离子对高效液相色谱法测定人体红细胞内核苷二磷酸激酶活性  

Determination of the activity of nucleoside diphosphate kinase in human red blood cell by reversed phase ion-pair HPLC

作　　者：沈如飞[1,2] 刘亚妮[1] 曾繁典[3] 师少军[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院药剂科,湖北武汉430022 [2]华中科技大学同济医学院2006级8年制 [3]华中科技大学同济医学院临床药理研究所,湖北武汉430030

出　　处：《中国医院药学杂志》2012年第4期252-255,共4页Chinese Journal of Hospital Pharmacy

基　　金：湖北省自然科学基金资助项目(编号:2009CDB380);中央高校基本科研业务费专项资金资助(编号:2011JC039)

摘　　要：目的:建立测定人体红细胞(RBC)内核苷二磷酸激酶(NDPK)活性的反相离子对高效液相色谱法。方法:稀释RBC中加入反应底物(dADP和dGTP)及其他反应混合液后,37℃孵育5 min,100℃灭活10 min,离心取上清液进样分析。以反应产物dATP定量分析NDPK活性。色谱柱为SymmetryShield TMRP18(150 mm×3.9 mm,5μm),检测波长为260 nm,柱温为25℃;采用梯度洗脱,流动相A为20 mmol.L-1 KH2PO4-K2HPO4缓冲液、加入离子对试剂5 mmol.L-1四丁基硫酸氢铵(TBAHS)溶液,流动相B为乙腈。结果:NDPK催化反应dADP+dGTPdATP+dGDP反应特异性良好,RBC内dATP的线性范围为4.33～43.34μmol.L-1(r2=1.000),定量下限为4.33μmol.L-1。日内及日间RSD分别小于5.53%和6.69%,准确度为98.4%～104.2%,平均绝对回收率大于83%;dATP样品在室温或4℃、-80℃保存30 d及反复冻融3次稳定性良好。结论:该法操作简便快速、灵敏、专属性强,可应用于人体RBC内NDPK活性测定,为研究NDPK活性与嘌呤类药物反应相关性提供基础。OBJECHVE To establish a reversed phase ion-pair HPLC method for the determination of the activity of nucleo- side diphosphate kinase （NDPK） in human red blood cells （RBC）. METHODS RBC with reaction substrates （dADP and dGTP） and other reaction mixtures were incubated at 37℃ for 5 min, then inactivated by heating at 100℃ for 10 min, and cen- trifuged with supernatant sample for analysis, dATP concentrations were quantified for the determination of the activity of ND- PK in human RBC. Separation was achieved by SymmetryShield TM RP18 （150 mm× 3.9 ram,5μm） with UV detection at 261） nm and column temperature of 25℃. Using gradient elution, the mobile phase A consisted of mixture of 20 mmol· L^-1 KH2 PO4 K2 HPO4 buffer solution and ion-pairing reagent 5 mmol. L ^-1TBAHS, and mobile phase B was acetonitrile. RESULTS NDPK could catalyze ADP ＋ dGTP←→dATP ＋ dGDP reaction specifically. The calibration curve was linear over the range of 4. 33 43.34 μmol.L^-1 （r2 = 1. 001）） and the limit of quantitation was 4. 33 μmol.L^-1. The intra- and inter-assay coefficients of varia- tion were less than 5.53% and 6. 69%, respectively. The mean analytical recoveries of dATP were 98. 4%- 104. 2% and the mean absolute recoveries were all more than 83 0%. A stability study showed that dATP samples in RBC were stable at room temperature or at 4℃ for at least 24 hours,as well as for at least 30 days at - 80℃ after three freeze-thaw cycles. CONCLU- SION This method is simple,rapid,sensitive, specific,and can be applied to study the relationship between activity of NDPK and efficacy and toxicity of thiopurines.

关 键 词：核苷二磷酸激酶 活性 红细胞 反相离子对高效液相色谱法 

分 类 号：R927.2[医药卫生—药学]