Pharmacokinetics And Tissue Distribution Study Of 3,4',5-transtrimethoxy-stilbene In Rats

Resveratrol?RES?is a well-studied natural small molecule that possesses a variety of biological benefits,however,also presents significant disadvant-ages,especially extremely low bioavailability.3,4',5-trans-trimethoxystilbene?TMB?is a derivative of resveratrol and retains various biological activity.However,systematic pharmacokinetics and tissue distribution information of TMB is lacking.It is important to evaluate its drug properties.Part One Pharmacokinetics study of TMB in ratsObjective:To establish HPLC-MS/MS method for determination of TMB in plasma,to plot the plasma drug concentration-time curve,and to calculate the pharmacokinetic parameters of TMB.Methods:1.Analysis condition:The separation of compound was carried out on a Phenomenex XB-C₁₈ column?3.0 mm×100 mm,2.6?m?at 40?C.The mobile phase was 80%?v/v?methanol and 20%?v/v?water?2 mmol ammonium acetate?at a flow rate of 0.4 ml/min.The injection volume was 5?L,and autosampler was set to 4?C.The total analysis time was 5.0 min for each run.the mobile phase from the column was introduced directly to the ESI source,which operated in positive mode with the following settings:ion spray voltage+5500 V;temperature?TEM?500?C;CAD,high;curtain gas?CUR?,nitrogen,20 psi;nebulizer gas?GS1?,nitrogen,40 psi;heater gas?GS2?,air,45 psi.Emodin methyl ether is an internal standard?IS?for TMB,as they share analogical solubility and approximate retention time.The multiple reactions monitoring?MRM?mode was performed,and transitions from precursor ions to product ions were optimized as m/z 271.2?147.1 for TMB,285.4?211.2 for IS,respectively.2.Analytical method validation including:specificity,matrix effect,recovery,accuracy,precision and stability.3.The sample treatment adopted a methanol precipitation protein method.4.Experimental protocol for pharmacokinetics:One group were orally given TBM?30 mg/kg?,and blood samples?approximately 0.5 ml?were collected at 0.25,0.5,0.75,1.5,2.0,2.5,3.0,5.0,8.0 and12 h after dosing;Another group were intravenously given TBM?10 mg/kg?,and blood samples were collected at 0.05,0.167,0.333,0.583,1.0,1.5,2.5,4.0,6.0,8.0 and 12 h after dosing.Results:1.The established HPLC-MS/MS method has high specificity,insignificant matrix effect,and good linearity?r>0.99?,precision,accuracy and stability.2.For oral administration in rats,the mean maximum peak concentration?Cmax?of TMB was 265.2±88.2 ug/L,which was observed about 1.13 h?Tmax?after administration.For intravenous administration in rats,the elimination half-life(T_1/2)and the volume of distribution?V/F?were 3.05 h and 198.3 L/kg,respectively.The mean absolute bioavailability?F?of the TMB was 32.1%obtained by comparing the means area under the plasma concentration–time curves?AUCs?in both oral and intravenous administrations.Conclusion:The established HPLC-MS/MS method is accurate and reliable,and can meet the needs of blood biological sample analysis.Pharmacokinetic parameters showed that absorption and metabolism rate of TMB were moderate,and oral bioavailability was higher than RES.Part Two Tissue distribution study of TMB in ratsObjective:To establish HPLC-MS/MS method for determination of TMB in different tissues,to detected content of TMB in different tissues after administration,and then to reveal distribution characteristics of TMB in the main tissues of rats.Method:1.Analysis condition:The separation of compound was carried out on a Phenomenex XB-C₁₈ column?3.0 mm×50 mm,2.6?m?at 40?C.The mobile phase was 80%?v/v?methanol and 20%?v/v?water?2 mmol ammonium acetate?at a flow rate of 0.4 ml/min.The injection volume was 10?L,and autosampler was set to 4?C.The total analysis time was 2.0 min for each run.the mobile phase from the column was introduced directly to the ESI source,which operated in positive mode with the following settings:ion spray voltage+5500 V;temperature?TEM?500?C;CAD,high;curtain gas?CUR?,nitrogen,20 psi;nebulizer gas?GS1?,nitrogen,40 psi;heater gas?GS2?,air,45 psi.Celecoxib is an internal standard?IS?for TMB,as they share analogical solubility and approximate retention time.The multiple reactions monitoring?MRM?mode was performed,and transitions from precursor ions to product ions were optimized as m/z 271.2?147.1 for TMB,382.1?362.2 for IS,respectively.2.For analytical method validation,a full method validation was performed on the liver,but other tissues only evaluate the specificity and linearity.3.Sample treatment:Tissue homogenate samples were treated with liquid-liquid extraction method by ethyl acetate.4.Experimental protocol for distribution:After oral administration of TBM at a single dose of 30 mg/kg,various tissues including heart,liver,lung,kidney,spleen,and brain were collected at 0.5,1,and 8 h.Subsequently,tissue samples were homogenized in ice-cold physiological saline solution.Result:1.The validated HPLC-MS/MS analytical method can satisfy the detection of various tissue biological samples.2.The highest concentrations of TMB in different tissues were observed at 0.5 h and gradually decreased over time.The concentration level of TMB was ranked as stomach>liver>lung>heart>spleen?brain>kidney at three different time points.Conclusion:The established HPLC-MS/MS method for the deter-mination of TMB in different tissues was accurate and reliable,and can meet the determination of TMB in different biological matrix.TMB was distributed widely throughout different tissues and had a large difference in content,and there was no long-term accumulation of TMB.