基于LC-MS技术测定人血浆中吉西他滨浓度方法学的建立与临床应用

Establishment and Application of the Method for Determination of Gemcitabine Concentration in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

目的 建立一种快速、灵敏度高、专属性强的液质联用(LC-MS)方法,用于监测人血浆中吉西他滨的浓度。方法 筛选我院确诊为非小细胞肺癌并采用含吉西他滨化疗方案治疗的患者,在吉西他滨给药5min后采血,离心,取血浆样品50μL,内标溶液100μL,加入水∶乙腈=4∶1混合液50μL沉淀蛋白,涡旋30s后,15000rpm离心5min,取上清液用0.22μm有机滤膜过滤,上样分析。采用AgilentZORBAXEclipsePlusC18(2.1×150mm1.8-Micron)色谱柱、0.1%甲酸乙腈和0.1%甲酸水流动相梯度洗脱、电喷雾电离(ESI)离子源质谱、多重反应监测(MRM)方式进行正离子检测,测定血浆中药物浓度。结果 LC-MS法的线性浓度范围5~500ng·mL^-1,定量下限位仪器检出限为0.052ng·mL^-1,线性方程为y=1272.93x+11213.5,r=0.999,三个浓度水平(10、50、400ng.mL^-1)的回收率分别为89.41%、92.38%、91.42%;日内相对标准差(RSD)分别为4.55%、3.09%、2.81%,室温和2~4℃放置24h后RSD为2.36%~4.78%,-20℃保存90d后RSD为7.98%~11.67%,-80℃保存180d后RSD为13.87%~16.52%,24例人血浆样本中吉西他滨浓度为(13.84±5.61)μg·mL^-1。结论 LC-MS法灵敏性高、专属性强、稳定性好,测定结果可靠,可用于临床血浆样本中吉西他滨血药浓度测定以及药代动力学研究。

Objective To establish a method for the determination of gemcitabine concentration in human plasma by liquid chromatographytandem mass spectrometry (LC-MS). Methods The sample plasma 200 μL was precipitated protein by adding acetonitrile. After 30 s vortex, the plasma was centrifuged at 9000 rpm for 5 min. The supernatant was filtered and 0.22 μm was taken to determine. The Agilent ZORBAX Eclipse Plus C18 (2.1×150 mm 1.8-Micron) chromatographic column was adopted, and acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid were used as mobile phase. Electrospray ionization (ESI) ion source mass spectrometry and multiple reaction monitoring (MRM) were used for positive ion detection. Results The calibration curve exhibited a linear dynamic range of 5~500 ng·mL^-1. The detection limit of quantitative lower limit was 0.052 ng·mL^-1. The linear equation was y=1272.93x+11213.5, r=0.999. The recovery rate of the three concentration levels (10, 50, 400 ng·mL^-1) were respectively 93.41%, 98.22% and 96.95%, and the intraday relative standard deviation (RSD) were 4.55%,3.09% and 2.81%, respectively. The RSD was 2.36%~4.78% after 24 h at room temperature and 2~4 ℃, 7.98%~11.67% after 90 days at -20 ℃, and 13.87%~16.52% after 180 days at -80 ℃. The concentration of gemcitabine in 24 human plasma samples was (13.84±5.61)μg·mL^-1. Conclusion An accurate, simple and sensitive method for determining the plasma concentration of gemcitabine by LC-MS was available. It can be used in the determination of plasma concentration and pharmacokinetics of gemcitabine.