肾移植术后患者西罗莫司血药浓度的测定

摘要

Objective To develop a HPLC-MS method for determination of blood sirolimus (SRL) concentrations in patients after renal transplantation.Methods We selected 20 patients who received a triple immunotherapy of FK506/CsA + Sirolimus + glucocorticoids after rental transplantation.The whole blood samples were collected.To determine the concentrations of SRL,danazol was used as the internal standard (IS),and SRL and IS were separated on a Zorbax SB-C18 Narrow Bore RR (100 × 2.1 mm,3.5 μm)with a mobile phase containing H20-acetonitrile-methanol (27:45:28,v/v) at a flow rate of 0.2 mL/min and temperature of the column is 60 ℃C following protein precipitation and a simple two-step liquid-liquid extraction procedure.The mass spectrometer was operated in the positive ion mode,and selective ion monitors (SIM) were at m/z 936.76 ([M+Na] +) for SRL and 338.26 ([M+H] +) for internal standard,respectively.At the same time,a microparticle enzyme immunoassay (MEIA) was also used to determine blood SRL concentrations.The consistency of results by the HPLC-MS method MEIA was analyzed.Results Calibration curves were linear (r =0.9992) between 0.2 and 100 μg/L of SRL and the sensitivity was 0.2 μg/L.Intraday and inter-day precision was less than 6％.Analytical recovery ranged from 92.93％ to 102.98％.The correlation coefficient of the two methods was 0.979 (P ＜ 0.05) suggesting a close correlation between the two methods (n =20).An average deviation of the two methods was 1.4 μg/L when blood SRL concentrations ranging from 1.9 to 11.1 μg/L.Conclusion The HPLC-MS method described herein had been successfully applied for TDM in adult renal transplant recipients due to its low limits of quantification and stability.There is a correlation between the HPLC-MS and MEIA in determination of blood RSL concentrations.However,the average concentration values of the same sample are higher using MEIA when compared to HPLC-MS method.%目的 建立肾移植术后患者西罗莫司血药浓度的液相色谱-质谱联用(HPLC-MS)测定方法.方法 选取服用FK506/CsA+西罗莫司+糖皮质激素三联免疫治疗的肾移植术后患者20例,采集西罗莫司谷浓度全血样本,以达那唑为内标,经沉淀蛋白及液液萃取处理后,经液相色谱分离,采用ESI离子源,以SIR正离子方式进行检测,检测离子为m/z 936.76(西罗莫司),m/z 338.26(内标).同时将所建立的HPLC-MS法和微粒子酶免疫法(MEIA)分别测定肾移植患者全血西罗莫司浓度,评价两种分析方法测定结果的一致性.结果 西罗莫司在0.2～100 μg/L范围内线性关系良好(r=0.9992),最低定量限为0.2 μg/L.日内、日间精密度均小于6％,方法学回收率为92.93％～102.98％.两种方法测得的数据相关系数为0.979(P＜0.05),两组数据间存在相关性(n=20).当西罗莫司浓度在1.9～11.1 μg/L时,两种方法测定的平均偏差为1.4 μg/L.结论 本文建立的HPLC-MS测定方法快速、准确、灵敏、选择性高,适用于临床对西罗莫司的血药浓度监测;HPLC-MS法和MEIA法测定西罗莫司全血浓度存在相关性,同一份样本用MEIA测定的浓度均值比HPLC-MS法测定的值高.