Analytical Sciences

A Simple HPLC-Fluorescence Detection of Nitric Oxide in Cultivated Plant Cells by in situ Derivatization with 2,3-Diaminonaphthalene

Mitsuhiro WADA,* Chieko MORINAKA,* Toshihiko IKENAGA,** Naotaka KURODA,* and Kenichiro NAKASHIMA*** 

*School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
**Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
***Graduate School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

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°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°C for 1 h was significantly higher than that treated at 25°C and 35°C for 1 h (n = 3, p < 0.05).

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