高效液相色谱法测定人红细胞中6-甲基巯基嘌呤核糖核苷酸的浓度

Determination of the concentrations of 6-methylmercaptopurine ribonucleotides(6-MMPR) in human red blood cells by HPLC

目的：建立测定人红细胞6-甲基巯基嘌呤核糖核苷酸（6-MMPR）浓度的高效液相色谱法。方法：红细胞首先经70％高氯酸沉淀蛋白，并在酸性条件下100℃加热45min，6-MMPR水解生成6-甲基巯嘌呤（6MMP）后进样分析。以6-MMP定量分析6MMPR浓度。色谱柱为HypersilGOLDC1H柱（4．6mm×250mm，5gm），检测波长290nm，柱温35℃；流动相为0．02mol·L“磷酸钾缓冲液（pH3．3）乙腈（95：5）；流速0．9mL.min-1.结果：红细胞中6-MMP在80．91～3236．36pmol.（8×10-8）-1。红细胞浓度范围内线性关系良好（r2=0．9985），最低定量浓度为80．91pmol·（8×10^8）-1。红细胞。日内及日间RSD分别小于2．09％，5．61％，方法回收率为100．27％～110．79％，平均提取回收率大于70％。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红细胞6一MMPR浓度的测定。

OBJECTIVE To establish an HPLC method for the determination of 6-methylmercaptopurine ribonucleotides （6- MMPR） in human red blood cells （RBC）. METHODS The RBC samples were deproteinated by 70% perehloric acid. 6-MM- PR were hydrolyzed to 6-methylmercaptopurine （6 MMP） by heating the sample for 45 rain at 100 ℃. Separation was achieved by Hypersil GOLD Clscolumn （4. 6 mm× 250 mm, 5 ptm） with UV detection at 290 nm. The mobile phase consisted of 0. 02 mol.L-1 potassium phosphate buffer （pH 3. 3） ： acetonitrile （95：5）. RESULTS The calibration curve was linear over the range of 80. 91 -3 236. 36 pmol- （8 × 10^8 ） -1 RBC 02 = 0. 998 5） and the limit of quantitation was 80. 91 pmol. （8 × 10^8 ）- 1 RBC; The intra-and inter-day coefficients of variation were less than 2. 09% and 5.61%, respectively. The mean analytical recoveries were 100. 27% - 110. 79%, and the mean extraction recoveries were more than 70%. CONCLUSION This method is simple, rapid, sensitive and specific, and it enables quantitation of 6-MMPR in RBC from patients under AZA therapy.