Determination of N-G,N-G-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

摘要

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 N-G,N-G-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 mu M (1.0 nmol per injection) using N-G-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 mu M (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. (c) 2006 Elsevier B.V. All rights reserved.