Establishment of a blood concentration detection system for everolimus in recipients after liver transplantation
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摘要:
目的 建立测定肝移植受者全血中依维莫司浓度的超高效液相色谱-串联质谱法(UPLC-MS/MS)检测体系。 方法 用甲醇和硫酸锌沉淀样本蛋白质,以依维莫司-D4为内标物。采用Phenomenex Kinetex PFP色谱柱;流动相A:水(含有2 mmol/L甲酸铵和0.1%甲酸),流动相B:甲醇(含有2 mmol/L甲酸铵和0.1%甲酸),梯度洗脱,流速1 mL/min,柱温50 ℃,进样量1 μL。在正离子模式下,以多反应监测方式进行定量测定。该UPLC-MS/MS检测体系只需100 μL全血,无需复杂的样品制备即可达到足够的定量下限,总运行时间4.5 min内。采用依维莫司峰面积/依维莫司-D4峰面积(y)和依维莫司浓度/依维莫司-D4浓度(x)进行线性回归(1/ x2)分析,计算校准函数,分析其准确度和线性关系。并应用UPLC-MS/MS检测5例肝移植术后受者血样本的依维莫司血药谷浓度。 结果 依维莫司在血药浓度1~100 ng /mL范围内的质控准确度都在15%以内,线性关系良好(R2 > 0.990)。测定的5例肝移植受者血样本的依维莫司血药谷浓度为3.77~9.27 ng/mL。 结论 本研究建立的UPLC-MS/MS检测体系测定肝移植受者全血中依维莫司血药浓度准确度高、样品处理方法简便、检测时间短,适用于肝移植术后受者血样本中依维莫司的药物浓度监测。 -
关键词:
- 依维莫司 /
- 超高效液相色谱-串联质谱法(UPLC-MS/MS) /
- 高效液相色谱法(HPLC) /
- 肝移植 /
- 血药浓度 /
- 钙调磷酸酶抑制剂(CNI) /
- 哺乳动物雷帕霉素靶蛋白(mTOR) /
- 西罗莫司
Abstract:Objective To establish a detection system of ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for everolimus concentration in whole blood of liver transplant recipients. Methods The proteins of samples were precipitated with methanol and zinc sulfate, and everolimus-D4 was used as the internal standard. Phenomenex Kinetex PFP column was used. The mobile phase A was water (containing 2 mmol/Lammonium formate and 0.1% formic acid), and the mobile phase B was methanol (containing 2 mmol/L ammonium formate and 0.1% formic acid). The gradient elution was performed with the flow rate of 1 mL/min, the column temperature of 50 ℃ and the injection volume of 1 μL. The multi-reaction monitoring mode was used to quantitatively analyze with electrospray positive ionization. The UPLC-MS/MS detection system required only 100 μL of whole blood, and could achieve a sufficient lower limit of quantification without complicated sample preparation. The total running time was within 4.5 min. Linear regression (1/x2) analysis was performed using peak area of everolimus / peak area of everolimus-D4 (y) and concentration of everolimus/concentration of everolimus-D4 (x) to calculate the calibration function and analyze its accuracy and linear relationship. UPLC-MS/MS was used to detect the trough blood concentration of everolimus in blood samples of 5 recipients after liver transplantation. Results The accuracy of quality control was within 15%, and the linear relationship of everolimus was good in the blood concentration range of 1-100 ng /mL(R2 > 0.990). Trough blood concentration of everolimus measured in blood samples of 5 liver transplant recipients ranged from 3.77 to 9.27 ng/mL. Conclusions The detection system of UPLC-MS/MS in this study is suitable for monitoring the concentration of everolimus in whole blood of liver transplant recipients because of its high accuracy, simple sample processing method and short detection time. -
表 1 依维莫司标准曲线质控的准确度结果
Table 1. Accuracy results of quality control of everolimus standard curve
样本序号 待测物峰面积 理论浓度(ng/mL) 内标物峰面积 实测浓度(ng/mL) 准确度(%) STD1 5.13×103 1.00 1.01×105 1.00 100.0 STD2 2.33×104 5.00 8.63×104 4.85 97.0 STD3 5.05×104 10.00 9.21×104 9.75 97.5 STD4 1.39×105 25.00 8.88×104 27.60 110.4 STD5 2.62×105 50.00 9.09×104 50.80 101.6 STD6 5.00×105 100.00 9.46×104 93.20 93.2 L-QC 1.68×104 3.31 8.91×104 3.43 103.6 M-QC 7.81×104 13.00 1.07×105 13.00 100.0 H-QC 2.18×105 41.50 9.19×104 41.80 100.7 表 2 5例肝移植受者的依维莫司血药谷浓度检测结果
Table 2. Test results of everolimus trough blood concentration in 5 liver transplant recipients
样本序号 待测物峰面积 内标物峰面积 实测浓度(ng/mL) Sample-1 4.06×104 9.11×104 8.05 Sample-2 3.28×104 8.99×104 6.60 Sample-3 2.43×104 1.17×105 3.77 Sample-4 4.07×104 9.59×104 7.67 Sample-5 4.96×104 9.66×104 9.27 -
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