HPLC测定炎症性肠病患者红细胞中6-硫鸟嘌呤核苷酸及6-甲巯基嘌呤的浓度  

Concentration Determination of 6-TGNs and 6-MMP in Red Blood Cells of Patients with Inflammatory Bowel Disease by HPLC

作　　者：苏涌[1] 杨春兰[1] 黄燕[1] 汪燕燕[1] 宋帅 夏泉[1] 许杜娟[1,2] Su Yong;Yang Chunlan;Huang Yan;Wang Yanyan;Song Shuai;Xia Quan;Xu Dujuan(Department of Pharmacy,the First Affiliated Hospital of Anhui Medical University,Third-grade Pharmaceutical Chemistry Laboratory of State Administration of Traditional Chinese Medicine,Hefei 230022,China;Fuyang Hospital of Anhui Medical University)

机构地区：[1]安徽医科大学第一附属医院药剂科国家中医药管理局中药化学三级实验室,合肥230022 [2]安徽医科大学附属阜阳医院

出　　处：《中国药师》2021年第1期194-198,共5页China Pharmacist

基　　金：安徽省自然科学基金青年基金项目(编号:1508085QH166)。

摘　　要：目的:建立HPLC法同时测定炎症性肠病(IBD)患者红细胞中硫唑嘌呤代谢物6-硫鸟嘌呤核苷酸(6-TGNs)及6-甲巯基嘌呤(6-MMP)浓度,为硫唑嘌呤(AZA)个体化治疗提供依据。方法:红细胞经高氯酸沉淀,6-TGNs在酸性条件下加热水解生成6-硫鸟嘌呤(6-TG),6-MMP水解生成4-氨基-5-(甲硫基)羰基咪唑(AMTCI),以5-溴尿嘧啶(5-BU)为内标,采用HPLC法测定其含量。色谱柱:Kromasil 100-5-C18(150 mm×4.6 mm,5μm),流动相:乙腈-20 mmol·L1磷酸二氢钾溶液(6∶94),流速:0.8 ml·min^(-1),柱温:30℃;6-TG、6-MMP及5-BU检测波长分别为340,303,280 nm。10例IBD患者,口服AZA 50~100 mg·d-11个月以上,检测其6-TGNs及6-MMP浓度,焦磷酸测序检测TPMT基因型。结果:6-TG在8~800pmol﹒(8×108)-1RBC浓度范围内线性关系良好(r=0.994 0);低、中、高3个浓度的绝对回收率为63.24%~69.09%(n=5)。AMTCI在250~16 000 pmol·(8×108)-1RBC浓度范围内线性关系良好(r=0.997 8);低、中、高3个浓度的绝对回收率为85.17%~92.28%(n=5)。10例患者TPMT均为野生型。1例患者6-TGNs浓度偏高,白细胞减少;2例患者6-MMP浓度较高,碱性磷酸酶轻度升高。结论:本方法专属性、精密度及稳定性较好,灵敏度高,适用于同时测定炎症性肠病患者红细胞中6-TGNs及6-MMP的浓度。Objective:To establish an HPLC method for determining the concentrations of 6-thioguanine nucleotides (6-TGNs)and 6-methylmercaptopurine (6-MMP) in patients with inflammatory bowel disease (IBD) to improve azathioprine (AZA) individualized treatment.Methods:The red blood cells were deproteinated by perchloric acid,6-TGNs was hydrolyzed to 6-thioguanine (6-TG)and 6-MMP was hydrolyzed to 4-amino-5-(methylthio) carbonyl imidazole (AMTCI) by heating under acidic conditions.5-Bromouracil(5-BU) was selected as the internal standard.A Kromasil 100-5-C18(150 mm×4.6 mm,5μm) column was used and the flow rate was 0.8 ml·min^(-1).The mobile phase consisted of acetonitrile and 20 mmol·L^(-1)potassium dihydrogen phosphate (6∶94).The column temperature was 30℃and the detection wavelength was 340 nm,303 nm and 280 nm for 6-TG,6-MMP and 5-BU,respectively.Totally 10 patients with IBD were orally given AZA 50-100 mg·d-1for more than one month,and their 6-TGNs and 6-MMP concentrations were detected.The TPMT genotype was detected by pyrosequencing.Results:The linear relationship range was 8-800 pmol·(8×108)-1RBC for 6-TG (r=0.994 0).The absolute recovery was between 63.24%and 69.09%(n=5).The linear relationship range was 250-16 000 pmol·(8×108)-1RBC for AMTCI (r=0.997 8).The absolute recovery was between 85.17%and 92.28%(n=5).The TPMT of all the 10 patients was wild type.One patient had leukopenia with high 6-TGNs concentration,and two patients had mildly elevated alkaline phosphatase with high 6-MMP concentration.Conclusion:The method is specific,accurate,stable and sensitive,which is suitable for the determination of 6-TGNs and 6-MMP concentrations in red blood cells of patients with IBD.

关 键 词：硫唑嘌呤 6-硫鸟嘌呤核苷酸 6-甲巯基嘌呤 高效液相色谱法 治疗药物监测 

分 类 号：R969.1[医药卫生—药理学]