高效液相色谱固相萃取法测定血浆及大肠癌细胞中5-Fu的含量

Determination of 5-Fluorouracil in plasma and colorectal cancer cell by high performance liquid chromatography with solid-phase extraction

采用高效液相固相萃取提取和分析大肠癌细胞SW480-B7和SW480-NC及血浆中5-Fu的含量,提高了萃取效率。优化色谱条件,在流动相中添加三氟乙酸改善了峰型,提高了理论塔板数。色谱柱:Diamonsil C18柱(5μm,4.6×250 mm),流动相:0.08%磷酸水溶液(含0.15%三氟乙酸)-甲醇溶液(体积比95:5),检测波长266 nm。血浆样本线性范围0.025~25μg/m L,检出限0.02μg/m L(S/N≥3),回归方程为Y=34783X+34562(R^2=0.9991);癌细胞线性范围0.03125~12.5μg/m L,最小检测浓度0.025μg/m L(S/N≥3),回归方程为Y=37963X+36842(R2=0.9993)(SW480-B7);Y=30244X+32421(R2=0.9998)(SW480-NC);所有样品回收率在86%~102%之间,日内RSD和日间RSD分别小于5%和8%。

HPLC solid-phase extraction was successfully developed for the extraction of 5-Fu and analysis of its content in plasma and colorectal cancer cell samples with improved efficiency.The peak shape and the number of theoretical plates were improved with optimized conditions by adding trifluoroacetic acid（ TFA） in mobile phase.Chromatographic conditions consisted of a Diamonsil C18 column（ 5 μm,4.6 × 250 mm） using a mixture of methanol and 0.08% phosphoric acid solution（ with 0.15% TFA）（ 5∶ 95 V/V） as mobile phase at a flow rate of 1.0μm L/min with UV detection at 266 nm.The calibration curve was linear over the concentration range of0.025 ～ 25 μg/m L and 0.03125 ～ 12.5 μg/m L with 0.02 μg/m L and 0.025 μg/m L（ S/N≥3） lowest detection limit for plasma and colorectal cancer cell,respectively.The linear equation were Y = 34783 x ＋ 34562（ R2= 0.9991） for plasma,Y = 37963 x ＋ 36842（ R2= 0.9993） for SW480- B7 and Y = 30244 x ＋ 32421（ R2=0.9998） for SW480-NC.The recoveries of all samples ranged from 86% to 102%.The intra-day and inter-day RSD were less than 5% and 8%,respectively.