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

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.