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建立了新鲜烟叶中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)3种磷酸腺苷同时测定的方法。烟草样品经过对氯苯酚提取、纯化后,采用三乙胺缓冲液作为流动相洗脱,经C18色谱柱分离,在259 nm波长对样品进行检测。与高氯酸(PCA)法,以及DNA常用的苯酚提取法进行了比较,结果均表明:低温下以对氯苯酚提取的效果较佳,使用三乙胺缓冲液流动相具有分离效率高、检出灵敏的优点。在线性范围(5~200μg/mL)内,磷酸腺苷浓度与峰面积具有很好的相关系数(r>0.9999),检出限达到纳克级水平,氯苯酚法对ATP,ADP和AMP的平均回收率分别为96.8%,97.2%和93.3%,RSD均小于5%。
A method for simultaneous determination of three adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in fresh tobacco leaves was established. The tobacco samples were extracted with p-chlorophenol, purified, and eluted with triethylamine buffer as the mobile phase. The samples were separated on a C18 column and detected at a wavelength of 259 nm. Compared with perchloric acid (PCA) method and phenol extraction method commonly used in DNA, the results showed that the extraction efficiency of p-chlorophenol was better at low temperature, and the mobile phase using triethylamine buffer had high separation efficiency Sensitive advantages. In the linear range (5 ~ 200μg / mL), there was a good correlation coefficient (r> 0.9999) between the concentration of adenosine triphosphate and the peak area, the detection limit reached the level of nanogram level, The average recoveries were 96.8%, 97.2% and 93.3%, respectively, with RSDs less than 5%.