超快速液相色谱法测定大鼠肝细胞中腺苷酸含量  

Determination of adenine nucleotides in rat hepatocytes by ultra fast liquid chromatography

作　　者：贾鹏禹[1] 孙蕊[1] 李良玉[1] 韩毅强[1] 周行 冯乃杰[1] JIA Peng-yu;SUN Rui;LI Liang-yu;HAN Yi-qiang;ZHOU Hang;FENG Nai-jie(Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学

出　　处：《中国生物制品学杂志》2019年第10期1138-1142,共5页Chinese Journal of Biologicals

基　　金：黑龙江省普通本科高等学校青年创新人才培养计划(UNPYSCT-2018086);大庆市指导性科技计划(zd-2017-34);黑龙江八一农垦大学“校内培育课题资助计划”(XZR2015-15)

摘　　要：目的建立大鼠肝细胞中3种腺苷酸[三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和一磷酸腺苷(AMP)]的超快速液相色谱测定法。方法制备3种腺苷酸混合标准工作液,同时将BRL大鼠肝细胞样品经超声波破碎仪处理提取目标化合物。液相色谱条件:色谱柱为Poroshell 120 SB-AQ C18(100 mm×3.0 mm,2.7μm),流动相为20 mmol/L磷酸氢二钠+20 mmol/L磷酸二氢钠缓冲溶液,流速为0.20 mL/min,柱箱温度为30℃,二极管阵列检测器光谱扫描范围为190~800 nm,定量波长为259 nm,进样量为5μL。同时对方法的线性范围、稳定性、重复性及准确性进行验证。结果色谱柱对标准品和待检样品的3种腺苷酸组分分离状况良好,样品目标峰位无杂峰干扰,色谱分离在15 min内完成。各目标组分在10~100μg/mL浓度范围内与峰面积呈良好的线性关系,检测限在0.652~0.793μg/mL之间;样品提取液于不同时间点进样所得ATP、ADP和AMP的峰面积RSD分别为2.06%、1.15%和1.26%;6份平行样品在同一检测流程下所得的ATP、ADP和AMP峰面积的RSD分别为2.13%、2.04%和1.89%;样品各组分平均回收率在97.05%~102.58%之间,RSD在2.16%~2.55%之间。结论建立了大鼠肝细胞中3种腺苷酸含量的超快速液相色谱测定法,且具有良好的线性、稳定性、重复性及准确性。Objective To establish an ultra fast liquid chromatography method for simultaneous determination of 3 kinds of adenine nucleotides(ATP,ADP and AMP)in rat hepatocytes.Methods Live cell samples from BRL rats were pretreated by ultrasonic-assisted fragmentation with perchloric acid solution for extracting target compounds.Separation was performed on a Poroshell 120 SB-AQ C18 column(100 mm×3.0 mm ID,2.7μm)with sodium phosphate buffer solution(20 mmol/L disodium hydrogen phosphate and 20 mmol/L monosodium orthophosphate)as mobile phase at a flow rate of 0.20 mL/min,in a column case at 30℃.The sample load was 5μL.The scanning spectrum of the diode array detection was set between 190~800 nm,and the quantitative wavelength of PDA was set at 259 nm.The linear range,stability and repeatability were also determined.Results The separation of 3 adenine nucleotides from both standard and test samples was successful,with no interference to target peaks.The separation could be completed within 15 min,with a linear range of 10~100μg/mL.The detection limit was in the range of 0.652~0.793μg/mL.The relative standard deviation(RSD)values for peaks of ATP,ADP,or AMP from different sampling times were 2.06%,1.15%and 1.26%respectively,and were 2.13%,2.04%and 1.89%respectively for 6 parallel repeats.The recovery rates were between 97.05%~102.58%,with RSD values between 2.16%~2.55%.Conclusion A protocol to detect 3 adenine nucleotides in rat hepatocytes through UFLC was developed,with good linearity,stability,repeatability and accuracy.

关 键 词：超快速液相色谱法 腺苷酸 肝细胞 大鼠 

分 类 号：S852.61[农业科学—基础兽医学]