High-performance liquid chromatographic assay of NG-monomethyl-l-arginine, NG,NG-dimethyl-l-arginine, and NG,NG′-dimethyl-l-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent

NG-Monomethyl-l-arginine (l-NMMA), NG,NG-dimethyl-l-arginine (ADMA), and NG,NG′-dimethyl-l-arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of l-NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nω-Propyl-l-arginine was used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for l-NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for l-NMMA, ADMA and SDMA were linear within the range of 50-5000 fmol. The method was applied to the quantitative determination of l-NMMA, ADMA and SDMA in 200 μl of rat plasma. The concentrations of l-NMMA, ADMA and SDMA in rat plasma were 0.16 ± 0.03, 0.80 ± 0.25 and 0.40 ± 0.21 μM, respectively (n = 5).