人全血他克莫司浓度高效液相色谱-质谱法检测方法学的建立及性能评价

目的 建立全血他克莫司高效液相色谱-质谱(HPLC-MS)的检测方法.方法 液液萃取的方法从血中提取出他克莫司,然后用高效液相色谱-质谱的方法分离并检测他克莫司的浓度;同时进行标准曲线、线性范围、最低检测限、精密度、回收率及比对实验.结果 方法标准曲线回归方程为Y=0.2464x+0.0082,R2=0.9996;线性范围为0.100～40.000 ng/ml,回归方程为Y=1.0294x-0.035,R2=0.9998;最低检测限为0.100 ng/ml;以浓度为0.300、4.004、8.008、16.016 ng/ml的4种浓度样本测定不精密度(CV),其批内CV分别为4.67%、2.78%、3.22%、3.58%,其总CV分别为5.67%、3.16%、3.97%、4.29%;用浓度为0.300、4.004、8.008、16.016 ng/ml 4种标准品进行回收实验,其回收率分别为95.67%、97.00%、95.88%、98.30%;与酶联免疫吸附试验(ELISA)方法进行比对,回归方程为y=1.0172x+0.3742,相关系数R2=0.9630,2种方法相关性较好,ELISA方法的测定值较HPLC-MS方法的测定值高,差异有统计学意义(P<0.01).结论 HPLC-MS方法具有方便、快速、准确和成本低的优点,可用于对全血中他克莫司的临床监测和实验研究.

Measurement of tacrolimus in blood with the method of high performance liquid chromatography-mass spectrometry

Objective To develop a simple, precise, and specific method for measurement of tacrolimus in whole blood. Methods Tacrolimus was extracted from the blood samples of 40 patients treated with tacrolimus by liquid-liquid extraction. Then high performance liquid chromatography-mass spectrometry (HPLC/MS) was used, with ascomycin as internal marker, to isolate the tacrolimus and measure the concentration thereof. The standard curve was drawn. The linearity of quantitative measurement was determined. Detection limits test, CV test, and recovery test were performed. ELISA was used simultaneously. The results of these 2 methods were compared. Results A good standard curve was drawn based on the results of HPLC/MS assay(y =0. 2464x +0. 0082,R2 =0. 9996). The detected data were highly related to the detected concentrations. The linearity range was 0. 100 -40. 000 ng/ml( y = 1. 0294x -0. 035, R2 =0. 9998). The detection limit of the assay was 0. 100 ng/ml. The CV values by this assay were basically 5%. The recovery rate ranged 95.67% -98. 30% for tacrolimus over the range 0. 300 - 16. 016 ng/ml. There was a linear correlation (y = 1. 0172x + 0. 3742, R2 = 0. 9630) between the assay results by HPLC/MS to ELISA in blood. The measurement value of ELISA was ( 19 ± 9), significantly higher than that of HPLC/MS ( 18±9, P0. 01 ). Conclusion This newly developed method of HPLC/MS is simple, precise, and specific and with lower cost, it can be used in the clinical practice and experimental study on tacrolimus.