超高效液相色谱-串联质谱法测定人血浆中安罗替尼浓度的方法学研究及应用

Quantitative analysis of anlotinib in human plasma by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry and assessment of clinical application

关键词:  超高效液相色谱-串联质谱法  安罗替尼  血药浓度  

Key words:  Ultra-high-performance liquid chromatography-mass spectrum/mass spectrum  Anlotinib  Human plasma  

摘要:

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-串联质谱法(UPLC-MS/MS)并评估临床应用。方法：以泽布替尼为内标物,血浆用乙腈蛋白沉淀,使用Ultimate XB-C18色谱柱 (100mm × 2.1mm, 3.0μm) 分离,流动相为10mM醋酸胺+0.1%甲酸和乙腈进行梯度洗脱,流速为0.6 mL/min,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、基质效应与稳定性及临床应用。结果：安罗替尼在1.0-100.0ng/ml线性关系良好,标准曲线为y=0.515148x+0.0144461(R₂^=0.9984),精密度RSD小于9%,回收率和基质效应分别是104.81-107.32% 和102.54%-104.26%,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。本研究考察了52名服用安罗替尼单药治疗的非小细胞肺癌患者,采集服用安罗替尼治疗第43天的谷浓度血浆,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高于1.0 ng/mL (最低定量下限),血药浓度均值±标准差是11.38± 4.29ng/mL,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。

Abstract:

Objective: To establish an 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. Method: The internal standard using zanubrutinib and the extraction process was performed through protein precipitation method using acetonitrile, followed by separation on an Ultimate XB-C18 column (100mm × 2.1mm, 3.0μm) using acetonitrile and 10mM ammonium acetate-0.1% formic acid step-elution gradient. The flow rate was 0.6 mL/min 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 408.1→339.1 for anlotinib and 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, the calibration curve was y=0.515148x+0.0144461 (R₂^=0.9984). The precision was less than 9%, the recovery and matrix effect were 104.81-107.32% and 102.54%-104.26%, respectively, and this method has good stability and was not affected by matrix effect. The method has been used for determined 52 advanced non-small cell lung cancer (NSCLC) patients treated with anlotinib. The trough plasma concentration (C_trough) was measured on day 43 after initiation of anlotinib treatment. Anlotinib C_trough were higher than LLOQ (1.0 ng/ml) from 52 patients. The plasma concentration of anlotinib C_troughwas 11.38 ± 4.29 ng/ml with 37.66% coefficients of variation, which were shown large inter-patient variability_.Conclusion: This method was high sensitivity, specificity and accurate, and suitable for determination of anlotinib C_troughin human plasma.