Analysis of hydroxylation and nitration products of D-phenylalanine for in vitro and in vivo radical determination using high-performance liquid chromatography and photodiode array detection.
D-phenylalanine is capable of trapping reactive oxygen species (ROS) and reactive nitrogen species (RNS) by forming three major hydroxylation (o-, m-, p-tyrosine) and two major nitration products (nitrophenylalanine, nitrotyrosine). Here, we show how a method for the analysis of these phenylalanine derivatives was established using isocratic HPLC (Nucleosil120, C18 column) coupled with photodiode array detection and validated for cell-free in vitro and in vivo determination of radical formation. An ideal separation was achieved using a mobile phase consisting of 5% acetonitrile, 50mM KH(2)PO(4), pH 3.0, a column temperature of 35 degrees C and a flow rate of 1.0 mL/min. Limits of detection were in the range of 5-100 nM. Linearity was given within 5 nM-100 microM (correlation coefficient >0.999). Retention times as well as peak heights exhibited a high precision (RSD: <or=0.1% and <1.5%, respectively). The feasibility of d-phenylalanine for ROS/RNS measurement was demonstrated in a cell-free in vitro assay using peroxynitrite and by analysis of brain samples of mice treated with the dopaminergic neurotoxin 6-hydroxydopamine.
Department CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach an der Riss, Germany.
Patrick
P
Oeckl
Boris
B
Ferger
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry*; D006900:Hydroxylation; D051379:Mice; D008810:Mice, Inbred C57BL; D010649:Phenylalanine / Q000737:chemistry*
D005609:Free Radicals; D010649:Phenylalanine