Determination of NG,NG-dimethyl-l-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of NG,NG-dimethyl-l-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) – 140 μM (1.0 nmol per injection) using NG-monomethyl-l-arginine (l-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82 ± 0.05 μM (n = 4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.