Study On The Pharmacokinetics And Dynamic Distribution Of Strychnine And Its Metabolites In Rats

Objective:1.To establish a method for the determination of strychnine（STR）and its metabolite strychnine nitrogen oxide（SNO）in rat plasma,tissues and organs by high performance liquid chromatography tandem mass spectrometry（HPLC-MS/MS）.2.To establish the pharmacokinetics model of strychnine and strychnine nitrogen oxide in male and female rats,and to study its pharmacokinetics law;to study the relationship between strychnine and strychnine nitrogen oxide and the time of administration,and to establish the inference method of strychnine administration time,so as to provide experimental basis for forensic identification of strychnine poisoning cases.3.To study the distribution of strychnine and strychnine nitrogen oxides in rats,so as to provide experimental basis for forensic identification of death related cases of strychnine poisoning.Methods:1.Pretreatment and detection:1.1 plasma samplesThe 180μL plasma sample was precisely transferred,and the internal standard working liquid（ephedrine hydrochloride was 100ng/mL）20μL was added,and the scroll was mixed for 15s.1ml ACN was added,the vortex was 20s,13000rpm/min was centrifuged for 10min,and the supernatant was removed to the clean tube,and the residue was dried by nitrogen at 35°C and the residue was dried with 200μL mobile phase（MEOH-10mmol/L ammonium formate（adjusted by formic acid pH=4）,40:60）was dissolved again,and the vortex was 30s.the supernatant was taken to pass0.22μm organic membrane and 5μL was injected.1.2 organ samplesTake 200mg of the tissue to be tested,add 200μL of high-purity water and 60μL（1mol/L）hydrochloric acid,and then cut it thoroughly;add 20μL of internal standard working solution（ephedrine hydrochloride 100ng/mL）,vortex for 15s,mix well,add 1ml of ACN,vortex for 20s,and centrifuge at 13000rpm/min for 10min,transfer the supernatant to a clean test tube,blow dry with nitrogen at 35°C,and use 200μL of mobile phase（MEOH-10mmol/L ammonium formate（adjusted by formic acid pH=4）,40:60）,vortex for 30s,take the supernatant through 0.22μm organic membrane,5μL injection.2.Model building:2.1 pharmacokinetics modelForty SD rats,half male and half female,weighing 200±20g（purchased from Beijing Changyang Xishan farm）,were fasted for 12h after adapting to the environment for one week,and then gavaged.Two male rats and two female rats were randomly selected as blank control group.Methods:18 male SD rats were randomly divided into three groups with 6 rats in each group;18 female SD rats were randomly divided into three groups with 6 rats in each group.STR solution was given by gavage according to1/2LD₅₀,1/4LD₅₀and treatment dose（0.27 mg/kg）.Blood was taken from orbit at 5,10,15,30,45,60,90,120,240,360,480,720,1440,2880 min after gavage,and plasma was separated by centrifugation at 5000 rpm/min for instrument analysis.2.2 distribution modelForty four SD rats,half male and half female,were fasted for 12 hours after one week of adaptation.Two male rats and two female rats were randomly selected as blank control group.Methods:20 male SD rats were randomly divided into four groups according to the time points,with 5 rats in each group;20 female SD rats were randomly divided into four groups according to the time points,with 5 rats in each group.STR solution was given by gavage with 1/4 LD₅₀.The rats were killed at 15,30,120 and 1440min after gavage,respectively.The heart,liver,spleen,lung,kidney and brain were separated,and the organs were washed with ultrapure water and dried with filter paper for instrumental analysis.Results:1.HPLC-MS/MS analysis and detection of strychnine and its metabolites:The linear ranges of strychnine and strychnine nitrogen oxides in rat plasma were0.25-300ng/mL and 0.05-100ng/mL,respectively.The minimum detection limits of strychnine and strychnine nitrogen oxides were 0.1ng/mL and 0.01ng/mL,respectively.The lower limits of quantitation were 0.25ng/mL and 0.05ng/mL,respectively.The linear ranges of strychnine and strychnine nitrogen oxides in rat liver were 1-1000ng/g and1-300ng/g,and the minimum detection limits of strychnine and strychnine nitrogen oxides were 0.25ng/g and 0.25ng/g,respectively.The lower limits of quantitation were0.75ng/g and 0.80ng/g,respectively.The correlation coefficients（r）of strychnine and strychnine nitrogen oxides in blood and liver of rats were greater than 0.999,the intra-day and inter-day precision were less than 20%,the extraction recoveries were more than 80%,and the matrix effects were in the range of 70%～120%.2.Pharmacokinetic:In this study,non atrioventricular model was used to analyze the data.The results showed that the peak concentration of strychnine and strychnine nitrogen oxide in plasma and the area under the time curve of plasma concentration of strychnine and strychnine nitrogen oxide in female and male rats increased with the increase of intragastric dose;Under the three doses,the peak time of strychnine and strychnine nitrogen oxide in the plasma of female rats was 30 min,and that of male rats was 45 min;At the same dose,the peak concentration of strychnine in the plasma of female rats was higher than that of male rats,and the peak concentration of strychnine nitrogen oxide in the plasma of female rats was similar to that of male rats;The elimination half-life of strychnine in male rats was longer than that in female rats;The plasma clearance rates of strychnine and strychnine nitrogen oxides in male rats were higher than those in female rats.3.Time inference based on metabolic dynamics:The average concentration time curves of strychnine and strychnine nitrogen oxides in male and female rats at different doses fit well,R²is mostly above 0.90.Some experimental data were randomly selected to calculate the error of medication time.The results show that the error of medication time is mostly less than 20%based on the average concentration of strychnine and strychnine nitrogen oxides in male and female rats’plasma and the corresponding curve equation.4.Distribution of strychnine and strychnine nitrogen oxides in rats:The rats were inoculated by oral gavage with a dose of 1/4LD₅₀,and the strychnine content in the tissues and organs of the dead animals from high to low was:Female group:15min death group:liver（29）kidney（29）lung（29）spleen（29）heart（29）brain;30min death group:liver（29）kidney（29）lung（29）spleen（29）heart（29）brain;120min death group:liver（29）kidney（29）spleen（29）lung（29）heart（29）brain;720min death group:kidney（29）liver（29）spleen（29）lung（29）brain（29）heart.Male group:15min death group:liver（29）kidney（29）lung（29）spleen（29）heart（29）brain;30min death group:liver（29）kidney（29）lung（29）spleen（29）heart（29）brain;120min death group:kidney（29）liver（29）spleen（29）lung（29）heart（29）brain;In the death group at 720min,a small amount of strychnine was detected in liver and kidney.The distribution of strychnine in female and male rats was basically the same;with the increase of time,the content of strychnine in various organs of male and female rats decreased gradually;at the same time,the content of strychnine in various organs of female group was significantly different from that of male group,and the concentration of strychnine in various organs of female rats was higher than that of male rats.Strychnine nitrogen oxide was only detected in the liver,kidney,lung and spleen of the 15min and 30min death groups,but the content of strychnine nitrogen oxide was relatively low,and some of the values were even lower than the lower limit of quantification.Conclusion:1.The HPLC-MS/MS method for the detection of strychnine and its metabolites was established,and the sample pretreatment process was simple and rapid.The method has high sensitivity and recovery rate,and can be used for the determination of strychnine poisoning and death cases.2.The pharmacokinetic model of strychnine and strychnine nitrogen oxide in male and female rats was established.The data were analyzed by non atrioventricular model.The pharmacokinetic parameters of strychnine and strychnine nitrogen oxide were obtained,which could provide experimental basis for forensic identification of strychnine poisoning and death cases.3.This study established a method to infer the duration of strychnine administration after oral exposure.The fitting equations between the mean plasma concentrations of strychnine and strychnine nitrogen oxides in blood and the medication time had a high fitting degree within 0～48h,with R²greater than 0.82.4.At the same time,the concentration of strychnine in female rats was higher than that in male rats;the distribution of strychnine in male and female rats was basically the same,and it was higher in liver and kidney.Strychnine nitrogen oxide can be detected in major organs,but its concentration is far lower than that of protostrychnine,indicating that Strychnine nitrogen oxide can be produced by in vivo transformation of strychnine,but it may not be the main metabolite of strychnine in vivo.