液相色谱-串联质谱法快速同时测定人血浆中卡培他滨及其活性代谢物

A Rapid and Simultaneous Determination of Capecitabine and Its Activated Metabolites in Human Plasma by LC-MS/MS

目的建立一种简单、快速、同时检测人血浆中卡培他滨及其活性代谢产物氟尿嘧啶(5-FU)浓度的液相色谱-串联质谱(LC-MS/MS)的方法,并用于临床实践。方法取血浆样本100μL,以乙腈沉淀蛋白后,高速离心后取上清液直接进样分析,以乙腈-25 mmol·L^(-1)乙酸铵溶液(含0.1%甲酸)(95:5)为流动相,通过Hilic硅胶柱进行液相分离,采用ESI负离子源以及多离子反应监测(MRM)对血浆中卡培他滨及5-FU进行定量分析,卡培他滨及5-FU离子对分别为m/z 358.1→154.1,m/z 129→42.1。接受卡培他滨化疗患者20例,监测血浆中卡培他滨及5-FU稳态谷浓度,并结合卡培他滨及5-FU单点浓度的相关性评价该检测方法的临床应用性。结果卡培他滨及5-FU的线性范围均为39.062 5～10 000 ng·mL^(-1)(R^2=0.999),最低定量限为39.062 5 ng·mL^(-1)。卡培他滨在低(78.125 ng·mL^(-1))、中(625 ng·mL^(-1))、高(5000 ng·mL^(-1))3种质控浓度的提取回收率分别为(78.22±3.15)%,(75.37±2.99)%及(81.05±2.78)%,5-FU低、中、高3种浓度的提取回收率分别为(70.45±2.19)%,(72.56±1.68)%及(72.02±1.72)%。日间和日内精密度差异均小于5%,各浓度间基质效应无显著差异。20例临床患者标本检测结果表明卡培他滨及其活性产物5-FU的血药浓度存在个体差异,且卡培他滨与其对应活性产物血药浓度间相关性较差。结论该实验建立测定卡培他滨及5-FU的方法灵敏度高,专属性强,样品处理过程简单快捷,适用于同时检测血浆中卡培他滨及5-FU的浓度,应用于临床以提高卡培他滨相关化疗方案的有效性和安全性。

Objective To develop a simple and rapid method for simultaneous quantification of capecitabine and its active metabolite(5-fluorouracil )in human plasma by liquid chromatography- tandem mass spectrometry (LC-MS/MS) and apply it to clinical practice. Methods After a protein precipitation using acetonitrile, the supernatant taken from the centrifugal plasma samples was analyzed directly. With a mobile phase comprising acetonitrile/0.1% aqueous formicacid and25 mmol·L^-1 ammonium acetate (95 : 5) and an electrospray ion (ESI) source, samples contained capecitabine and 5-fluorouracil were quantitative analyzed. The MS/MS detection was performed using multiple reaction monitoring (MRM), in positive mode. The mass transitions of the protonated precursor/product ion pairs were used to record the selected ion mass chromatograms of capecitabine and 5-fluorouracil were m/z358.1→154.1 and m/z129→42.1, respectively.20 patients were receiving chemotherapy with capecitabine were then the steady state trough concentration of capecitabine and 5-fluorouracil in the patients’ plasma were monitored. The clinical application of the detection method was evaluated through the correlation of single point concentration of capecitabine and 5-fluorouracil. Results The linear calibration curve was obtained in the concentration range from39.062 5-10 000 ng·mL^-1 ( R2 =0.999)for both capecitabine and 5-fluorouracil with the limit of quantification was39.062 5 ng·mL^-1 . The extraction recoveries of capecitabine at three concentration levels(78.125,625 and 5000 ng·mL^-1 ) and that of the 5-fluorouracil were (78.22±3.15)%,(75.37±2.99)%,(81.05±2.78)% and (70.45±2.19)%,(72.56±1.68)%,(72.02±1.72 )%, respectively. The intra-and inter-run precisions,expressed as the relative standard deviation(RSD)were less than 5% for both of capecitabine and 5-fluorouracil. The detection result acquired from the plasma samples indicated that there was differences for the plasma concentration of capecitabine and 5-fluorouracil between individuals and that there was a poor correlation of plasma concentration between the capecitabine and its active metabolite. Conclusion The method is sensitive,specific and validate rapid for simultaneous quantification of capecitabine and 5-fluorouracil in human plasma by liquid chromatography-tandem mass spectrometry and will be successfully applied for clinical practice to improve the effectiveness and security of chemotherapy regimens with capecitabine.