Determination of free SN-38 concentration in rat plasma by equilibrium dialysis combined with LC-MS/MS and its pharmacokinetic application
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.
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