HPLC法间接测定肿瘤患者外周血DPD的活性

目的 建立高效液相色谱法(HPLC)间接测定肿瘤患者外周血二氢尿嘧啶脱氢酶(DPD)活性的方法.方法 采用HPLC法测定肿瘤患者血浆中内源性物质二氢尿嘧啶(H2U)和尿嘧啶(U)的比值,间接估算DPD的活性.色谱柱:Xterra RPC18 (250 mm×4.6 mm,5μm)；流动相:甲醇-0.02 mol/L磷酸二氢钠(pH4.0),比例为5∶95;流速为1.0 mL/min,H2U和U检测波长分别为204 nm、260 nm.结果 H2U线性范围为62.5 ～2 000μg/L,线性方程为:Y=7.48×10 -4X+ 1.24×10-1,r=0.999 7(n=6),批内RSD小于7.7％,批间RSD小于13.8％,方法学回收率为99.63％ ～ 103.02％;U线性范围为15.625 - 500 μg/L,线性方程为:Y=3.42×10-3X+2.58×10-2,r=0.999 3(n=6),批内RSD小于8.3％,批间RSD小于13.4％,方法学回收率为98.38％～109.08％.结论 本方法可用于肿瘤患者外周血DPD活性的常规监测.

Determination of Dihydropyrimidine Dehydrogenase Activity in Blood of Cancer Patients by HPLC

Objective To establish an HPLC method for determining dihydropyrimidine dehydrogenase activity in blood of cancer patients.Methods We determined the ratio of dihydrouracil(H2U)and uracil(U) in blood of patients with cancer by HPLC,and predicted the activity of dihydropyrimidine dehydrogenase(DPD) indirectly.The liquid chromatography column was Xterra RPC18(250 mm×4.6 mm,5 μm);the mobile phase was composed of 0.02 mol/L phosphate buffer(pH 4.0)and methanol(95:5)with a flow rate of 1.0 mL/min.The detection wavelength for H2U was 204 nm,and for U was 260 nm.Results A linearity was obtained from 62.5 to 2 000 μg/L for H2U.The calibration equation of H2U was Y=7.48×10-4X+1.24×10-1,r=0.999 7(n=6).The RSD within day and between days were less than 7.7 % and 13.8 % for H2U.The recoveries of H2U were 99.63%～103.02%.A linearity was obtained from 15.625 to 500 μg/L for U.The calibration equation of U was Y=3.42×10-3X+2.58×10-2,r=0.9993(n=6).The RSD within day and between days were less than 8.3% and 13.4% for U.The recoveries of U were 98.38%～109.08%.Conclusion This method can be used for routine clinical determination of DPD activity in blood of cancer patients.