HPLC法同时测定人血浆中5-氟尿嘧啶及其活性代谢物5-氟-2′-脱氧尿嘧啶核苷的浓度

HPLC simultaneous determination of 5-fluorouracil and its metabolite 5-fluoro-2'-deoxyuridine in human plasma

目的:建立一种同时测定人体血浆中5-氟尿嘧啶及其活性代谢物5-氟-2′-脱氧尿嘧啶核苷浓度的HPLC法。方法:以肌苷为内标,血浆样品用硝酸银(10%)沉淀蛋白质,采用C_(18)柱,检测波长为266nm,流动相:0.01mol·L^(-1)磷酸盐(用2mol·L^(-1)氢氧化钠溶液调pH为7.0)-甲醇(96∶4),流速1.0mL·min^(-1),柱温为45℃。结果:5-氟尿嘧啶和5-氟-2′-脱氧尿嘧啶核苷分别在0.1～20μg·mL^(-1)(r=0.9997)和0.2～40μg·mL^(-1)(r=0.9997)浓度范围内线性关系良好,最低检测浓度分别为5和10ng·mL^(-1),方法回收率分别为98.2%～101.2%、99.5%～102.3%,日内、日间RSD均小于6%。结论:方法灵敏、快速、准确,适用于临床上测定5-氟尿嘧啶及其活性代谢物5-氟-2′-脱氧尿嘧啶核苷的血药浓度及药动学的研究。

Objective： To establish an HPLC method for the simultaneous determination of 5 - fluorouracil （5 - FU） and its active metabolite 5 - fluoro - 2＇ - deoxyuridin （FdUrd） in human plasma. Method： Proteins in plasma sam- ple were precipitated by silver nitrate（ 10% ） after addition of the internal standard inosine. After centrifugation, the supernatant was injected for the analysis by HPLC. Separation was achieved on a Zorbax - ODS C18 （4. 6 mm ×250 mm,5 μm） column at 45 ℃ ,and detected at 266 nm. The mobile phase consisted of 0. 01 mol·L^-1 sodium phosphate buffer （adjusted pH to 7.0 with 2 mol·L^-1 NaOH solution） -methanol（96： 4）at a flow rate of 1.0 mL ·min^-1. Results： The linear ranges （ n = 7 ） for the determination of 5 - FU, FdUrd were 0. 1 - 20 μg·mL^-1（ r = 0. 9997） ,0.2 - 40 μg·mL^-1 （ r = 0. 9997 ）, respectively. The detection limits of the assay were 5 and 10 ng ·mL^-1 ,respectively. The method recovery were 98.2% - 101.2% and 99.5% - 102. 3%, respectively; intra - day RSD and inter - day RSD were less than 6%. Conclusion： This method is sensitive, convenient and accurate, and is suitable for determination of 5 - FU and its active metabolite 5 - FdUrd in human plasma and phannacokinetics studies.