| Background:Semen Strychni,officially listed in Chinese Pharmacopoeia,has good research values and application prospect.As the predominant active constituents and the main toxic constituents of Semen Strychni,strychnine and brucine are research objects in this study.The purpose of this paper is to compare the pharmacokinetics of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and three toxic doses,to investigate the effects of different form(namely,the total strychnos alkaloids,strychnine monomer and brucine monomer.)on the toxicity and pharmacokinetics of strychnine,brucine and their metabolites in rats,and to further study the tissue distribution of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats.The results of the present study might illustrate the rule of occurrence and development of the toxicity of strychnine and brucine in vivo and provide a basis for toxicity mechanism of strychnine and brucine,which is of great significance for the rational use of Semen Strychni in clinical and can lead to a better understanding on the toxicity and pharmacokinetics of Semen Strychni.Objective:1.A rapid,specific and sensitive liquid chromatography-tandem mass spectrometric(LC-MS/MS)method was developed and validated for the simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rat plasma.2.To investigate the pharmacokinetics of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and three toxic doses by means of the established analytical method-LC-MS/MS.3.To compare the pharmacokinetics of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni,strychnine monomer and brucine monomer at normal dose and high toxic dose by means of the established analytical me t ho d-LC-MS/MS.4.A rapid,specific and sensitive liquid chromatography-tandem mass spectrometric(LC-MS/MS)method was developed and validated for the simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide in tissue samples.5.To study the tissue distribution of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and toxic doses by means of the established analytical method-LC-MS/MS.Methods:1.LC-MS/MS method for simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide:Ephedrine hydrochloride was used as the internal standard(IS).The protein precipitation by methanol was used to extract the analytes from plasma.The HPLC separation was conducted on an SHIMADZU LC-20A HPLC system(SHIMADZU,Japan)equipped with a ZORBAX Eclipse XDB-C18 column(2.1×150 mm,3.5?m).The mobile phase consisted of phase A(water,10 mM ammonium acetate,adjusted to pH 4.0 with formic acid)and B(methanol).The gradient program started as follows:0?1 min,5%B;1?6.5 min,5%?70%B;6.5?7.5 min,70%B;7.5?7.6 min,70%?5%B;7.6?11 min,5%B.The flow rate was 0.2 mL · min--1.The column temperature was set at 30?.The injection volume was 5?L.The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization(ESI)inlet in the positive ion multiple reaction monitoring(MRM)mode.The AutoMS operation parameters are described as follows:positive ion mode(ESI+);IonSpray voltage,5500 V;turbo gas temperature,350?;curtain gas,40 psi;collision gas,12 psi;nebulizer,20 psi.2.The pharmacokinetic of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and three toxic doses:Rats were divided randomly into four groups(n=6 in each group).The total alkaloids from Semen Strychni at a normal dose of 1.2 mg·kg-1 and three toxic doses(1.8 mg · kg-1,2.4 mg · kg-1 and 3.0 mg · kg-1)were administered orally to six rats at each group.Blood samples were withdrawn at different times.The blood samples were determined by the established LC-MS/MS.DAS 2.0 software was applied to calculate the pharmacokinetic parameters while the SPSS 19.0 software was used for statistical analysis.3.The pharmacokinetic of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni,strychnine monomer and brucine monomer at normal dose and high toxic dose:Rats were divided randomly into four groups(n=6 in each group).The total alkaloids from Semen Strychni,Strychnine monomer and brucine monomer were administered orally at a normal dose of 0.4 mg · kg-1 for strychnine and 0.2554 mg · kg-1 for brucine as well as at a toxic dose of 1 mg · kg-1 for strychnine and 0.6385 mg · kg-1 for brucine to six rats at each group.Blood samples were withdrawn at different times.The blood samples were determined by the established LC-MS/MS.DAS 2.0 software was applied to calculate the pharmacokinetic parameters while the SPSS 19.0 software was used for statistical analysis.4.LC-MS/MS method for simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide:Ephedrine hydrochloride was used as the internal standard(IS).The liquid-liquid extraction with chloroform was used to extract the analytes from tissue samples.The HPLC separation was conducted on an SHIMADZU LC-20A HPLC system(SHIMADZU,Japan)equipped with a ZORBAX Eclipse XDB-C18 column(2.1×150 mm,3.5?m).The mobile phase consisted of phase A(water,10 mM ammonium acetate,adjusted to pH 4.0 with formic acid)and B(methanol).The gradient program started as follows:0?1 min,5%B;1?6.5 min,5%?70%B;6.5?7.5 min,70%B;7.5?7.6 min,70%?5%B;7.6?11 min,5%B.The flow rate was 0.2 mL · min-1.The column temperature was set at 30?.The injection volume was 5?L.The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization(ESI)inlet in the positive ion multiple reaction monitoring(MRM)mode.The AutoMS operation parameters are described as follows:positive ion mode(ESI+);IonSpray voltage,5500 V;turbo gas temperature,350?;curtain gas,40 psi;collision gas,12 psi;nebulizer,20 psi.5.The tissue distribution of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and high toxic dose:Rats were divided randomly into two groups.The total alkaloids from Semen Strychni at a normal dose of 1.2 mg · kg-1 and a high toxic dose of 3.0 mg · kg-1 were administered orally to rats at each group.Rats were executed at different times and blood,heart,liver,spleen,lung,kidney and brain were all taken out.The tissue samples were determined by the established LC-MS/MS.Results:1.LC-MS/MS method for simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide:The calibration curves of strychnine and brucine in plasma were linear in the range of 0.510?306.3 ng · mL-1.The calibration curves of strychnine N-oxide and brucine N-oxide in plasma were linear in the range of 0.102?306.0 ng · mL-1.The accuracy of strychnine,brucine,strychnine N-oxide and brucine N-oxide were(89.44±3.43)%?(113.01±5.01)%.The extraction recovery were(73.60±9.70)%?(90.50±1.64)%.The inter-day and intra-day RSDs were less than 14.88%.The stability were(88.92±2.92)%?(116.90±3.30)%.2.The pharmacokinetic of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and three toxic doses:Over the dose range(strychnine 0.4?1 mg · kg-1,brucine 0.2554?0.6385 mg · kg-1)studied,the mean Cmax and AUC of strychnine and brucine increased and proportional to the doses.The SPSS 19.0 software was used for statistical analysis and no significant difference in Cmax?t1/2 and AUC of strychnine and brucine was found between normal dose and three toxic doses.3.The pharmacokinetic of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni,strychnine monomer and brucine monomer at normal dose and high toxic dose:the mean Cmax? AUC of strychnine and brucine in the total alkaloids groups are lower than those in the monomer groups.The SPSS 19.0 software was used for statistical analysis and no significant difference in Cmax?t1/2 and AUC of strychnine and brucine was found between monomer and the total alkaloids from Semen Strychni at corresponding dose.4.LC-MS/MS method for simultaneous determination of strychnine,brucine,strychnine N-oxide and brucine N-oxide:The calibration curves of strychnine and brucine in plasma were linear in the range of 1.02?405 ng · mL-1.The calibration curves of strychnine N-oxide and brucine N-oxide in tissue were linear in the range of 0.306-135 ng · mL-1.The accuracy of strychnine,brucine,strychnine N-oxide and brucine N-oxide were(85.94± 1.43)%?(113.77±5.19)%.The extraction recovery were(79.20±8.54)%?(110.00±6.48)%.The inter-day and intra-day RSDs were less than 14.9%.The stability were(89.13±2.19)%?(110.81±3.86)%.5.The tissue distribution of strychnine,brucine,strychnine N-oxide and brucine N-oxide in rats after oral administration of the total alkaloids from Semen Strychni at normal dose and high toxic dose:the distribution of strychnine in normal dose and toxic dose were listed from largest to smallest:kidney>spleen>liver>lung>heart>brain>blood,kidney>liver>lung>spleen>heart>brain>blood.Brucine:kidney>liver>spleen>lung>heart>blood>brain,liver>kidney>spleen>lung>heart>blood>brain.Strychnine N-oxide:spleen>liver>kidney>lung.Brucine N-oxide was only detected in liver after oral administration of the total alkaloids in high toxic dose.Conclusion:In this study,a rapid,sensitive,accurate and reliable LC-MS/MS method was developed and validated to quantify strychnine,brucine,strychnine N-oxide and brucine N-oxide in rat biological samples simultaneously.The results showed that strychnine and brucine were both fast absorbed in vivo.Over the dose range(strychnine 0.4?1 mg · kg-1,brucine 0.2554?0.6385 mg · kg-1)studied,the mean Cmax and AUC of strychnine and brucine increased and proportional to the doses.The similar general trends of plasma concentration-time curves reveal the similar ADME system of strychnine and brucine in different doses.In comparative pharmacokinetics studies,the experimental results demonstrated that some other strychnos alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine.The lower toxicity of the total alkaloids from Semen Strychi in comparison to strychnine monomer and brucine monomer is further supported by the main pharmacokinetics parameters.In tissue distribution study,strychnine,brucine,strychnine N-oxide and brucine N-oxide distribute inordinately in rats.The four detected components in liver and kidney were more than that in other tissues,but the least in brain.Both strychnine and brucine can cross BBB(Blood Brain Barrier)to the brain,which affect the central nervous system. |
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