Pharmacokinetics Of Compound Luotuopengzi Ointment And Hydrogel Patches In Rats

Objective:To establish a HPLC content determination method for simultaneous determination of 6 main active ingredients(vasicine,scopolamine hydrobromide,atropine sulfate,harmaline,harmine,colchicine)in the Compound Luotuopengzi Ointment and Hydrogel patches,to lay the foundation for the improvement of the quality control method and and research of the intracorporal pharmacokinetics.To establish a UPLC-MS/MS determination method for simultaneous determination of the 6 main active ingredients in rat plasma,dialysate and skin dialysate after the administration of Compound Luotuopengzi Ointment and Hydrogel patches,and establish a pre-extraction method for plasma sample.On this basis,study the intracorporal and topical pharmacokinetic processes of the two preparations in rats by the Plasma Concentration method and microdialysis method respectively,and preliminarily compare the pharmacokinetic parameters obtained by the two methods.Evaluate the applicability of Plasma Concentration method and microdialysis method in studying the topical and intracorporal pharmacokinetics of transdermal thrapeutic systems.Methods:1)Use the HPLC method to simultaneously determine the contents of 6 main active ingredients in the Compound Luotuopengzi Ointment and Hydrogel patches.2)Use UPLC-MS/MS analysis method to determine the contents of the 6 main active ingredients in the plasma,dialysate and skin dialysate samples of the rats after transdermal administration of the Compound Luotuopengzi Ointment and Hydrogel patches.3)Study the pharmacokinetics of 6 main active ingredients in the in the Compound Luotuopengzi Ointment and Hydrogel patches in rats by the Plasma Concentration method and microdialysis respectively.Microdialysis method requires self-made microdialysis probe to screen and evaluate the effects of factors such as perfusate and perfusion speed on the percent recovery in vivo and in vitro.Calculate the concentration of drug in plasma,dialysate and skin dialysate of rats after transdermal administration of the CompoundLuotuopengzi Ointment and Hydrogel patches by the standard curve of each test substance,and draw the drug concentration-time curve.Use the DAS 3.2.7pharmacokinetics software to analyze the non compartment model.Use statistical moment method to calculate the pharmacokinetic parameters of each rat in each group.Obtain the average pharmacokinetic parameters of 6 rats in each group,including peak time(Tmax),peak concentration(Cmax),mean residence time(MRT0-t 、 MRT0-∞),elimination half-life(T1/2z),and area under plasma concentration curve(AUC0-t 、 AUC0-∞).Express all experimental data and pharmacokinetic parameters in the form of mean,and calculate relative bioavailability.Use SPSS 21.0 for t-test of pharmacokinetic parameter AUC0-t、AUC0-∞、MRT0-t、MRT0-∞、T1/2z、Cmax after logarithmic transformation,Use Tmax for rank sum test,and use single factor analysis of variance among high,medium and low dosage groups to preliminarily investigate the intracorporal pharmacokinetic process of each ingredient to be measured.Results:1)The method for the determination of the content of the Compound Luotuopengzi Ointment and Hydrogel patches was determined by Agilent Eclipse Plus C18 column(4.6 mm × 250 mm,5 μm),mobile phase 0.1% ammonium acetate buffer solution-acetonitrile gradient,detection wavelength 250 nm and 264 nm,flow rate of 1.0 m L/min,column temperature:27 °C,injection volume:10 μL.The linear relationships of the six ingredients were good in their linear ranges,and the precision,repeatability,stability and percent recovery met the requirements.The content results of vasicine,hydrobromide,atropine sulfate,harmaline,harmine,colchicine were0.2835,0.4439,0.9940,1.1530,1.4871,0.1531 mg/g respectively.The contents results of self-made ointment determination vasicine,scopolamine hydrobromide,atropine sulfate,harmaline,harmine,colchicine were 0.5583,0.8882,1.9665,2.3187,2.9533,0.3219mg/grespectively.2)The results of plasma sample pretreatment method showed that the best pretreatment method was to take 200 μL of plasma,add 400 μL of methanol to precipitate protein,vortex and mix for 1 min,centrifuge(4 ° C,15000 r/min)for 10 min;take quantitative supernatant,N2 blow Dry,add 200 μL of methanol to reconstitute,vortex for 1 min,centrifuge(4 ° C,15000 r/min)for 10 min,take the supernatant UPLC-MS/MS analysis.UPLC-MS/MS detection method using ACQUITY UPLC BEH C18 column(2.1× 100 mm,1.7 μm);mobile phase: 0.1% formic acid water-acetonitrile;gradient elution,flow rate 0.4 m L/min;injection volume:1 μL;column temperature: 40 °C;mass spectrometry conditions: ionization method is electrospray ionization source(ESI+);multi-reaction monitoring MRM.The linear relationships of the six ingredients were good in their linear ranges,and the precision,repeatability,stability and percent recoverymet the requirements.3)Compound Luotuopengzi Ointment and Hydrogel patches after skin administration,the plasma concentration-time curve of 6 kinds of the high medium low dosage groups to be measured in the ointment and hydrogel patches showed that the concentrations of the compound tested in the hydrogel patches group were relatively stable relative to the ointment.In the hydrogel patches group,the absorption and elimination were slower,and the area under the curve was larger,showing a certain slow release trend.Most of the compounds in the hydrogel patches were dose-dependent and consistent with the linear kinetic process.The dialysate concentration time curve of 6compounds of the medium and low dose group in ointment and hydrogel patches was similar to the plasma concentration time curve.The skin dialysate concentration time curves of the 6 compounds of the medium and low dose groups in the ointment and hydrogel patches showed that the absorption of the concentration of the dialysate of the vasicine and colchicine in the hydrogel patches was slower than that in the ointment,but the area under the curve was larger and the elimination was slower.The absorption of the concentration of the skin dialysate of the Scopolamine Hydrobromide,Atropine Sulfate,Harmaline,Harmine in the gelata was slower than that in the ointment,and the peak concentration was lower,but the elimination process was longer and the area under the curve was similar to that of the ointment group.Conclusion: The established HPLC determination method for simultaneous determination of 6 compounds in the Compound Luotuopengzi Ointment and Hydrogel patchesis accurate and reliable,and it can effectively improve the quality control methods of the two preparations.The established UPLC-MS/MS analysis method for simultaneous determination of 6 compounds of 3kinds of biological samples after transdermal administration of the Compound Luotuopengzi Ointment and Hydrogel patches is is accurate and reliable.It can provide a scientific basis for the pharmacokinetic study of the two preparations.The results of pharmacokinetics showed that the Compound Luotuopengzi Ointment and Hydrogel patches.Hydrogel patches had slower absorption than ointment but had a certain release effect.The bioavailability of the compounds in gelata was higher than that of ointment,and the concentration of drug in topical skin was larger.It will last longer and be expected to produce better curative effect.The results of intracorporal pharmacokinetics of the two preparations by the plasma concentration method and microdialysis method are basically similar.Comprehensive evaluation shows that plasma concentration method is suitable for intracorporal pharmacokinetics of transdermal thrapeutic systems,and microdialysis method is more suitable for the topical pharmacokinetics of the transdermal thrapeuticsystems.