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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 method for the simultaneous determination of thioguanosine-1, diphosphate and triphosphate (TGMP, TGDP, TGTP) in erythrocytes (RBCs) and apply them to patients with azathioprine (AZA) Metabolite determination. METHODS: Single thioguanine nucleotides in RBC were first extracted with methylene chloride and then oxidized with potassium permanganate solution for fluorescence detection (excitation at 315 nm and emission at 390 nm). Column: Nucleosil C_ (18) column (150 mm × 4.6 mm, 5 μm); column temperature: 30 ℃; mobile phase: 20 mmol·L -1 potassium phosphate buffer ~ (-1) tetrabutylammonium hydrogen sulfate) -acetonitrile (80:20); flow rate: 1.0 ml · min -1. Results: The concentration of TGMP, TGDP and TGTP in the range of 50 ~ 500 pmol · ml -1 RBC, 50 ~ 1 000 pmol · ml -1 RBC and 100 ~ 5000 pmol · ml -1 RBC The linear relationship within the good. The lowest limit of quantitation of TGMP, TGDP and TGTP were 50, 50, 100 pmol · ml -1 RBC. The RSDs of intra-and inter-day precision of the three metabolites were <7.0%. The recoveries of the three metabolites were 95.0% ~ 103.6%. The average recoveries were both> 90%. TGTP was the main metabolite in RBC of 30 renal transplant recipients in AZA. Conclusion: The method is simple, rapid, sensitive and specific and can be applied to the determination of single thioguanine nucleotide concentration in patients undergoing renal transplantation with AZA.