High-performance liquid chromatographic assay of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G)'-dimethyl-L-arginine using 4-fluoro-7-nitro-2, 1,3-benzoxadiazole as a fluorescent reagent.
N(G)-Monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), and N(G),N(G)'-dimethyl-L-arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of L-NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nomega-Propyl-L-arginine was Used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for L-NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for L-NMMA, ADMA and SDMA were linear within the range of 50-5000 fmol. The method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in 200 microl of rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.03, 0.80 +/- 0.25 and 0.40 +/- 0.21 microM, respectively (n = 5).
Nonaka
Satoko
S
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Imai
Kazuhiro
K
Funatsu
Takashi
T
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005456:Fluorescent Dyes; D008297:Male; D011786:Quality Control; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D019323:omega-N-Methylarginine / Q000032:analysis* / Q000097:blood
D005456:Fluorescent Dyes; C487735:dimethylarginine; D019323:omega-N-Methylarginine; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D001120:Arginine; D009327:4-Chloro-7-nitrobenzofurazan