Analysis of Citrulline, Arginine, and Methylarginines Using High‐Performance Liquid Chromatography

Citrulline is a product of arginine degradation by nitric oxide synthase and is a precursor for arginine synthesis in animal cells. After arginine is incorporated into proteins, it may undergo methylation to form N(G)-monomethylarginine, which may be converted to asymmetric dimethylarginine and symmetric dimethylarginine. The degradation of these methylated proteins produces free methylarginines. This chapter focuses on the analysis of these amino acids in biological samples (including plasma/serum, urine, cell culture medium, and tissues) using high-performance liquid chromatography that involves precolumn derivatization with o-phthaldialdehyde. Fluorescence is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 5 nM for amino acids. The assays are linear between 1 and 100 microM for citrulline and arginine and between 0.1 and 10 microM for methylarginines. These chromatographic methods are highly sensitive, specific, accurate, and easily automated and provide a useful tool to study the regulation of the arginine-nitric oxide pathway.