title: 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.
abstract: 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.
authors.affiliation: Faculty of Pharmaceutical Life Sciences, Kitasato University, Tokyo, Japan; Pfizer Worldwide Research & Development, Tokyo Laboratories, Tokyo, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Nonaka
authors.forename: Masae
authors.initials: M
authors.lastname: Sekine
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsunoda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ozeki
authors.forename: Kumiko
authors.initials: K
authors.lastname: Fujii
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Akiyama
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Shimoda
authors.forename: Takemitsu
authors.initials: T
authors.lastname: Furuchi
authors.forename: Masumi
authors.initials: M
authors.lastname: Katane
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Saitoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Homma
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D064368:Healthy Volunteers; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012822:Silicon Dioxide / Q000737:chemistry*; D052616:Solid Phase Extraction; D019323:omega-N-Methylarginine / Q000097:blood*
chemical_list: D005456:Fluorescent Dyes; C000598073:N(G),N(G')-dimethylarginine; D019323:omega-N-Methylarginine; C018524:N,N-dimethylarginine; D012822:Silicon Dioxide; D001120:Arginine
keywords: Arginine; Methylated arginines; Monolithic silica columns; Monolithic spin columns; Nitric oxide