title: A simple HPLC-fluorescence detection of nitric oxide in cultivated plant cells by in situ derivatization with 2,3-diaminonaphthalene.
abstract: An HPLC method with fluorescence detection for the determination of nitric oxide (NO) in cultivated plant cells (Agave pacifica, Agavaceae) was developed. NO was derivatized in situ with 2,3-diaminonaphthalene (DAN) as a labeling reagent and converted to 1(H)-naphthotriazole. The maximum peak height of the derivative was observed by incubation for 3 h at 25 degrees C with 0.2 mM DAN. Excess reagent in cells was removed by washing 3 times with 5 ml of water. The calibration curve for authentic standard of DAN-NO spiked to cultivated plant cells showed a good linearity (r = 0.995) in the range of 5.0 to 50 pmol/g cell. The detection limit at a signal-to-noise ratio of 3 was 3.4 pmol/g cells. The proposed method was successfully applied to the monitoring of NO concentration with cell growth. The effect of thermal treatment on the concentration of NO in plant cells was also examined. The concentration of NO in cells treated at 5 degrees C for 1 h was significantly higher than that treated at 25 degrees C and 35 degrees C for 1 h (n = 3, p < 0.05).
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Wada
authors.forename: Chieko
authors.initials: C
authors.lastname: Morinaka
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ikenaga
authors.forename: Naotaka
authors.initials: N
authors.lastname: Kuroda
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Nakashima
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000032:analysis*; D027882:Agave / Q000737:chemistry* / Q000166:cytology* / Q000254:growth & development; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D009569:Nitric Oxide / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; C026373:2,3-diaminonaphthalene; D009569:Nitric Oxide; D015081:2-Naphthylamine