高效液相色谱法测定人红细胞硫鸟嘌呤核苷酸的浓度

Quantitation Determination of 6-thioguanine Nucleotides in Red Blood Cells by HPLC

目的建立测定人红细胞（RBC）硫鸟嘌呤核苷酸（6-TGNs）浓度的高效液相色谱法。方法 RBC首先经70%高氯酸沉淀蛋白,并在酸性条件下100℃加热45 min,6-TGNs水解生成6-硫鸟嘌呤（6-TG）后进样分析。HypersilGOLD C18柱（4.6 mm×250 mm,5μm）,检测波长342 nm,柱温35℃;流动相为20 mmol.L-1磷酸二氢钾溶液（pH3.3）-乙腈（95：5,V/V）;流速：1.0 mL.min-1。结果 6-TG在8.09-809.89 pmol.（8×108）-1RBC浓度范围内线性关系良好（r=0.997 8）,最低定量浓度为8.09 pmol.（8×108）-1RBC。日内及日间RSD分别〈7.71%,6.64%,方法回收率为98.07%-108.08%,平均提取回收率〉60%。结论该法操作简便快速、灵敏,专属性强,可应用于患者服用硫唑嘌呤后RBC 6-TGNs浓度的测定。

Objective To establish a HPLC method for determination of 6-thioguanine nucleotides（6-TGNs）in red blood cells（RBC）. Methods The RBC samples were deproteinated by 70% perchloric acid.6-TGNs were hydrolyzed to 6-thioguanine（6-TG）by heating for 45 min at 100 ℃.Separation was carried out on Hypersil GOLD C18 column（4.6 mm×250 mm,5 μm）with UV detection at 342 nm.The mobile phase consisted of 20 mmol·L-1 KH2PO4（pH 3.3）-acetonitrile（95：5,V/V）with flow rate of 1.0 mL·min-1. Results The linear relationship was occurred over the range of 8.09-809.89 pmol·（8×108）-1RBC for 6-TG（r= 0.997 8）and the limit of quantitation was 8.09 pmol·（8×108）-1 RBC.The intra-and inter-assay coefficients of variation were less than 7.71% and 6.64%,respectively.The mean analytical recoveries were 98.07%～108.08%,and the mean extraction recoveries were all more than 60%. Conclusion This method is simple,rapid,sensitive and specific,which could be used for quantitation of 6-TGNs in RBC from patients under AZA therapy.