HPLC-荧光检测法同时测定单个硫鸟嘌呤核苷酸浓度及临床应用  

Simultaneous Determination and Clinical Application of Individual Thioguanine Nucleotides in Red Blood Cells by an HPLC-fluorescence Method

作　　者：万景[1] 杨春晓[1] 周嘉黎[1] 刘亚妮[1] 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,武汉430022

出　　处：《中国药师》2017年第7期1185-1187,1217,共4页China Pharmacist

基　　金：湖北省自然科学基金项目(编号:2009CDB380)

摘　　要：目的:建立同时测定红细胞(RBC)中硫鸟嘌呤核苷一、二和三磷酸(TGMP,TGDP,TGTP)浓度的HPLC-荧光检测法,并应用于口服硫唑嘌呤(AZA)肾移植患者体内代谢物测定。方法:RBC中单个硫鸟嘌呤核苷酸首先经二氯甲烷提取,随后加入高锰酸钾溶液进行氧化后荧光检测(激发波长315 nm;发射波长390 nm)。色谱柱:Nucleosil C_(18)柱(150 mm×4.6 mm,5μm);柱温:30℃;流动相:20 mmol·L^(-1)磷酸钾缓冲液(pH 6.8,加入5 mmol·L^(-1)四丁基硫酸氢铵)-乙腈(80∶20);流速:1.0 ml·min^(-1)。结果:TGMP,TGDP和TGTP在50~500 pmol·ml^(-1)RBC,50~1 000 pmol·ml^(-1)RBC和100~5 000 pmol·ml^(-1)RBC浓度范围内线性关系良好。TGMP、TGDP、TGTP最低定量限分别为50,50,100 pmol·ml^(-1)RBC。3种代谢物的日内及日间精密度RSD均<7.0%,方法回收率为95.0%~103.6%,平均提取回收率均>90%。服用AZA的30名肾移植患者RBC内TGTP为主要代谢物。结论:该法操作简便、快速、灵敏、专属性强,可应用于AZA治疗肾移植患者体内单个硫鸟嘌呤核苷酸浓度的测定。Objective： To establish an HPLC-fluorescence detection method for the determination of thioguanosine-monophosphate （TGMP）, thioguanosine-diphosphate （TGDP） and thioguanosine-triphosphate （TGTP） in red blood cells （RBC）, as well as quantify the individual thioguanine nucleotides metabolites in kidney transplant recipients with azathioprine （AZA） therapy. Methods： The in- dividual thioguanosine phosphates were extracted from RBC by dichloromethane and subsequently oxidized by potassium permanganate. The separation was achieved on a Nucleosil Clscolumn （150 mm × 4.6 mm,5 μm） with an ion pairing reagent and detected by a fluorescence detector （excitation at 315 nm, emission at 390 nm）. The mobile phase consisted of 20 mmol·L^-1 potassium phosphate buffer （ pH was adjusted to 6.8 by 5 mmol·L^-1 tetrabutylammonium hydrogensulfate） -acetonitril （ 80：20） with the flow rate of 1.0 ml·min^-1. Results： TGMP, TGDP and TGTP were quantified from RBC within the range of 50-500, 50-1000 and 100-5 000 pmol·ml-1, respectively. The limit of quantification （LOQ） was 50, 50 and 100 pmol·ml-1 RBC for TGMP, TGDP and TGTP, respectively. The intra- and inter-day RSDs were below 7.0% with the method recovery between 95.0% and 103.6%. The mean extraction recovery was above 90%. The assay was applied in the blood samples of 30 kidney transplant recipients with AZA therapy, and the resuits indicated that TGTP was the predominant phosphate metabolite in RBC. Conclusion： The method is simple, rapid, sensitive and specific, and it can quantitatively determine the individual thioguanosine phosphates in RBC of kidney transplant recipients with AZA therapy.

关 键 词：硫唑嘌呤 肾移植患者 硫鸟嘌呤核苷一、二和三磷酸 高效液相色谱法 荧光检测 

分 类 号：R969.1[医药卫生—药理学]