Determination of methylated arginines by column-switching high-performance liquid chromatography-fluorescence detection

A column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method for the determination of three methylated arginines, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric dimethyl-L-arginine, ADMA), and N(G),N(G)'-dimethyl-L-arginine (symmetric dimethyl-L-arginine, SDMA), which are endogenous nitric oxide synthase inhibitors, was developed. After fluorescence derivatization of plasma samples with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized methylated arginines were trapped on a cation exchange column with filter to remove proteins, separated within 42 min on a reversed-phase column, and detected at an emission wavelength of 530 nm with excitation at 470 nm. The detection limits were 10 fmol for L-NMMA and 20 fmol for ADMA and SDMA with a signal-to-noise ratio of 3. A good linearity for calibration curves for each methylated arginine was observed within the range of 50-5000 fmol using homoarginine as an internal standard. The proposed method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.01, 0.73 +/- 0.02 and 0.41 +/- 0.05 micromol l(-1), respectively (n= 5).