HPLC法测定炎症性肠病患者红细胞中硫鸟嘌呤核苷酸浓度

摘要

OBJECTIVE:To establish a method for determining the concentrations of 6-thioguanine nucleotides (6-TGNs) in RBC,and to provide reference for improving clinical efficacy and reducing adverse drugs reaction.METHODS:The red blood cell samples were deproteinated by perchloric acid,6-TGNs were hydrolyzed to 6-thioguanine (6-TG) by heating under acidic conditions.HPLC method was adopted.The separation was performed on a Hypersil ODS2 colum with mobile phase consisted of acetonitrile-20 mmol/L potassium dihydrogen phosphate (pH adjusted to 3.3 using phosphoric acid)(5:95) at the flow rate of 1.0 ml/min.The detection wavelength was set at 340 nm and column temperture was 30 ℃.RESULTS:The linear relationship range was 7.87-787.40 pmol/(8×108) RBC for 6-TG (r=0.999 7); the RSDs of intra-day and inter-day were between 0.46％ and 7.64％;mean recoveries were 99.10％-102.07 ％.CONCLUSIONS:The method is accurate,specific,stable and sensitive,which could be applied to monitor 6-TGNs concentration in red blood cells from patients under azathioprine or 6-mercaptoparine therapy.%目的:测定硫唑嘌呤与6-巯基嘌呤的活性代谢产物硫鸟嘌呤核苷酸(6-TGNs)在红细胞内的浓度,为提高临床疗效和降低副作用提供参考.方法:红细胞稀释液经高氯酸沉淀,6-TGNs在酸性条件下加热水解生成6-硫鸟嘌呤(6-TG),采用高效液相色谱法测定其含量.色谱柱为Hypersil ODS2,流动相为乙腈-20 mmol/L磷酸二氢钾溶液(磷酸调节pH 3.3)(5:95)；流速为1.0 ml/min;柱温为30℃；检测波长为340 nm,以外标法定量.结果:6-TGNs在上述条件下水解生成6-TG,在7.87～787.40 pmol/(8×108)红细胞浓度范围内线性关系良好(r=0.999 7)；日内及日间RSD在0.46％～7.64％之间；平均方法回收率在99.10％～102.07％之间.结论:本方法准确,专属性和稳定性较好,灵敏度高,适用于接受硫唑嘌呤或6-巯基嘌呤治疗的炎症性肠病患者红细胞中6-TGNs浓度监测,可为临床在实施个体化治疗时提供参考.