啤酒酵母转化腺苷生成ATP的HPLC测定方法的研究  

Separation and Determination of AR,AMP,ADP and ATP by High Performance Liquid Chromatography

作　　者：王金涛 邱蔚然 黄凤杰[1] WANG Jin-tao;QIU Wei-ran;HUANG Feng-jie(School of Life Sciences and Technology,China Pharmaceutical University,Nanjing 211198,China;Nantong QZU Biological Technology Co.,Ltd,Nantong 226236,China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京211198 [2]南通秋之友生物科技有限公司,江苏南通226236

出　　处：《药物生物技术》2021年第2期149-156,共8页Pharmaceutical Biotechnology

摘　　要：为了建立和优化啤酒酵母转化腺苷生成三磷酸腺苷反应液中AR、AMP、ADP、ATP的分离及含量的测定方法。采用HPLC法,色谱柱:Welch Ultimate AQ-C18(250 mm×4.6 mm,5μm);流动相A:0.05 mol/L磷酸盐缓冲溶液(p H 6.8)-0.6%甲醇溶液(V/V=89∶11);流动相B:甲醇。梯度洗脱:0~15 min,A相100%;15~20 min,A相100%~85%,B相0%~15%;20~35 min,A相85%,B相15%;体积流量:1.0 m L/min;检测波长260 nm;柱温:室温;进样量:20μL;洗脱时间:35 min。AR在0.198~7.920μg、AMP在0.252 5~10.10μg、ADP在0.500~20.0μg、ATP在0.400 2~16.008μg范围内线性关系良好,且上述4个物质间的分离度大于1.5,分离效果较好。4℃保存,12 h内四组分峰面积的RSD小于2.0%,低温稳定性较好。多次进样,4组分峰面积RSD小于2.0%,精密度较好。上述成分的回收率分别为97.73%,99.04%,95.36%,99.68%,对应的RSD分别为1.50%,1.91%,1.43%,1.95%。表明建立的方法准确、灵敏、简便,可应用于啤酒酵母转化腺苷生成三磷酸腺苷反应的多组分的含量测定及反应转化率计算。The objective of this study is to establish and optimize the method used for the separation and determination of AR,AMP,ADP and ATP from the reaction solution,in which the Bear yeast is used to transform adenosine into adenosine-5’-triphosphate. The HPLC column used in the method is Welch Ultimate AQ-C18( 250 mm ×4. 6 mm,5 μm). The mobile phase is a complex mobile phase:the phase A is the combination of 0. 05 mol/L phosphate buffer solution( p H6. 8) and 0. 6% methanol solution( V/V = 89∶ 11) and the phase B is the pure methanol. The HPLC method is operated followed a gradient elution program: 0 ~ 15 min,phase A 100%;15 ~ 20 min,phase A 100% ~ 85%,phase B 0% ~ 15%;20 ~ 35 min,phase A 85%,phase B 15%. Besides,during the whole operation,the flow rate is 1. 0 m L/min;The detection wave length is 260 nm;The column temperature is at room temperature;The injection volume is 20 μL;The elution time is 35 minutes. The linear range of AR,AMP,ADP,ATP is 0. 198 ~ 7. 920 μg,0. 252 5 ~ 10. 10μg,0. 500 ~ 25. 0 μg and 0. 400 2 ~ 16. 008 μg,and the resolution between the four substances is greater than 1. 5,meant the separation effect is well;Hold the samples at 4 ℃,within 12 hours,the RSD of the peak-area of each component is under 2%,meant the sample is stabilized at low temperature;After multiple sampling,each RSD of the peak-area of the four components is less than2. 0%,meant the precision is great;The corresponding recovery is 97. 73%,99. 04%,95. 36%,99. 68%,and the corresponding RSD is 1. 50%,1. 91%,1. 43% and 1. 95%,respectively. These results showed that the method is accurate,sensitive and simple,thus it can be used for the determination of the four components in the reaction solution and the computation of conversion efficiency.

关 键 词：生物合成 腺苷 单磷酸腺苷 二磷酸腺苷 三磷酸腺苷 高效液相色谱法 梯度洗脱 

分 类 号：O657.72[理学—分析化学]