LC-MS/MS法同时分析血液中几种免疫抑制剂浓度

目的：建立LC-MS/MS法同时测定人血浆及全血中霉酚酸（MPA）、环孢素（CsA）、他克莫司（TAC）及西罗莫司（SRL）等几种常用免疫抑制剂的浓度。方法：分别采用蛋白沉淀法与液液萃取法处理血浆与全血：100 μL血浆加入含内标乙腈,涡旋振荡, 离心后取上清液进入LC-MS/MS分析。100 μL全血加入硫酸锌破裂血细胞,加入乙醚萃取,分离有机相,以氮气吹干后流动相复溶分析。色谱柱为 Agilent Eclipse XDB-C₁₈柱（3.5 μm,2.1 mm×100 mm）,流动相为 2 mmol·L^-1 甲酸铵水溶液和甲醇,采用梯度洗脱的方式,流速0.3 mL·min^-1,多反应监测定量,ESI 正离子方式进行检测,用于定量分析的检测离子对分别为m/z→274.4/159.2（MPA）,m/z→1 219.4/1 202.4（CsA）,m/z →821.7/768.5（TAC）,m/z →931.6/864.5（SRL）,内标m/z→809.6/756.5（子囊霉素）,m/z→356.3/312.0（吲哚美辛）。回顾性收集常规监测肾移植患者的全血标本,比较LC-MS/MS 法与 EMIT法的检测结果。结果：CsA、TAC、SRL、MPA的线性范围分别为：4~1 000 ng·mL^-1 （r=0.999 7）、0.2~50 ng·mL^-1 （r=0.999 5）、0.2~50 ng·mL^-1 （r=0.999 8）、0.204~51 μg·mL^-1 （r=0.997 6）,血浆和全血日内和日间相对标准差（RSD）均小于15%,提取回收率以及基质效应血浆中为72.38%~98.52%、65.61%~99.19%,全血中为85.34%~99.76%、75.94%~97.67%。不同法检测全血中与血浆中CsA及TAC浓度相关性良好,而血浆中CsA与TAC显著低于全血中水平,MPA血浆浓度大约为全血浓度的2倍。结论：本研究建立的方法具有方便快速的特点,可以同时测定几种免疫抑制剂浓度,对同时监测多种免疫抑制剂具有一定价值。

Establishment of LC-MS/MS assay for the determination of blood immunosuppressive agents level simultaneously

OBJECTIVE To establish an LC-MS/MS method for determination of concentrations of several common used immunosuppressive agents, including mycophenolic acid (MPA), cyclosporin (CsA), tacrolimus (TAC) and sirolimus (SRL), in human plasma and whole blood, simultaneously. METHODS Plasma and whole blood were treated by protein precipitation method and liquid extraction method respectively:200 μL of acetontrile containing internal standard was added to 100 μL plasma and vortexed, centrifuged and the supernatant was taken into LC-MS/MS analysis. Blood cells in 100 μL whole blood was lysed with zinc sulphate, and the immunosuppressive agents was extracted by ethyl ether and the organic phase was separated and evapored with N₂. The chromatographic column is Agilent Eclipse XDB-C₁₈ column (3.5 μm, 2.1 mm×100 mm), and the flow phase is 2 mmol·L^-1 ammonium formate aqueous solution and methanol. The gradient elution method was adopted. The flow rate is 0.3 mL·min^-1, and the multi-reaction monitoring (MRM) modes with the transition of m/z 274.4→159.2(MPA),m/z 274.4/159.2(MPA), m/z 1 219.4/1 202.4(CsA), m/z to 821.7/768.5 (TAC), m/z to 931.6/864.5(SRL), internal standard m/z to 809.6/756.5(ascomycin), m/z to 356.3/312.0(indomethacin) were used for the quantification. The residual whole blood samples after conventional TDM of patients received renal transplantation were collected retrospectively. The immunosuppressive agents level were determined by the established assay and results were compared with EMIT methods. RESULTS The linear ranges of CsA, TAC, SRL and MPA in this method were:4-1 000 ng·mL^-1(r=0.999 7), 0.2-50 ng·mL^-1 (r=0.999 5), 0.2-50 ng·mL^-1(r=0.999 8), 0.204-51 μg·mL^-1(r=0.997 6), The inter-day and between-day relative standard deviation (RSD) of various immunosuppressive agents in plasma and whole blood were all less than 15%. Extraction recovery rate and matrix effect was 72.38%-98.52%, 65.61%-99.19% in plasma, 85.34%-99.76%, 75.94%-97.67% in whole blood. A good correlation was observed between the concentration of CsA and TAC in whole blood and plasma detected by different methods, while CsA and TAC in plasma were significantly lower than that in whole blood, and the plasma concentration of MPA was about twice that of whole blood. CONCLUSION The method established in this study is convenient and rapid. It can be used to determine the concentration of several immunosuppressive agents simultaneously.