肝移植术后受者依维莫司血药浓度检测体系的建立

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。

Establishment of a blood concentration detection system for everolimus in recipients after liver transplantation

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.