Study On Crystal Forms Of HQ

HQ is a natural flavonoid which widely exists in flowers,leaves and fruits.Mordern pharmacological studies showed that HQ had many certain therapeatical effects such as anticancer,anti-inflammatory,antibacterial,anti-hypertension and preventing cardiovascular disease.However,HQ dihydrate is almost insoluble in water,which results in the low oral bioavailability and limits its applications in pharmacological effects.In order to increase the water-solubility and blood concentration of HQ,polymorph and cocrystal technology were used.The aim of this study was to investigate the phenomenon of HQ polymorphism and HQ-urea cocrystal.The preparation methods of different crystal forms were established and evaluation of crystal forms was also developed in vitro.We developed a rather sensitive and selective LC-MS/MS method to determine HQ in beagle plasma.The method was applied to pharmacokinetics of beagle after oral medication of HQ polymorphic form B and HQ dihydrate.The obtained results would be helpful for screening an excellent crystal form of HQ.Part one Preparation and identification of HQ polymorphism and HQ-urea cocrystalObjective:To investigate the phenomenon of HQ polymorphism and prepare HQ polymorphism and HQ-urea cocrystal.Methods:HQ polymorphic form A was prepared by drying method and HQ polymorphic form B was prepared by using suspension method.HQ-urea cocrystal was prepared by suspension and solvent assisted grinding method and then HQ polymorphism and HQ-urea cocrystal were identified using XRPD,TG and DSC.Results:The result of DSC showed that HQ dihydrate,HQ polymorphic form A and HQ polymorphic form B were of high crystalline purity.The HQ polymorphic form B was identified as a new crystal form by XRPD.The crystal purity of HQ-urea cocrystal prepared by suspension method was higher than prepared by grinding method.Conclusions:This research has successfully verified that HQ persents the phenomenon of HQ polymorphism,and the HQ polymorphic form B was reporded for the first time.By using grinding method with absolute ethanol,the trend of forming cocrystal with urea was found.Part two Study of HQ polymorphism and HQ-urea cocrystal on solubility and stabilityObjective:To investigate the solubility and stability of four kinds of quecertin crystal forms?HQ dihydrate,HQ polymorphic form A,HQ polymorphic form B and HQ-urea cocrystal?.Methods:The medium was 0.2%SDS aqueous solution and the revolving speed was 50r/min.The samples were collected from the tester at 1,3,5,15 and 60 min and determined by UV at 372 nm.High humidity test were designed for HQ crystal forms and the trans-crystal behavior was investigated by XRPD.Results:The solubility of the four kinds of HQ crystals was different in 60min.HQ polymorphic form B has the fastest dissolution rate and the highest solubility.Under the condition of high humidity,HQ polymorphic form B is stable while HQ polymorphic form A is easy to convert to HQ dihydrate.Conclusions:The solubility test and high humidity test showed that HQ polymorphic form B could be applied for its excellent character.Part three Pharmacokinetic study of HQ polymorphic form B in beagles by using HPLC-MSObjective:To develop an HPLC-MS/MS method to determine the concentrations of HQ in beagle plasma after the oral administrating of HQ form B and HQ dihydrate.Methods:Six beagles were divided into two groups at random and then were given HQ form B capsule and HQ dihydrate capsule respectively.Blood samples were collected from the vein of the hind leg at 5,15,30,45,60,90,120,180,300 min after oral administration.The plasma samples were pretreated and extracted by a simple liquid-liquid extraction method by ethyl acetate.Kaempferol was used as internal standard.Analysis was performed on a Diamonsil C₁₈ column?150 mm×4.6 mm,5?m?eluted with acetonitrile and water?0.05%formic acid?in an isocratic elution.The flow rate was 1 mL/min,the injection volume was 20?L and the column temperature was 30?.The multiple-reaction monitoring scanning?MRM?was employed for the quantification in negative mode.The ion spray voltage was set at-4500 V and the turbo spray temperature was maintained at 650?.Results:HQ showed a good linearity in the ranges of 0.05-5ng/mL.Full validation of the assay including specificity,accuracy and precision was acceptable.The bioavailability of HQ polymorphic form B is higher than HQ dihydrate and the relative bioavailability is 569.3%.Conclusions:A selective,rapid,simple HPLC-ESI-MS/MS method was developed.It could be used as a quantitative determination method for HQ in beagle plasma.