Pharmacokinetics Of Main Metabolites Of Epimedium Extract In Rats

Objective:To establish an accurate,sensitive and reliable liquid chromatography–tandem mass spectrometric?LC/MS/MS?method for the simultaneous determination of four major metabolites of Epimedium flavanoids:sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II in rat plasma,tissues,feces and urine.To investigate the pharmacokinetics characteristics,the distribution of different tissues and the rule of excretion of four major metabolites of Epimedium flavanoids in rats.Methods:The methods for determination of rats plasma and tissues homogenate:The plasma and tissue samples of rats were pretreated by liquid-liquid extraction ethyl acetate.The chromatographic separation was carried out on Agilent Eclipse XDB-C18?2.1×150 mm,5?m?column.The mobile phase consisted of0.1%acetic acid-water?solvent A?and ACN?solvent B?.The linear gradient elution program was as follows:0-1 min:30%B,1-6 min:30%B-95%B,6-8.5min:95%B,8.5-9 min:95%B-30%B,9-12 min:30%B.The flow rate was 0.3mL·min^-1 and the column temperature was maintained at 40?.The quantification of the analytes was performed by mass spectrometry with an ESI source in the negative ion multiple reaction monitoring?MRM?scan mode.After a single oral administration of epimedium extract aqueous solution(0.69 mg·Kg^-1),the plasma and tissues homogenate of normal rats were treated with established LC/MS/MS method for the determination of sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II.Draw the concentration-time curve and calculate the main pharmacokinetic parameters,then investigated its distribution in rats.The methods for determination of rats feces and urine:the faecal samples?1 g?were mixed in 5 mL methanol were ultrasonic extraction for 90 min.The urine samples were diluted 1:1 with methanol.The chromatographic separation was carried out on Agilent Eclipse XDB-C 18?2.1×150 mm,5?m?column.The mobile phase consisted of 0.1%acetic acid–water?solvent A?and ACN?solvent B?.The linear gradient elution program was as follows:0-1 min:30%B,1-6min:30%B-95%B,6-8.5 min:95%B,8.5-9 min:95%B-30%B,9-12 min:30%B.The flow rate was 0.3 mL·min^-1 and the column temperature was maintained at 40?.The quantification of the analytes were performed by mass spectrometry with an ESI source in the negative ion multiple reaction monitoring?MRM?scan mode.After a single oral administration of epimedium extract aqueous solution(0.69 mg·Kg^-1),the feces and urine of normal rats were treated with established LC/MS/MS method for the determination of sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II.Calculate the main drainage dynamics parameters,and analysis of excretion characteristics in rats.Results:The linear range of sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II in rats plasma were 0.5²00 ng·mL^-1,and the linearity was good.The intra-day precision relative standard deviation?RSD?was less than 4.8%and the accuracy ranged from 102.4%to 109.3%.The intraday precision relative standard deviation?RSD?was less than 12.8%and the accuracy ranged from 93.3%to 109.3%.The extraction recovery ranged from102.4%-109.3%,relative standard deviation?RSD?was less than 1.9%.The matrix effects ranged from 108.3%to 112.7%,relative standard deviation?RSD?was less than 3.5%.The AUC_0-t of metabolites of the four epimedium flavonoids were 103.25±43.83 ng/L·h,121.97±63.52 ng/L·h,1376.99±776.33 ng/L·h and 71.11±28.28 ng/L·h.The T_max were 11.70±4.55 h,14.40±5.42h,14.40±5.42 h and 12.80±2.19 h.The t_1/2 were 9.74±4.18 h,8.19±3.12 h,8.87±3.23 h,9.28±3.00 h.The linear range of sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II in rats tissues were 0.5²00 ng·mL^-1,and the linearity was good.The intra-day precision relative standard deviation?RSD?was less than 7.7%and the accuracy ranged from 92.8%to 107.6%.The intraday precision relative standard deviation?RSD?was less than 12.5%and the accuracy ranged from 90.1%to 111.6%.The extraction recovery ranged from 66.0%-110.8%,relative standard deviation?RSD?was less than 7.5%.The matrix effects ranged from 91.2%to 113.8%,relative standard deviation?RSD?was less than 8.6%.The accuracy of dilution factors in stomach and small intestine were ranged from 98.2%to 105.8%and 97.3%to 106.1%,the relative standard deviations?RSD?were less than 4.5%and 5.3%.After a single oral administration,the metabolites of four flavonoid glycosides rapidly distributed to various tissues of the whole body at 0.25 h and persisted for a long time.At 24 h,four flavonoid glycoside metabolites were still detectable in all tissues.The vast majority of flavonoid metabolites wrere distributed in the small intestine and stomach,followed by the liver and lung.The distribution of flavonoid metabolites in the kidney were relatively small,and in other tissues were almost the same.The linear range of sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II in rats feces and urine were 0.5²00 ng·mL^-1,and the linearity was good.The intra-day precision relative standard deviation?RSD?of feces was less than 5.2%and the accuracy ranged from 94.2%to 108.3%.The intraday precision relative standard deviation?RSD?was less than 12.8%and the accuracy ranged from 93.4%to 109.0%.The intra-day precision relative standard deviation?RSD?of urine was less than 5.1%and the accuracy ranged from 94.5%to 108.5%.The intraday precision relative standard deviation?RSD?was less than 10.9%and the accuracy ranged from 93.9%to 108.8%.After 36 hours of administration of epimedium extract,the cumulative excretion of rats were 665240.500±296234.200 ng,622723.375±184210.063 ng,8486770.833±2490226.380 ng and 1363643.833±536522.772ng.The ratios of cumulative excretion to administration were 45.500±22.383,39.730±11.389%,47.702±13.733%,26.889±10.301%.The mean accumulated urine excretion in urine were 76.710±82.800 ng,70.148±78.818 ng,2274.088±2962.171 ng,67.974±54.035 ng,and the ratio of cumulative excretion to administration was 0.201±0.204‰,0.164±0.173‰,0.462±0.563‰and 0.049±0.037‰.Conclusion:In this study,an accurate,sensitive and reliable LC/MS/MS method was established and applied to quantify sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II in rat plasma,tissues,feces and urine simultaneously.According to the plasma pharmacokinetic parameters C_max and AUC,indicated that four flavonoid glycosides metabolites metabolized poorly in rats.The t_1/2/2 and MRT showed that the elimination rate of each metabolite in rats is slow,probably due to the intestine was the main route of excretion.After a single intragastric administration of epimedium extract,sagittatoside A,sagittatoside B,2''-O-Rhamnosyl icariside II and icariside II was widely distributed in various tissues.The contents of stomach and small intestines was highest,which may be the plasma concentration-time curve of the main reasons for the double peak.Moreover,the content of four flavonoid glycosides metabolites were low in urine,while were high in feces,the result of excretion indicated that the kidney was not the main excretion pathway of four flavonoid glycosides metabolites,and the main route of excretion was the intestine.