Fang Yang-Il, Ohata Hisayuki, Honda Kazuo
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
J Pharmacol Toxicol Methods. 2009 May-Jun;59(3):153-5. doi: 10.1016/j.vascn.2009.01.001.
In this study, a simple and sensitive fluorometric HPLC method was described for the determination of nitrite, an indicator of nitric oxide production in biological samples.
Nitrite was reacted with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole (NAT). Because NAT has higher fluorescence intensity at alkaline conditions, a reverse phase HPLC method employing a polymer-based column was applied to separate NAT using an alkaline mobile phase system.
NAT was well separated from DAN and other fluorescent substances with increased detection sensitivity under alkaline conditions. A linear relationship (r=0.999) between the fluorescence intensity of NAT and the concentration of nitrite was observed in the range from 0 to 800 nM with the detection limit of about 25 nM.
The present HPLC method appears suitable for routine analysis of nitrite in crude biological samples, since the polymer-based column can withstand rigorous cleaning with either acid or base.
在本研究中，描述了一种简单且灵敏的荧光高效液相色谱法，用于测定生物样品中一氧化氮产生的指标亚硝酸盐。
亚硝酸盐与2,3-二氨基萘（DAN）反应形成荧光性的2,3-萘三唑（NAT）。由于NAT在碱性条件下具有更高的荧光强度，因此采用基于聚合物的色谱柱的反相高效液相色谱法，使用碱性流动相系统来分离NAT。
在碱性条件下，NAT与DAN及其他荧光物质得到了良好分离，检测灵敏度提高。在0至800 nM范围内观察到NAT的荧光强度与亚硝酸盐浓度之间呈线性关系（r = 0.999），检测限约为25 nM。
目前的高效液相色谱法似乎适用于粗生物样品中亚硝酸盐的常规分析，因为基于聚合物的色谱柱能够耐受用酸或碱进行的严格清洗。
