| Objective GuBishu Tablet is a proprietary Chinese medicine for the treatment ofrheumatoid arthritis. Mainly contain Semen Strychni, fleece-flower root, mulberryparasitism, astragalus root and other Chinese native medicine ingredient. SemenStrychni is involving GuBishu Tablet in one of the main medicinal materials, the SemenStrychni of brucine and strychnine are effective ingredients and toxic ingredients, inorder to ensure the quality of involving GuBishu Tablet of controllable,Clinicalmedication safety, effective, the establishment of involving the GuBishu Tablet in thecontent determination brucine and strychnine then the method; Further establish twoingredients in the determination of biological sample analysis method, Study of brucineand strychnine pharmacokinetic process of compound preparations, understanding thefactors that affect system of Semen Strychni prescription, experiments to SemenStrychni, ReBiqing Tablet for certification as a comparison, for clinical rational drug useand strychnine and people’s better in the body’s metabolism research to provide certainreference basis.Methods1.GuBishu Tablet in the brucine and strychnine establishment of the contentdetermination method. With the content of brucine and strychnine as indicators, ofextraction solvent and extraction method of sample preparation, methods of extractingtime were investigated; HPLC methodology for further investigation, the establishmentof measurement involving the bone shu in the strychnine and then better contentdetermination method.2.Brucine and strychnine in biological samples and the levels of the establishmentof the measurement method. The preparation of biological samples by usingliquid-liquid extraction and high-speed centrifugal separation and purification in the form of peak area as indicators, the chloroform, methyl tertiary butyl ether and n-hexane-methylene chloride-isopropyl alcohol extraction solvent, three and further examinethe condition such as extraction solvent dosage, extraction times. Mass spectrum-theestablishment of the chromatographic conditions, with clenbuterol hydrochloride as theinternal standard, with peak area values and ionic strength as evaluation index, theoptimum Cone Voltage hole voltage, collision parameters to establish mass spectrometryanalysis method; Based on types of mobile phase and mobile phase pH value, optimizingthe parameters of the ratio of mobile phase components and methodologies,chromatographic analysis method is established.3.Research involving the GuBishu Tablet and Semen Strychni in rats in vivopharmacokinetic process. Using rats lavage for medicine and the blood, tail byUPLC-MS-MS method was developed for the determination of brucine and strychninelevels, pharmacokinetic model is set up, discuss with the same dose of brucine andstrychnine involving the GuBishu Tablet and Semen Strychni in rats in vivo metabolicregularity of the absorption of differences.Results1. To establish HPLC method for determination of involving GuBishu Tablet inthe brucine and strychnine method for the determination content. Sample preparation usealkalescent chloroform as solvent, the ultrasonic extraction for60min. Chromatographiccolumn for Agilent C18column (4.6mm*250mm,5microns); Mobile phase of methanol25%: buffer (water, acetic acid, triethylamine=230:2.4:0.3) by75%; The flow rate of1.0mL/min; Detection wavelength of254nm; Column temperature25℃; Samplequantity10μL.Brucine in0.0948~1.1376μg(r2=1), Strychnine in0.1208~1.4496μg(r2=0.9999)within the scope of linear is good. The average recovery strychninewas97.37%, RSD was1.26%. Strychnine is99.01%, RSD was0.91%.2.Establish UPLC-MS-MS determination of brucine and strychnine in biologicalsamples and then levels were determined. The preparation of biological samples bymethyl tert-butyl ether liquid liquid extraction and high-speed centrifugal method forseparation and purification. With clenbuterol hydrochloride as the internal standard,mass spectrometry conditions: ionization methods: electrospray (ESI), ion polarity:positive (+); Working parameters: Capillary (KV)3.50, Extractor (V)4.00, the SourceTemperature (110℃), DesolvationTemperature (300℃), Cone Voltage Gas Flow (L/Hr),50Desolvation Gas Flow (L/Hr)300, Gas Cell Pirani Pressure3.26e-3(mbar);Chromatographic conditions: chromatographic column for Agela Technologies VenusilASB C18column, mobile phase of acetonitrile:0.15%formic acid solution (40:60); Column temperature35℃; The flow rate of0.1mL/min,5mu L sample quantity. In thelinear range of brucine in biological samples from19~0.074ng/mL(r2=0.991171), thelinear range of strychnine0.198~50.75ng/mL(r2=0.996467). Brucine and strychnine thebetter average recovery are87.25%and87.76%, RSD was7.29%and7.29%respectively; The average matrix effect was93.48%and93.48%, RSD were5.87%and6.24%respectively.3.Rat oral lavage for the same content of the brucine and strychnine involvingGuBishu Tablet and Semen Strychni, the determination of strychnine in analysis of twokinds of samples brucine and strychnine in rats in vivo pharmacokinetic process are thesecond chamber model. GuBishu Tablet in the brucine and strychnine tmax27.5min and37.5min, respectively, up to peak concentrations were5.467ng/mL and22.483ng/mLand CL/F were4205.938L/min/kg and654.955L/min/kg, AUC(0-∞)were973.58ng/L·minand4526.795ng/L·min, Ka were0.068min-1and0.046min-1, t1/2kawere14.726min and9.285min, t1/2β285.299min and285.299min, t1/2α25.849min and45.958min.SemenStrychni of brucine and strychnine tmax40min and42.5min, respectively, up to the peakconcentration were4.667ng/mL and15.9ng/mL; CL/F were86.991L/min/kg and49.042L/min/kg, AUC(0-∞)were759.734ng/L·min and2135.14ng/L·min, Ka were0.235min-1and0.063min-1, t1/2kawere5.681min and9.285min, t1/2β306.33min and306.33min,t1/2α74.974min and45.958min.Conclusion1.This experiment to establish the determination of brucine and strychnineat the same time, better quantitative analysis method of high performance liquidchromatography, the method is simple, quick, complete, accurate and reliable, can beused as strychnine and method for the determination of the brucine and strychninecontent.2.This experiment in order to further study involving GuBishu Tablet in vivoabsorption, metabolism, build UPLC-MS-MS determination of brucine and strychnine inbiological samples and then levels were determined. This method is of high sensitivity,selectivity, short analysis time, suitable for in rat plasma concentration determination ofbrucine and strychnine. Biological sample pretreatment with liquid-liquid extraction,tert-butyl methyl ether with clenbuterol hydrochloride as the internal standard, adopt theway of high-speed centrifugal purification, biological sample not only pure nointerference, and extraction rate reached more than80%, in line with the requirements ofbiological sample determination.3.Pharmacokinetic experiment results show that involving GuBishu Tablet, ReBiqing Tablet and Semen Strychni of brucine and strychnine in rats model conformsto pharmacokinetic fannie and Freddie room, three in rats with fast absorption, fastelimination of pharmacokinetic characteristics. GuBishu Tablet and ReBiqing Tablet inthe brucine and strychnine bioavailability, absorption and elimination rate weresignificantly higher than that of Semen Strychni. |
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