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

N-G-Monomethyl-L-arginine (L-NMMA), N-G,N-G'-dimethyl-L-arginine (ADMA), and N-G,N-G-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. N-w-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 mul 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 +/- 10.21 muM, respectively (n = 5). (C) 2005 Elsevier B.V. All rights reserved.