Improved method for simultaneous determination of l-arginine and its mono- and dimethylated metabolites in biological samples by high-performance liquid chromatography

An improved method has been developed for the determination of l-arginine and its methylated metabolites, NG-monomethyl-l-arginine (l-NMMA), NG,NG-dimethyl-l-arginine (asymmetric DMA, ADMA) and NG,NG′-dimethyl-l-arginine (symmetric DMA, SDMA) in biological samples. Extraction of these compounds with a strong cation-exchange resin AG50W-X8 with l-homoarginine (2-amino-6-guanidinohexanoic acid) as an internal standard gave a recovery of more than 70% except for SDMA from plasma samples. After extracted samples were converted to fluorescent derivatives with o-phthalaldehyde (OPA) in an alkaline medium, the following high-performance liquid chromatographic separation with a ODS column (wide-pore size, 300 Å) was successfully performed with an isocratic mobile phase system. The method permits quantitative determination of l-arginine and its methylated metabolites at concentrations as low as 4 μM and 0.18 μM, respectively. Using this method, the levels of l-arginine, l-NMMA, ADMA and SDMA in human plasma, urine and rat tissue were determined.