Study On Residues And Tissue Distribution Of Free Gossypol And Its Metabolite Gossypolone In Animal

Objective: 1.To establish a HPLC method to determine the free gossypol and its metabolite gossypolone in different tissue of rat,crucian and chicken,which were feed with homemade cottonseed fodder,contained gossypol.And a UPLC-MS/MS method to analysis the tissue distribution of gossypol and its metabolite in rat.2.To bulid a HPLC method to detect free gossypol and its enantiomers in milk.Using these methods to determine the free gossypol and its metabolite gossypolone in commercial animal derived food.Method: 1.Select rat,crucian and chicken as experimental animals,fed with homemade cottonseed fodder,which contained certain amount of gossypol.After a certain period,experimental animals' oragan and tissue were collected.Including heart,liver,lung,kidney,meat,plasma.When the organ and tissue were taken,0.2% formic acid acetonitrile was used as extract solvent.Glycyrrhetic acid was internal standard substance.The organ and tissue were analysized by HPLC and UPLC-MS/MS after pretreatment of removing protein and centrifugal.Then process the data to determine the distribution and content of gossypol and its metabolite in organ and tissue.2.(R)-(-)-2-amino-1-propanol was chiral selectors.Glycyrrhetic acid was internal standard substance.Precolumn derivatization-HPLC was used to detect free gossypol and gossypol enantiomers in milk.Result: 1.HPLC and UPLC-MS/MS was suitable for detecting gossypol and its metabolite in animal.The linear range of gossypol and gossypolone of rat and chicken was 0.5-20?g/mL,crucian carp was 0.1-10?g/mL.The quantitative limit was 0.1?g/mL.The detection limit was 0.20?g /mL.The concentration range of gossypol and gossypolone in rat tissue and organ was 0.01-10?g/mL detected by UPLC-MS/MS.The quantitative limit was 0.01?g/mL.The content of gossypol and gossypolone in rats was increasd with feeding time.The content of gossypol from high to low was liver,plasma,spleen,lung,heart,testis,kidney,muscle.The gossypolone was only detected in liver.Gossypol accumulated more in female rat than male.2.The detection range of gossypol and its enantiomer was 2-10?g/mL in milk.3.58 samples purchased at market were detected by the method established.The detection rate was 51.7%.Conclusion: This study established a method to detect free gossypol and gossypolone by HPLC in animal and dairy products.UPLC-MS/MS was more sensitivie than HPLC.This research confirmed that gossypol's metabolite was gossypolone.This study provided an experimental basis for gossypolone in vivo.