苯酚提取-超高效液相色谱法测定烟草中的磷酸腺苷

摘 要: 建立了苯酚提取超高效液相色谱法（UPLC）同时测定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种磷酸腺苷的分析方法。苯酚提取样品溶液中的ATP、ADP和AMP较稳定，不易水解，样品在2 h以内各磷酸腺苷变化率小于10 %。采用6 ‰三乙胺（pH=6.6）和甲醇作为流动相梯度洗脱，3种磷酸腺苷在10分钟内实现了较好的分离，线性范围（5～200）μg/mL内，磷酸腺苷浓度与峰面积具有很好的相关系数（r>0.9999），3种磷酸腺苷检测限分别为0.5 μg/mL、0.4 μg/mL、0.3 μg/mL，回收率在91.68 %～110.79 %之间，相对标准偏差RSD在1.53 %～3.67 %之间。该方法可快速、准确检测烟草样品中ATP含量，进而为研究烟草能量代谢尤其不育系的产生机理，提供重要的检测指标。 

Measurement of adenosine phosphate in Phenol-TEA Pretreatment tobacco by UPLC

Abstract:The simultaneous analysis method of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in fresh tobacco leaves was established by ultra high performance liquid chromatography (UPLC) with phenol extraction. ATP, ADP and AMP in solution extracted with phenol were stable and hard to be hydrolyzed, its value changed by less than 10 % within 2 h. A better separation of ATP, ADP and AMP was achieved with the linear range (5~200) μg.mL-1 in 10 minutes with a gradient elution system of 6 ‰ triethylamine (pH = 6.6)-methanol as mobile phase, the adenosine concentration and peak area has a good correlation coefficient (r> 0.9999). The detection limits for ATP, ADP and AMP was 0.5 μg.mL-1, 0.4 μg.mL-1, 0.3 μg.mL-1 respectively, the average recovery were 91.68 % ~ 110.79 %, RSD were 1.53 %~3.67 %. This method can quickly and accurately detect ATP content of tobacco samples, and thus for the study of energy metabolism, especially tobacco CMS generation mechanism, providing important test indicators.