Studies On Key Technologies Of The Industrialization Process Of Conjugated Estrogens From The Pregnant Mare Urine In Xinjiang

Objective:Conjugated estrogens extracted from the pregnant mare urine(CEEPMU)is widely used as the drug in estrogen replacement therapy,which provides both favorable therapeutic effects and preventive effects for osteoporosis,cardiovascular diseases and improving women's menopause symptoms.There are a number of critical technologies need to be addressed during the process of developing conjugated estrogen by the full use of the advantages of local resources.These study focus on the following aspects:1)Establish new assessment methods on quality of the pregnant mare urine(PMU)and expecting to solve the problems during PMU acquisition process.2)Establish evaluation methods for quality of active pharmaceutical ingredients(API)from CEEPMU in Xinjiang,and evaluating the pharmaceutical equivalence between API from CEEPMU in Xinjiang and Premarin,a similar medicine produced by the United States.Provide the basis for API from CEEPMU in Xinjiang to obtain the international certification.3)Analysis of the chemical structure of the unknown ingredient of API from CEEPMU in Xinjiang,provide the basis for further revealed and confirmed the material basis of the combined effects of the multi-target in the pregnant mare CE.Methods:1.Study on quality evaluation of the Pregnant Mare urine(PMU)Homemade macroporous adsorptive resins solid phase extraction column HPD-400 were used to purify the PMU,5%NaCl solution and 1.5%NaOH solution were used to remove the impurities in PMU.Ethanol was carried out as eluent,using ethyl acetate-dichloromethane-methanol(30:90:22)as developing solvent,silica gel plate and silver silica gel plates were used to isolate the main conjugated estrogen in PMU,such as:estrone sodium(ES),sodium equilin sulfate(EqS)and sodium 17?-dihydroequilin sulfate(17a-EqS),Take 20%sulfuric acid in ethanol as a reagent to qualitatively identify the main CE in PMU.The macroporous adsorptive resins HPD-400 were used to purify the PMU.The quantitative assay methods were established by HPLC,UPLC and LC-MS,respectively,and the precision and accuracy of three methods were studied.2.Establishment of quality evaluation methods of API from CEEPMU in XinjiangUsing 0.025mol/L potassium dihydrogen phosphate solution and acetonitrile as the mobile phase to gradient elution,Agilent Zorbax ODS column(4.6mmX 250mm,5?m)as analytical column,and detection wavelength 215nm,established HPLC-UV fingerprints methods of API from CEEPMU in Xinjiang.The total numbers of peaks,relative retention time and relative peak area in the HPLC-UV fingerprints of 10 batches of API from CEEPMU in Xinjiang.The mean relative retention times of common peaks and the range of relative peak area served as evaluation index of HPLC-UV fingerprints of API from CEEPMU in Xinjiang and compared with three batches of similar products Premarin produced by the United States.Sulfatase were adopted to hydrolysis pregnant mare CE,then derivatization by 1%trimethyl-chlorosilane,(trimethylsilyl)tri fluoroacetamide,separated by a 15m DB-225 capillary column and analyzed in a constant voltage mode;MS conditions:El source;ion source temperature:200?;multiplier voltage:lkv;mass spectral libraries:NIST.Established GC-MS pharmaceutical equivalence method of API from CEEPMU in Xinjiang.The total numbers of peaks,relative retention time and relative peak area in the GC-MS fingerprints of 10 batches of API from CEEPMU in Xinjiang were studied.The mean relative retention times of common peaks and the range of relative peak area served as evaluation index of GC-MS pharmaceutical equivalence of API from CEEPMU in Xinjiang,and compared with three batches of Premarin.Using 12%acetonitrile-10mmol/L ammonium acetate buffer and 60%acetonitrile-10mmol/L ammonium acetate buffer as the mobile phase to gradient elution,YMC ODS-AM S3 120A as analysis column,detected by electrospray ionization source(ESI source)and the ion trap mass spectrometer,using negative ion detection mode,the ion source ejection voltage:4.50kV,capillary temperature:200?.Capillary voltage:-10V;sheath gas flow rate:70 flow units(au),auxiliary gas flow rate:lOau,detection method:full-scan manner(scan),the scanning range:150 to 600m/z,established API from CEEPMU in Xinjiang LC-MS pharmaceutical equivalence.Using a 56m/z extracted ion spectra to analysis numbers of fragment ions of which the relative peak area>0.1%and>1%.Compared LC-MS pharmaceutical equivalence of 10 batches of API from CEEPMU in Xinjiang with three batches of premain.3.Chemical structure analysis of unknown-components of API from CEEPMU in XinjiangBased on API from CEEPMU in Xinjiang GC-MS pharmaceutical equivalence method studied in the second part of the experiment and use the qualitative features of the MS in the GC-MS method,through the characteristic spectrum of the fragment ions acquired by electron impact source(EI)and chemical ionization(CI),combined with EI source split law and the information of the standard map,we analysis chemical structures of 40 common peaks corresponding compounds in GC-MS fingerprints of API from CEEPMU in Xinjiang.Result:1.Study on quality evaluation of the pregnant Mare urine(PMU)The macroporous adsorptive resins solid phase extraction column HPD-400method is a suitable method to purify the PMU.Without interference of the bland solution,three main conjugated estrogens were efficiently separated by the AgNO3-TLC method.The accuracy and precision of HPLC,UPLC and LC-MS method conform to the testing requirements.What's more,UPLC method and LC-MS method can greatly reduce the time of sample determination when compared with HPLC method and there are no statistically differences on the results among three methods.2.Establishment of quality evaluation methods of API from CEEPMU in XinjiangHPLC fingerprint method showed that 10 batches of API from CEEPMU in Xinjiang are in good consistency and found 27 common peaks of which relative peak areas are greater than 0.1%could be determined.What's more,there existed preferably consistency between the API from CEEPMU in Xinjiang and Premarin tablets,except that an extra peak in 58minute of API from CEEPMU in Xinjiang was found.GC-MS pharmaceutical equivalence method showed that 40 common peaks of which relative peak areas are greater than 0.1%could be inspected.Similarly,there existed preferably consistency between API from CEEPMU in Xinjiang and Premarin tablets,except that four differences of API from CEEPMU in Xinjiang were found.LC-MS characteristic ions method displayed that obvious peaks could be observed at 56 EIC m/z,including 230 peaks of which areas were greater than 0.1%at 50-54m/z under the EIC map,and 42 peaks of which areas were greater than 0.1%at 19-23m/z under the EIC map,which basically accord with industrial directory of FDA.Similarly,there existed preferably consistency between the API from CEEPMU in Xinjiang and Premarin tablets.3.Chemical structure analysis of unknown-components of API from CEEPMU in XinjiangFour components of API from CEEPMU different from Premarin were analyzed their chemistry structures by mass spectrum method.The chemical structure are:3-hydroxypregn-16-en-20-one,3-hydroxy-1,3,5(10)-steroid triene-6,17-dione,3-hydroxy-16-methoxy-pregn-20-one and 3-hydroxy-1,3,5(10)-estr-triene-17-one.These chemical structure were proved correctly by Reference substance.Chemical structure of two kinds of components in the raw drug is determined by LC-MS.One kind is with sodium sulfate form,another kind is free form.The structure information of 18 kinds of common components was obtained by standard map retrieval.The ten kinds of the chemical structure of the common components is obtained by mass spectrometry analysis.Mass analysis of the components showed that there are 40 common peaks in CEEPMU in Xinjiang.The estrogen is 14 kinds,five kinds of progesterone,androgen 10 kinds,fatty acid esters 2 kinds.8 kinds of compound corresponding to the chromatographic peaks of smaller area failed to identify.Conclusion:1.The macroporous adsorptive resins solid phase extraction(SPE)is a suitable method for pre-treatment of PMU by the qualitative identification and quantitative assay,because of its fast speed,low cost and high performance.AgNO3-TLC method and UPLC and LC-MS method can be used as the quick qualitative identification and quantitative detection of CE from PMU,respectively.2.The establishment of HPLC-.UV fingerprinting method,GC-MS pharmaceutical equivalence method and LC-MS pharmaceutical equivalence method provide important methods to evaluate the quality of API from CEEPMU in Xinjiang.Our results showed API from CEEPMU in Xinjiang and Premarin are very similar in composition.What is more,there is pharmaceutical equivalence between them.3.32 kinds of chemical structure of the main ingredient were identified by GC-MS fingerprint analysis for API from CEEPMU in Xinjiang and Premarin.11 components of CE have been reported,including the internal standard.The remaining 21 are for the first time by mass spectrometry to determine the chemical structure.The determination of the chemical structure of these ingredients laid the foundation for further study of the material basis and active ingredient of the pregnant mare CE.