benga MD, PhD, The “Gheorghe Benga” Foundation, Aleea Muscel No. 19, Ap. 11, 400076 Cluj-Napoca, Romania, E-mail: gbgbenga@gmail.com