Determination of methylated arginines by column-switching high-performance liquid chromatography-fluorescence detection.
A column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method for the determination of three methylated arginines, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric dimethyl-L-arginine, ADMA), and N(G),N(G)'-dimethyl-L-arginine (symmetric dimethyl-L-arginine, SDMA), which are endogenous nitric oxide synthase inhibitors, was developed. After fluorescence derivatization of plasma samples with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized methylated arginines were trapped on a cation exchange column with filter to remove proteins, separated within 42 min on a reversed-phase column, and detected at an emission wavelength of 530 nm with excitation at 470 nm. The detection limits were 10 fmol for L-NMMA and 20 fmol for ADMA and SDMA with a signal-to-noise ratio of 3. A good linearity for calibration curves for each methylated arginine was observed within the range of 50-5000 fmol using homoarginine as an internal standard. The proposed method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.01, 0.73 +/- 0.02 and 0.41 +/- 0.05 micromol l(-1), respectively (n= 5).
Tsunoda
Makoto
M
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan. makotot@mol.f.u-tokyo.ac.jp
Nonaka
Satoko
S
Funatsu
Takashi
T
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D006709:Homoarginine / Q000097:blood; D008297:Male; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019323:omega-N-Methylarginine / Q000097:blood
C487735:dimethylarginine; D006709:Homoarginine; D019323:omega-N-Methylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase