测定血液病患者血浆Venetoclax浓度的HPLC-MS/MS方法

目的建立测定血液病患者血浆Venetoclax浓度的HPLC-MS/MS法。方法患者血浆经甲醇(含内标Venetoclax-d8)沉淀蛋白处理。色谱柱为Ultimate@XB-C18(4.6×50 mm,5μm),柱温60℃。流动相为含0.1%甲酸和2 mmol·L^-1乙酸铵的水溶液和含0.1%甲酸的甲醇溶液,流速0.7 m L·min^-1,梯度洗脱。Venetoclax与Venetoclax-d8质谱检测方式均为电喷雾正离子模式,多反应监测(MRM),同时监测Venetoclax m/z 868.5~321.7(定量离子),Venetoclax m/z 868.5~177.6(定性离子)及Venetoclax-d8m/z 876.6~329.4(定量离子)。以Venetoclax和Venetoclax-d8峰面积比为定量依据,计算血浆Venetoclax浓度。并对该方法进行性能评价。结果Venetoclax在10~2 000 ng·mL^-1范围内线性良好,三批回归方程进行线性拟合后的标准曲线方程为Y=0.001 87 X+0.003 15,r=0.996 7;携带污染、重复性、实验室内精密度、回收率、基质效应和稳定性均符合性能验证要求。结论所建立的HPLC-MS/MS方法可准确、灵敏、快速地检测血液病患者血浆Venetoclax浓度,可应用于Venetoclax治疗药物监测及其体内药动学分析。

HPLC-MS/MS method for the determination of plasma Venetoclax concentration in patients with hematological diseases

Objective To establish an HPLC-MS/MS method for the determination of plasma Venetoclax concentration in patients with hematological diseases.Methods The patient.s plasma was treated with methanol(containing the internal standard Venetoclax-d8)to precipitate protein.The chromatography column was Ultimate@XB-C18(4.6×50 mm,5μm),and the column temperature is 60℃.The mobile phase is an aqueous solution containing 0.1%formic acid and 2 mmol·L^-1 ammonium acetate and a methanol solution containing 0.1%formic acid,with a flow rate of 0.7 m L·min^-1,and gradient elution.Both Venetoclax and Venetoclax-d8 mass spectrometry detection methods are electrospray positive ion mode,multiple reaction monitoring(MRM),and simultaneous monitoring of Venetoclax m/z 868.5~321.7(quantitative ion),Venetoclax m/z 868.5~177.6(qualitative ion)and Venetoclax-d8 m/z 876.6~329.4(quantitative ion).The peak area ratio of Venetoclax and Venetoclax-d8was used to calculate the plasma Venetoclax concentration.The performance of HPLC-MS/MS method for the determination of plasma Venetoclax concentration in patients with blood diseases was tested.Results Venetoclax has good linearity in the range of 10~2 000 ng·m L^-1.The standard curve equation after linear fitting of three batches of regression equations is Y=0.001 87 X+0.003 15,r=0.996 7.Carrying pollution,repeatability,precision in the laboratory,recovery rate,matrix effect and stability all meet the performance verification requirements.Conclusion The established HPLC-MS/MS method can accurately,sensitively and rapidly detect the plasma Venetoclax concentration in patients with blood diseases,and can be applied to the monitoring of Venetoclax therapeutic drugs and their in vivo pharmacokinetic analysis.