Pharmacokinetics,Tissue Distribution And Plasma Protein Binding Rate Study Of Carnosol

Objective:Carnosol is a kind of diterpenoids with extensive pharmacological effects found from rosemary extract.However,data on its in vivo studies are very limited.Therefore,this study aimed to establish a simple,exclusive and sensitive quantitative analysis method to determine the content of Carnosol in biological samples and further reveal the pharmacokinetic and tissue distribution characteristics of Carnosol in rats.Methods:Ultra-high performance liquid chromatography-tandem mass spectrometry（UHPLC-MS/MS）was developed.The content of Carnosol in rat plasma and tissue samples was determined and verified.Chromatographic separation was carried out on a WATERS HSS C₁₈column with the mobile phase of 0.1%formic acid aqueous solution-acetonitrile（20:80,v/v）by equivalent elution.Triple quadrupole tandem mass spectrometry was used as detector.Multiple reaction ion monitoring,anion ESI source detection mode was applied.Carnosol,the quantitative ion pair is m/z329.1→285.0,the qualitative ion is m/z 201.0;the internal standard ethyl m-hydroxybenzoate,the quantitative ion pair is m/z 165.1→92.1,the qualitative ion is m/z 137.0.Plasma and tissue samples were pretreated with simple and reproducible precipitation protein method,acetonitrile was selected as the precipitation reagent.Ethyl m-hydroxybenzoate was selected as the internal standard.Results:A simple,fast and accurate LC-MS/MS method has been developed and validated to determine the content of carnosol in rat plasma and tissues.The specificity was good,with the endogenous substances in rat plasma and tissues not interfering with the determination of carnosol and internal standard.Among the concentration range of 5.0-1000 ng/m L,the linear relationship was good.The precision and accuracy of the three concentration control samples were good,with the inter-day and intra-day precision RSD less than 15%and the accuracy RE within±15%.The average recovery was>90%;the mean matrix effect was 85.0-115.0%.The established analytical method conforms to the biological sample detection standard.After the intravenous administration of 2.0,5.0,10.0 mg/kg carnosol,the pharmacokinetic results showed that the C_maxwere 2170.00±918.20,3596.67±928.99,8996.67±2035.78 ng/m L,respectively;the AUC_0-twere 1872.40±382.49,2224.03±492.03,13241.43±1986.87 ng.h/m L,respectively.The C_maxand AUC_0-twere not strongly correlated with dose.The AUC_0-tincreased rapidly with the increase of dose,it might be caused by nonlinear elimination.After intravenous administration of 5 mg/kg carnosol,it was widely distributed in various tissues of SD rats.The content was highest in the live,followed by the lung and kidney;it could also cross the blood-brain barrier.The plasma protein binding rate of CA in low,mid and high blood concentration was non-concentration dependent,and the binding rate was about 70%.Conclusion:In this study,we established an accurate,sensitive and efficient UHPLC-MS/MS method for the quantification of carnosol in plasma and tissue samples,and successfully applied to evaluate the pharmacokinetics,tissue distribution and plasma protein binding rate of rat after gavage and intravenous injection of carnosol.