目的 建立大鼠血浆中游离SN-38浓度的LC-MS/MS检测方法.方法 采用平衡透析法模拟SN-38在人体内与血浆蛋白结合的过程,在透析完成后分离出游离SN-38,以[2H5]SN-38为内标,用蛋白沉淀法预处理透析液侧(F侧)样品,取上清液进行LC-MS/MS分析;总SN-38及透析后血浆侧(T侧)SN-38样品的预处理方法与游离SN-38相同;分别考察两种方法的专属性、线性关系、精密度、准确度、提取回收率、基质效应、稳定性.结果 F侧SN-38在0.04~80 ng·mL-1内与峰面积线性关系良好(r>0.99);提取回收率为98.6%~101.6%,基质效应为96.2%~97.2%;日内、日间RSD均<15%,且分别在不同条件下稳定性良好.大鼠血浆样品中的总SN-38在0.5~1000ng·mL-1内与峰面积线性关系良好(r>0.99);提取回收率为97.3%~99.5%,基质效应为100.5%~101.2%;日内、日间RSD均<15%,且分别在不同条件下稳定性良好.结论 该方法简单易行、准确、灵敏、专属性好,适用于大鼠血浆中游离SN-38浓度的测定.
Objective To establish an LC-MS/MS method to determine free SN-38 concentration.Methods Equilibrium dialysis was used to simulate the process of SN-38 binding with plasma protein,and the free SN-38 was isolated after the dialysis.The concentration of free SN-38 in dialysate side(F side)was determined by protein precipitation with[2H5]SN-38 as the internal standard and the supernatant was used for LC-MS/MS analysis.The total SN-38 and the SN-38 in the plasma(T side)after the dialysis were prepared by the same method as free SN-38.The specificity,linearity,precision,accuracy,extraction recovery,matrix effect and stability of both methods were determined respectively.Results SN-38 on the F side was well separated with good linear relationship(r>0.99)at 0.04~80 ng·mL-1.The extraction recovery was 98.6%~101.6%,and the matrix effect was 96.2%~97.2%.Both inter-day and intra-day RSDs were lower than 15%,showing good stability in different conditions.The total SN-38 in the rat plasma samples was well separated by this method,with good linearity at 0.5~1000 ng·mL-1(r>0.99).The extraction recovery were 97.3%~99.5%,and the matrix effect was 100.5%~101.2%.RSDs of both inter-day and intra-day were lower than 15%,and also had good stability in different conditions.Conclusion The method is simple,accurate,sensitive and specific,which is suitable for the determination of free SN-38 concentration in rat plasma.