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

Abstract

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 µmol l⁻¹, respectively (n = 5).