Study On Extraction And Purification Of Alkaloids From Aconiti Radix Cocta And Pharmacokinetics In Rats

Objective:A high sensitivity and accuracy method was established for the determination of monoester alkaloids in Aconiti Radix Cocta and the extraction and purification process was optimized.To establish and verify a method for the determination of the concentration of benzoylmesaconine in rat plasma,the pharmacokinetic characteristics of benzoylmesaconine in rats was studied,which provided basis for the pharmacokinetic study of preparation of Aconiti Radix Cocta.Methods:High performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）was used detect levels of benzoylaconine,benzoylmesaconine,and benzoylhypaconine.The chromatographic separation was performed on an Ultimate AQ-C18 column（3.0μm,2.1×100mm）under MRM mode detection mass spectrometry,column temperature was 40℃,mobile phase was 0.1%formic acid water-methanol=55:45,flow rate was 0.4m L·min^-1.The method to determine the concentration of three kinds of monoester alkaloids in Aconiti Radix Cocta was established.Through single factor test and orthogonal test,the factors such as grinding particle size,extraction times,ethanol concentration,ethanol dosage,extraction time and so on were investigated to determine the best extraction process.In order to determine the best purification process of monoester alkaloids,the factors such as the concentration of loading solution,p H value of loading solution,desorption solution concentration,flow rate of loading solution and maximum loading volume were investigated.The HPLC-MS/MS method for the determination of the concentration of benzoylmesaconine in rat plasma samples was establishedand verified.Under the MRM mode,m/z 590.5→540.4 and m/z 646.7→587.0were taken as the components to be measured and the internal standard mass spectrometry detection conditionsrespectively.The separation was performed on an Ultimate AQ-C18column（3.0μm,2.1×100mm）at 40℃with acetonitrile（A,containing 0.1%formic acid）-0.1%formic acid solution（B）as mobile phase at the flow rate of 0.4m L·min^-1and sample volume of 10μL.Rats in the intragastric group were given 5,10 and 20 mg·kg^-1neocaconitine solution,and rats in the intravenous group were given 1 mg·kg-1neocaconitine intravenously.Blood samples were collected at 0h,0.5h,1h,1.5h,2h,2.5h,3h,4h,6h,9h,12h and 24h after administration in the gavage group.Blood samples were collected at 0h,0.05h,0.167h,0.33h,0.5h,1h,2h,4h,6h,9h,12h,24h and 36h in the intravenous injection group.The plasma samples were extracted by liquid extraction with methyl tert-butyl ether and the concentration was determined.DAS 2.0 was used to calculate the pharmacokinetic parameters.Results:The optimum extraction parameters for monoester alkaloids were as follows:25 times 75%ethanol reflux extraction for two times,each extraction time was1.5 h;The purification process parameters of macroporous resin were as follows:the loading solution concentration was 0.50g·m L^-1,p H value was 3.5,the flow rate of the loading solution was 2BV/h,and the maximum loading volume was 36m L.The linear range of the mass concentration of benzoylmesaconine in plasma samples was 0.1～1 000ng·m L^-1.The lower limit of quantitation was 0.1 ng·m L^-1.The recoveries of benzoylmesaconine and internal standard were>93%,and intra-day and inter-day RSDs were lower than 10.7%.When the plasma concentrations were 0.2,5.0 and 200.0 ng·m L^-1,the extraction recoveries of the benzoylmesaconine and the internal standard were both high and stable,and the matrix effect was low.The change rate of the benzoylmesaconine sample at room temperature was<4.6%.The Tmax of the rats was about 2 h after oral administration of benzoylmesaconine,the t_1/2is about 5 h,but the oral absorption was poor and the bioavailability was less than 1%.The AUC was positively correlated with the dose.Conclusions:The optimized extraction and purification methods for monoester alkaloids in Aconiti Radix Cocta were stable and feasible,which provided theoretical support for future industrial production.The experiment established a method for detecting the concentration of benzoylmesaconine in rat plasma.The method was easy to implement,and highly sensitive,which provided support for the pharmacokinetic study of monoester alkaloids.