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

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.