The Analysis Of Fat-soluble Extracts From Arnebia In Biological Samples By High Performance Liquid Chromatography

Objective: To establish the HPLC method for detection of Arnebia euchroma lipid soluble components,and apply to determination of drug concentration in vitro and vivo,Investigate disposal situation of Arnebia euchroma lipid soluble components in vivo.Methods: By comparing the separation effect of different mobile phases and its influencing factors,established HPLC method for the determination of Arnebia euchroma lipid soluble components in vivo and virto concentration.(2)Investigate methodological indicators.(3)Comparative analysis of the drug extraction effect in different organic solvent extraction,acidification and alkalization treatment in blood.(4)Investigated the applicability and effect of plasma and serum samples.(5)study on Arnebia euchroma lipid soluble components in different pH environment,simulated gastric fluid and intestinal fluid simulation stability test;(6)Administration of gastric to rats,collected blood samples at different time before administration and after administration respectively,and Separation of serum and plasma,collected the urine and feces before and after administration for 48 hours,and HPLC analysis,determination of the concentration of these samples in shikonin,acetylshikonin,?-? dimethyl acryloyl shikonin pigment or single component.Results: The Chromatographic conditions is : C18-AR-COSMOIL II spectrum column(4.6mm x 5?m,250mm);flow: 1.0mL/min;column temperature: 25?;detection wavelength: 275 nm,mobile phase A liquid acetonitrile,B liquid-formic acid: water(0.5:300)(v/v)solution,sample volume 20?m,gradient elution0~10 min(60-40)%;15~30min(70-30)%;30~35min(60-40);Detection time asked 35 min.Under this chromatographic condition,extract of Arnebia euchroma lipid solubility shikonin,acetylshikonin and ?-? dimethyl acryloyl shikonin factor was get better separation.(2)The methodology indexes meet the requirements: The retention time of each target peak was RSD<1% in the precision test.The relative peak area of 12 h in the sample was RSD < 3%,The relative peak area of repeatability test RSD<3%(3)The conditions for the extraction and separation of drug in plasma and serum samples were: Samples were acidification first diluted with equal volume of hydrochloric acid,Extraction with ethyl acetate for 3 times,Acetonitrile precipitation protein.Plasma samples showed a good linear relationship with peak area in the range of 5?g/ml ~500?g/ml concentration.Regression equation of shikonin: y=20.613 x +979.88;acetylshikonin: y=24.254x-57.209;?-? 2 methyl acryloyl alkannin: y=48.406 x +3180.5;Correlation coefficients were 0.997,0.9957 and 0.9935 respectively.(4)The applicability of the sample,plasma samples is more suitable for Arnebia euchroma lipid soluble extract composition determination,in serum samples to add the right amount of heparin,peak shape and peak area was improved.(5)arnebia lipid soluble components(L-shikonin,acetylshikonin,? ? two methylacryloyl shikonin)relative stability stable in pH2.5 phosphate buffer solution and artificial gastric juice,But with the extension of time,the content showed a slow downward trend,in pH5.8 phosphate buffer showed a clear downward trend,with the increase of pH value,the content decreased rapidly.(6)The rats by gavage to drug after 0-48 hours to collect samples of blood and urine are not detecte the arnebia euchroma fat soluble extract contains prototype material specific peak,but there were found in the feces which L-shikonin,acetylshikonin,?-? two methylacryloyl shikonin content peak.Conclusion: after intragastric rats Arnebia euchroma lipid extract has not detecte in blood samples and urine,and the extract was found to be related to the extract.Explain to extract in the gastrointestinal tract almost does not absorb or absorb very little Arnebia euchroma fat soluble,the absorption,distribution,metabolism,excretion process to be further research in the future.