UPLC-MS/MS测定人血浆中安罗替尼浓度及临床应用

Determination of Anlotinib in Human Plasma by UPLC-MS/MS and Its Clinical Application

目的建立一种快速测定人血浆中安罗替尼浓度的超高效液相色谱-串联质谱(ultra-high-performance liquid chromatography-mass spectrum/mass spectrum,UPLC-MS/MS)法,并对其临床应用进行评估。方法以泽布替尼为内标物,血浆用乙腈蛋白沉淀,使用Ultimate XB-C_(18)(100 mm×2.1 mm,3.0μm)色谱柱分离,流动相为10 mmol·L^(-1)醋酸胺+0.1%甲酸和乙腈进行梯度洗脱,流速为0.6 mL·min^(-1),进样量为5μL。应用电喷雾离子化,正离子模式下用多反应模式监测安罗替尼(m/z 408.1→339.1)和内标物(m/z 472.2→290.1)的浓度,考察该方法学的专属性、定量下限与标准曲线、精密度与回收率、基质效应与稳定性。结果安罗替尼在1.0~100.0 ng·mL^(-1)线性关系良好,R^(2)=0.9984;精密度RSD<9%,萃取回收率和基质效应分别是104.81%~107.32%和102.54%~105.26%,该方法稳定性良好,血浆基质对安罗替尼测定结果影响较小。采集52例服用安罗替尼单药治疗的非小细胞肺癌患者第43天的血浆谷浓度,并对其血药浓度进行测定,发现所有患者安罗替尼的血药浓度均>1.0 ng·mL^(-1)(最低定量下限),血药浓度为(11.38±4.29)ng·mL^(-1),变异系数是37.66%,表现出较大的个体间差异。结论本方法灵敏度高、专属性强、定量准确,适用于人血浆中安罗替尼的浓度监测。

OBJECTIVE To establish a ultra-high-performance liquid chromatography-mass spectrum/mass spectrum(UPLC-MS/MS)method for the determination of anlotinib in human plasma and assessment of clinical application.METHODS Zanubrutinib was used as internal standard and the extraction process was performed through protein precipitation method using acetonitrile,followed by separation on an Ultimate XB-C_(18)(100 mm×2.1 mm,3.0μm)column using acetonitrile and 10 mmol·L^(-1)ammonium acetate-0.1%formic acid step-elution gradient.The flow rate was 0.6 mL·min^(-1)and injection volume was 5μL.The mass analysis was performed by positive ion electrospray ionization in multiple-reaction monitoring mode,and the mass spectrometer was set at m/z 408.1→339.1 for anlotinib and m/z 472.2→290.1 for internal standard,respectively.The specificity,standard curve and lower limit of quantification,precision and recovery,matrix effect and stability of the method and clinical application were investigated.RESULTS The method was validated over the concentration range of 1.0-100.0 ng·mL^(-1),with R^(2)=0.9984.The precision RSD was<9%,the recovery and matrix effect were 104.81%-107.32%and 102.54%-105.26%,respectively,and this method had good stability and was not affected by matrix effect.The method had been used for determined 52 advanced non-small cell lung cancer patients treated with anlotinib.The trough plasma concentration(Ctrough)was measured on day 43 after initiation of anlotinib treatment.Anlotinib Ctrough were higher than lower limit of quantitation(1.0 ng·mL^(-1))from 52 patients.The plasma concentration of anlotinib Ctrough was(11.38±4.29)ng·mL^(-1)with 37.66%coefficients of variation,which were shown large inter-patient variability.CONCLUSION This method is high sensitivity,specificity and accurate,and suitable for determination of anlotinib in human plasma.