| Objective:1.To develope and validate an accurate,reliable and efficient liquid chromatography-tandem mass spectrometric?LC-MS/MS?method for the determination of mangiferin in rat plasma and phosphate buffer saline;2.The equilibrium dialysis method combined with LC-MS/MS was employed to determine the plasma protein binding rate of mangiferin in diabetic rats induced by STZ and in normal rats,and then compared the differences from each other;3.To develope and validate a rapid,specific and sensitive liquid chromatography-tandem mass spectrometric?LC-MS/MS?method was developed and validated for the determination of mangiferin in tissue and plasma samples;4.To study the tissue distribution of mangiferin in diabetic rats induced by STZ and in normal rats by means of the established LC-MS/MS method.To understand the difference of distribution characteristics and regularity of mangiferin in various tissues after the normal rats were following single intragastric administration of free mangiferin,Zhimu decoction and Zhimu--Huangbai decoction and the diabetic rats were following Zhimu-Huangbai decoction.Methods:1.LC-MS/MS method for determination of mangiferin in rat plasma and phosphate buffer saline:Rutin was used as the internal standard?IS?.The protein precipitation was used to extract the analytes from plasma.The extractant was acetonitrile:acetic acid?9:1?.The chromatographic separation was carried out on Shiseido CAPCELL PAK C18 column?150 mm×2.0 mm,5?m?.by isocratic elution.Analysis time:5 minutes;the flow rate:0.25 mL · min-1;the column temperature:30?;the injection volume:10?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 MRM transitions of m/z 423.2 ?303.1 and m/z 611.5?303 were used to quantify for mangiferin and rutin.2.The investigation of the protein binding rates of mangiferin with plasma from diabetic rats induced by STZ and normal rats:Rats were randomly divided into two groups?normal rats,diabetic rats?.The equilibrium dialysis method combined with LC-MS/MS was employed to determine the concentration of mangiferin to calculated the plasma protein binding rates according to the formula,and analyze the data statistically.3.LC-MS/MS method for determination of mangiferin in rat plasma and tissue:Rutin was used as the internal standard?IS?.The protein precipitation was used to extract the analytes from plasma and tissue.The extractant were acetonitrile:acetic acid?9:1?for plasma and acetonitrile:acetic acid?40:1?for tissue.The chromatographic separation was carried out on Shiseido CAPCELL PAK C18 column?150 mm×2.0 mm,5 um?.Using gradient elution,analysis time:7 minute;the flow rate:0.3 mL · min-1;the column temperature:35?;the injection volume:10?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.4.The tissue distribution of mangiferin in diabetic rats and normal rats:Rats were divided randomly into four groups.The normal rats following single intragastric administration of free mangiferin,Zhimu decoction and Zhimu-Huangbai decoction?the dose was 35 mg mangiferin/kg body weight?;the diabets rats following single intragastric administration of Zhimu-Huangbai decoction.Rats were executed at different times and blood,various tissues were all taken out.The tissue samples were determined by the established LC-MS/MS to calculate the concentration of mangiferin.To investigate the distribution of free mangiferin in normal rats and to compare the differences of the distribution of free mangiferin group and Zhimu decoction and Zhimu-Huangbai decoction.Meanwhile,After administration of the same dose of mangiferin in Zhimu-Huangbai decoction,compared the distribution of mangiferin in normal rats with diabetic rats.Results:1.LC-MS/MS method for determination of mangiferin in rat plasma and phosphate buffer saline:The linear concentration range of mangiferin was 200?3000 ng · mL-1 in rat plasma and 50?2000 ng · mL-1 in PBS;the accuracy of mangiferin was 94.7%?108.6%,the inter-day and intra-day relative standard deviation?RSD?was less than 11.3%;the recovery and the matrix effect of mangiferin were 73.3%?73.7%and 67.2%?73.5%.2.The protein binding rates of mangiferin:The plasma protein binding rate of mangiferin in diabetic rats plasma at the concentrations of 100,200 and 500 ng · ml-1 were?84.5±1.4?%,?82.6±1.4?%and?82.4±1.7?%,respectively;while the plasma protein binding rate of mangiferin in normal rats plasma at the above concentrations were?65.5±0.6?%,?66.6±1.0?%and?68.4±0.7?%.The difference was statistically significant from diabetic rats and normal rats.The average plasma protein binding rate of mangiferin in diabetic rats was higher than that in normal rats.3.LC-MS/MS method for determination of mangiferin in rats plasma and tissue:The linear concentration range of mangiferin was 1?600 ng· mL-1 in rat plasma and 2?500 ng · mL-1 or 5?2000 ng · mL-1 in rat tissue;the accuracy of mangiferin was 97.2%?111.7%,the inter-day and intra-day relative standard deviation?RSD?was less than 14.0%;the recovery and the matrix effect of mangiferin were 30.2%?84.2%and 30.6%?78.0%.4.The tissue distribution of mangiferin in diabetic rats and normal rats:After oral administration of free mangiferin,mangiferin could be detected at just 10 minutes in heart,liver,spleen,lung,kidney,stomach,intestine,skeletal muscle and pancreas tissues except brain.The distribution in each tissue in free mangiferin group was lower than that in the Zhimu group and Zhimu-Huangbai group.The contents in many tissues in the Zhimu-Huangbai group were lower than that in the Zhimu group.However,in pancreas,the distribution of mangiferin in Zhimu-Huangbai group were significantly higher than that in Zhimu group.After oral administration of Zhimu-Huangbai decoction,the distribution of mangiferin in pancreas and small intestine in the diabetic rats was lower than that in the normal rats,but still significantly higher than that in other tissues.In liver,spleen,lung,kidney,stomach or blood,the distribution was significantly higher in diabetic rats than in the normal rats.Conclusion:In this study,a rapid,sensitive,accurate and reliable LC-MS/MS method was developed and validated to quantify mangiferin in rat biological samples.The results showed that the protein binding rates of mangiferin with diabetic rats plasma was significantly higher than normal rats plasma.The distribution of mangiferin in normal rats was rapid and extensive,but there were less tissue distribution and no distribution in the brain following single intragastric administration of free mangiferin,meanwhile,the contents in stomach and small intestine were great.In comparative tissue distribution studies,the experimental results demonstrated that the distribution of mangiferin in the Zhimu decoction group were significantly higher than that in the free mangiferin group,suggesting that other active ingredients in Zhimu may promote the distribution of mangiferin.Combined with Huangbai the distribution of mangiferin in pancreas was significantly increased,suggesting that some of the components in Huangbai may promote the distribution in pancreas tissue.The microenvironment of rat disease may also affected the absorption of mangiferin.The distribution of mangiferin in liver,spleen,lung,kidney and stomach in diabetic rats was significantly higher than that in normal rats.At the same time,these organs are also the main lesion of diabetic rats and their complications.According to the characteristics of drug distribution,the target organ of drug effect can be effectively predicted,which is of great significance to expand the clinical use of mangiferin. |
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