Separation of the Structural Isomers of Monomethylarginine in Human Plasma by 2-D-HPLC and MS–MS Detection

Abstract

Elevated plasma concentrations of the methylated metabolites of the amino acid l-arginine, especially asymmetric and symmetric dimethylarginine, are long established cardiovascular risk markers. The role of the two structural isomers of monomethylated l-arginine, N G-monomethyl-l-arginine and N δ-monomethyl-l-arginine, is not so well understood, probably because of the low concentrations of these compounds in human plasma and difficulties in separating them by chromatographic or mass spectrometric means. In this work, we provided a method for the interference-free baseline separation of the two substances, together with l-arginine, by derivatization with o-phthalaldehyde/2-mercaptoethanol and 2-D chromatographic separation on C18- and phenyl-modified stationary phases followed by MS–MS detection. Stable isotope labeled arginine served as an internal standard. Linear calibration functions were obtained in the ranges of 7.5–150 µmol L−1 for l-arginine, 25–1000 nmol L−1 for N G-monomethyl-l-arginine, and 5–350 nmol L−1 for N δ-monomethyl-l-arginine. Relative standard deviations were less than 9% (precision) for all compounds in all concentration levels and the accuracy was 97.48–106.53% of the theoretical value.