Simultaneous determination of N~G-monomethyl-L-arginine, N~G,N~G-dimethyl-L-arginine, N~G,N~G'-dimethyl-L-arginine, and L-arginine using monolithic silica disk-packed spin columns and a monolithic silica column

摘要

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N~G-monomethyl-L-arginine, N~G,N~G-dimethyl-L-arginine, and N~G,N~G' -dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-Arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2–1.0 μM for methylated arginines and 40–200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r~2). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3–4.8% (intraday) and 3.0–6.8% (interday).When this method was applied to samples from six healthy donors, the detected concentrations of N~G-monomethyl-L-arginine, N~G,N~G-dimethyl-L-arginine, N~G,N~G'-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.