Pharmacokinetic Study Of Sustained-release Microspheres Huperzine A In Vivo

AIM: This study will establish a quantitative method of low concentration of huperzine A in human plasma with LC-MS/MS,which was fast,exclusive,accurate and sensitive.Then we will observe how does the concentration of sustained-released microspheres huperzine A for injection in plasma change,estimate the human pharmacokinetic parameters,evaluate pharmacokinetic characteristics of sustained-released microspheres huperzine A in healthy subjects and the effect of sustained-release,explore how huperzine A metabolizes,find potential active metabolites to reveal the changes in human bodies more clearly and provide theoretical basis for new drug development and clinical analysis of toxic and side effects after administration of huperzine A.METHOD: The method of liquid-liquid extraction was developed to prepare the human huperzine A plasma samples,the simultaneous quantitative analytical assay was developed by LC-MS/MS.Chromatographic system was performed using a reversed-phase Ascentis C18 column(10 cm×2.1 mm,5 mm)with a gradient elution involved 10 m M ammonium acetate as solvent A and methanol as solvent B.Positive electrospray ionization mass spectrometry with multiple reaction monitoring(MRM)mode was applied to achieve 10-3000 pg/m L linear range in human plasma.Transition channels of the protonated molecular ions were m/z 243.1? m/z 210.1,and m/z 285.2? m/z 193.1 for huperzine A and diazepam.Our study is designed as a randomized,double-blind,placebo-controlled trial.Healthy adult subjects are selected to be injected a single dose of the drug in five doses(0.75 mg,1.5 mg,3.0 mg,4.5 mg and 6.0 mg).1 m L plasma samples is extracted by 7 m L Ethyl ether-dichloromethane(3:2,v/v),and then dissolved by methanol-water(50:50,v/v)100 ?l,of which 50 ?l was used for LC-MS/MS analysis.Diazepam was selected as internal standard,which was analyzed with huperzine A through the mass spectrum detector after separation in the chromatographic system via Ascentis-C18 column(10 cm x 2.1 m M,5?m).we choose electrospray ionization(ESI)source,positive ion mode test,and the multiple reaction monitoring(MRM)mode,which was used for quantitative analysis of m/z 243.1 to m/z 210.1(huperzine A)and m/z 193.1 to m/z 285.2(diazepam)respectively.After all,we established a quantitative method of huperzineA from every time point in human plasma with(LC-MS/MS),then estimate the pharmacokinetic parameters with DAS 3.0 software,evaluate pharmacokinetic characteristics and the effect of sustained-releasing of huperzine A on the base of the data.After administration of huperzine A we collected the excrement(urine and feces)from the subjects,which was processed by Oasis HLB SPE column and then analyzed by mass spectrometry.We combine the chromatograms,MS,MS/MS and the structure of huperzine A to infer what metabolites may occur.RESULTS: The method for the quantification of huperzine A with strong specificity,excellent quality accuracy and superior sensitivity was.suitable for pharmacokinetic analysis of huperzine A sustained-released microspheres.The LLOQ was 10 pg/m L,the analysis time was 8.50 min for each sample,,accuracy was between 85.33% and 112.33%,intra-and inter-day precisions was terms of relative standard deviation(RSD)were ?9.73% and ?9.63%.The extraction recovery was high and the matrix had little effect for determination of huperzine A and internal standard diazepam.Results of stability study showed that the plasma samples of huperzine A were stable after placed in-80°C refrigerator for 360 days,repeated freezing and thawing three times in-80°C,placed 4 h in room temperature and vial placed after extracted for 4 h.The AUC0-t,AUC0-? and Cmax of huperzine A sustained-released microspheres in this study had linear correlation with the doses,the CL does not rely on the doses,huperzine A had linear pharmacokinetics among doses of 0.75mg~6.0mg.The peak time and half-life were prolonged obviously.Huperzine A can be sustained-released,the time of sustained-releasing prolonged greatly and the effect of sustained-releasing are best compared with other sustained-released formulations.In combination with the data of metabolism experiment,we speculated desaturation,oxidation,and desaturation after deamination may occur in vivo.