Breviscapine Orally Disintegrating Tablets In Beagle Dogs Pharmacokinetics And Relative Bioavailability Research

Objective:The HPLC method for establishing tandem mass spectrometry（LC-MS/MS）detects the concentration of scutellarin aglycone in Beagle dog plasma and verify it.Beagle dogs were given the test preparation breviscapine orally disintegrating tablets and the reference preparation to study the pharmacokinetics of breviscapine tablets.To analyze the pharmacokinetic parameters and evaluate the relative bioavailability of breviscapine orally disintegrating tablets.And the main pharmacokinetic parameters AUC_0-t,AUC_0-∞,C_max of the test preparation and the reference preparation were statistically analyzed.The setting of blood sampling points and cleaning period in animal experiments is examined whether it is reasonable,and the repeatability,stability,and applicability of the detection method are verified,which provides a reference for the following clinical trials.Method:1.The plasma samples were hydrolyzed by sulfatase.Quercetin was used as the internal standard,and 0.1%formic acid-0.1%formic acid methanol was used as the mobile phase gradient to elute.The LC-MS/MS method was established for the detection of the concentration of scutellarin aglycone in plasma samples,and verify methodology.2.The animal experiment adopts a two-preparation,two-period,self-double-crossover,fasting single-dose test protocol.6 Beagle dogs,half male and half female,were divided into two groups,and two groups were given test and reference preparations.After an interval of 5 days,they were cross-administered to the reference preparation and the test preparation.The blood samples were collected from the designed blood sampling points,and the concentration of scutellarin aglycone in the dog plasma samples was detected by the established LC-MS/MS method.the corresponding pharmacokinetic parameters were calculated by DAS 3.3.0 software.The relative bioavailability of the test preparation breviscapine orally disintegrating tablets was calculated,and the paired T test and non-parametric test（Wilcoxon signed rank test）were used to evaluate and judge whether the test preparation and the reference preparation have similar bioavailability.Results:1.Methodological verification results:endogenous substances in plasma do not affect the determination.The linear regression equation of scutellarin is Y=0.0104x+0.00823 r=0.9992;the linear range is 7.60 ng·m L^-1-488 ng·m L^-1.The lower limit of quantification is 7.60 ng·m L^-1.The intra-day and inter-day accuracy ranges of quality control samples with low,medium and high(13.5 ng·m L^-1,108 ng·m L^-1,432ng·m L^-1)concentrations are 93.24%-100.89%,96.30%-103.32%;intra-day and inter-day precision RSD%are less than 8.46%;average normalized matrix effect is（110.47±1.89）%,RSD%value is 1.72%;average extraction recovery is（107.09±3.19）%,RSD%The value is 2.98%;RE stored at room temperature for 24 hours,2-8°C for 24hours,-20°C for 30 days,-20°C for 6 days,and freeze-thaw 3 times.After treatment,the sample solution is stored at room temperature for 48 hours.The results of%value are all within±15%,and the stability is good.2.The pharmacokinetic results showed that after 6 Beagle dogs took the two preparations orally,the main pharmacokinetic parameters of the test preparation and the reference preparation were as follows:C_max（235.08±112.53）ng·m L^-1,（267.90±111.04）ng·m L^-1,T_max（6.33±6.05）h,（4.33±1.92）h,AUC_（0-t）（1554.58±548.49）ng·m L^-1·h,（1628.08±408.78）ng·m L^-1·h.The relative bioavailability of the tested preparation breviscapine orally disintegrating tablets was 102%.Conclusion:1.The established LC-MS/MS method for detecting the concentration of scutellarin aglycone in dog plasma samples is highly specific,rapid and sensitive.This method meets the requirements of plasma sample analysis and detection.2.The pharmacokinetic parameters of breviscapine orally disintegrating tablets were obtained in the experiment.The relative bioavailability of the tested preparation breviscapine orally disintegrating tablets was 102%.Breviscapine orally disintegrating tablets and breviscapine tablets have similar bioavailability.