高效液相色谱法测定人红细胞6-硫鸟嘌呤核苷酸和6-甲基巯基嘌呤核苷酸的浓度

Simultaneously determination of 6- thioguanine nucleotides and 6- methlymercaptopurine nucleotides concentrations in human red blood cells by HPLC

目的建立同时测定人红细胞内硫嘌呤药物活性毒性代谢产物6-硫鸟嘌呤核苷酸(6-TGNs)和6-甲基巯基嘌呤(6-MMP)浓度的高效液相色谱法(HPLC)。方法色谱柱:Waters Xterra MS C18,流动相:甲醇-20 mmol·L^(-1)磷酸二氢钾溶液-三乙胺(磷酸调节pH=3.2)=5∶95∶0.1,流速:1.0 m L·min^(-1),柱温:25℃,6-TGNs和6-MMP检测波长分别为348,303 nm。考察该方法的专属性、标准曲线和定量下限、精密度与回收率、稳定性。结果红细胞中6-TGNs在1.25×10^(-2)~1.25μg·m L^(-1)内线性关系良好(r=0.999 6),6-MMP在0.04~5.00μg·m L^(-1)内线性关系良好(r=0.999 4)。6-TGNs和6-MMP定量下限分别为1.25×10^(-2),0.04μg·m L^(-1)。6-TGNs和6-MMP日内及日间精密度(RSD)均≤8.9%,6-TGNs回收率为60.99%~72.04%,6-MMP为85.62%~95.13%。在稳定性考察中RSD值均≤6.08%。结论本方法简单快速、准确、灵敏、专属性强和稳定性好,适用于服用硫嘌呤类药物治疗的炎症性肠病患者血药浓度监测。

Objective To establish a high performance liquid chroma- tography （HPLC） method for determining thiopurine active metabolites 6 - thioguanine nucleotides （ 6 - TGNs） and 6 - methlymercaptopurine （6-MMP） simultaneously in red blood cells （RBC） of human. Methods The separation was performed on a Waters Xterra MS C18 colum with mobile phase consisted of methanol -20 mmol × L-1 potas- sium dihydrogenphosphate -triethylamine （pH adjusted to 3.2 using phosphoric acid） （ 5 ： 95 ： 0. 1 ） with the flow rate of 1.0 mL ~ min - 1 The column temperature was 25℃. The detection wavelength was set at 348 nm for 6 - TGNs and 303 nm for 6 - MMP. Results The linear relationship range was 1.25 × 10 -2 - 1.25μg · mL-1 for 6 - TGNs （r =0. 999 6） and 0. 04 -5.00 μg · mL-1 for 6 - MMP （r =0. 999 4）. The lowest limit of quantification was 1.25 ×10-2μg · mL-1 for 6 - TGNs and 0. 04μg · mL-1 for 6 - MMP. The RSDs of intra - day and inter - day of 6 - TGNs and 6 - MMP were less than 8. 9%. The extraction recoveries were 60. 99% - 72. 04% for 6 - TGNs, 85.62% - 95. 13% for 6 - MMP. The RSDs of 6 - TGNs and 6 - MMP stability were less than 6. 08%. Conclusion The method is simple, rapid, sensitive, specific and stable, which could be applied to monitor thiopurine metabolites concentrations in red blood cells from inflammatory bowel disease and provide reference for personal medicine.