佟志强 吴东媛 张娟 程梦飞 张硕 董梅

摘 要 目的：建立同時测定人血浆中5-氟尿嘧啶（5-FU）及其代谢产物5-氟-5，6-二氢尿嘧啶（5-FUH2）浓度的方法，并应用于临床。方法：血浆样品经乙酸乙酯二次萃取处理后，以5-溴尿嘧啶（5-Bru）为内标，采用超高效液相色谱-串联质谱技术（UPLC-MS/MS）测定。色谱柱为Acquity UPLC HSS T3，流动相为甲醇-水（30 ∶ 70，V/V），流速为0.3 mL/min，柱温为20 ℃，进样量为5 μL。采用电喷雾离子源，以多反应监测进行负离子扫描，毛细管电压为1.5 kV，锥孔电压为20 V，脱溶剂温度为450 ℃，脱溶剂气流为850 L/h，锥孔气流速为50 L/h;用于定量分析的离子对分别为m/z 129.00→41.90（5-FU）、m/z 130.87→82.92（5-FUH2）、m/z 189.00→42.10（5-Bru）。选择2020年8-10月哈尔滨医科大学附属肿瘤医院接受5-FU持续静脉滴注46 h为基础化疗方案治疗的晚期结直肠癌患者10例，测定其持续静脉滴注18～30 h时的5-FU稳态血药浓度及5-FUH2血药浓度，计算5-FU药-时曲线下面积（AUC）和5-FUH2/5-FU浓度比值。结果：5-FU、5-FUH2检测质量浓度的线性范围均为20～1 000 ng/mL（R 2均大于0.990），定量下限均为20 ng/mL;精密度试验的RSD均小于20%，相对误差在±10%范围内;提取回收率、基质效应均不影响待测物的测定。10例晚期结直肠癌患者中，5-FU稳态血药浓度为180.04～622.83 ng/mL，AUC为8.28～28.65 mg·h/L;5-FUH2血药浓度为336.48～948.43 ng/mL;5-FUH2/5-FU浓度比值为0.93～4.21;10例患者的5-FU AUC存在约3～4倍的个体差异。结论：所建方法精密度、准确性均较好，灵敏度较高且操作简便，可用于晚期结直肠癌患者体内5-FU血药浓度的监测。

关键词 超高效液相色谱-串联质谱法;5-氟尿嘧啶;5-氟-5，6-二氢尿嘧啶;血药浓度

ABSTRACT   OBJECTIVE： To establish the method for simultaneous determination of 5-fluorouracil （5-FU） and its metabolites 5-fluoro-5，6-dihydrouracil （5-FUH2） in human plasma， and apply it in the clinic. METHODS： After plasma samples were processed twice by ethyl acetate， UPLC-MS/MS method was adopted using 5-bromouracil （5-Bru） as internal standard. The determination was performed on Acquity UPLC HSS T3 column with mobile phase consisted of methanol-water （30 ∶ 70， V/V） at the flow rate of 0.3 mL/min. The column temperature was 20 ℃， and sample size was 5 μL. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring. The capillary voltage was 1.5 kV; the taper hole voltage was 20 V; the desolvent temperature was 450 ℃; the desolvent air flow was 850 L/h; the cone hole gas velocity was 50 L/h. The ion transitions for quantitative analysis were m/z 129.00→41.90（5-FU），m/z 130.87→82.92（5-FUH2）， m/z 189.00→42.10（5-Bru）， respectively.  From Aug. to Oct. 2020， 10 patients with advanced colorectal cancer were treated with continuous intravenous drip of 5-FU for 46 hours were collected from Harbin Medicinal University Cancer Hospital. Steady-state plasma concentration of 5-FU and plasma concentration of 5-FUH2 were determined at 18-30 h of continuous intravenous drip. The area under the curve （AUC） for 5-FU and concentration ratio of 5-FUH2/5-FU were calculated. RESULTS： The linear range of 5-FU and 5-FUH2 were 20 to 1 000  μg/mL （R 2>0.990）. The quantification limits were 20 ng/mL. RSDs of precision test were all lower than 20%， and relative error ranged ±10%. The extraction recovery and matrix effects didnt affect the determination of substance to be measured. Among 10 patients with advanced colorectal cancer， the steady-state concentration of 5-FU were 180.04-622.83 ng/mL， and AUC of 5-FU ranged from 8.28 to 28.65 mg·h/L. The concentration of 5-FUH2 ranged 336.48-948.43 ng/mL， and concentration ratio of 5-FUH2/5-FU ranged 0.93-4.21. AUC of 5-FU in 10 patients had about 3-4 fold individual differences. CONCLUSIONS： The established method has good precision and accuracy， high sensitivity， and simple operation. It can be used for plasma monitoring of 5-FU in patients with advanced colorectal cancer.

KEYWORDS   UPLC-MS/MS; 5-FU; 5-FUH2; Plasma concentration

氟尿嘧啶（5-fluorouracil，5-FU）可同时抑制肿瘤细胞DNA和RNA的合成，是晚期结直肠癌患者化疗的基石[1-3]。其作为抗代谢类细胞毒性药物，治疗窗相对较窄，能在浓度暴露水平重叠的情况下发挥疗效并产生毒性[4]。在……