Study On The Quality Of AOH And Its Preparation

Objective:AOH is a new kinds of chemicals drug in the category 1.1 that are being developed.Based on the quality analysis of AOH active pharmaceutical ingredient,the formulation optimization and preparation of AOH freeze-dried powder preparation were carried out.We also developed a quality determination method for the AOH frozen powder and related substances assaying.Finally,the studying of the quality standard and stability of AOH freeze-dried powder was conducted.In summary,our research has laid down a solid foundation for the AOH freeze-dried powder's application for the new drug.Methods:The physicochemical characteristics of the four batch of AOH materials were measured and the inorganic impurities were investigated.High performance liquid chromatography(HPLC)was adopted to determine the content and related substances of AOH drug substance,and conducted methods validation.Determination of residual solvents in AOH raw material was performed by gas chromatography.This research has establish the quality standard of the AOH raw material,and designed the preparation process of AOH freeze-dried powder.Then,the identification and examination of AOH freeze-dried powder injection were carried out.To set up the quality control standard of AOH freeze-dried powder,a high performance liquid chromatography(HPLC)method was developed to determine the content and related substances of AOH freeze-dried powder,and has been evaluated.Finally,the stability test was carried out,that included impact factor experiment,acceleration experiment and long term experiment.Results:Raw material of AOH appeared to be white powder,odorless and bitter taste.The melting point is 175~183 C,chloride less than 0.012%,sulfate less than0.1%,loss on drying less than 5%,residue on ignition less than 0.1%,heavy metals less than 20ppm,arsenic less than 2ppm.We has determine the preparation process of AOH freeze-dried powder,twain 80 and mannitol were added moderately into the reaction material AOH,and obtained AOH freeze-dried powder after freeze-drying.The content of AOH in the synthetic AOH freeze-dried powder is 90%-110%.We established three methods of identification of AOH freeze-dried powder as Liebermann-Burchard reaction,Salkowski reaction and high performance liquid chromatography.The three batches of samples of AOH lyophilized powder showed the same phenomenon as those of the reference substance identified by Liebermann-Burchard reaction,Salkowski reaction and high performance liquid chromatography.The reaction was negative when take the auxiliary material to operate with the method.It shows that the auxiliary material does not interfere with this identification.This methods were highly exclusive and can be used for the identification of AOH freeze-dried powder.AOH freeze-dried powder dosage difference,dry weight loss in line were complied with the relevant provisions.A method for the determination of the content and related substances of AOH and AOH freeze-dried powder was established and chromatographic conditions as blow:chromatographic instrument:Waters 2795/2996;chromatographic column:Diamonsil C18(250mm×4.6mm,5um);the ultraviolet detection wavelength:206nm;mobile phase:acetonitrile-water(45:55);the flow rate:1.0ml/min;the sample concentration was 0.5mg/ml;the column temperature was room temperature and the injection quantity was 20?l.Under the chromatography condition,the number of the theoretical plate AOH was not less than 5000 and the separation ratio was larger than1.5.Through the methodology test,this product with the corresponding peak area in the range of 0.2mg/ml-0.8mg/ml(r=0.9993)concentration showed a good linear relationship,the linear equation was:A=1×10~7x-6×10~4(n=7);limit of detection(LOD)and limit of quantification(LOQ)were 50ng and 200ng;RSD of the instrument precision test was 0.61%(n=6),less than 1%.RSD of the repeatability test was 0.98%(n=9),less than 1%.RSD of the interal-day and infra-day precision was less than1%.The procedure was tested in terms of accurate,sensitivity,precision,repeatability and linearity range.The results shows that such procedures were applicable to AOH,s assaying.We have established a headspace capillary GC method for the determination of residual solvents in AOH materials,including ethanol.The chromatographic conditions are as follows:gas chromatograph:A g i l e n t 6 8 2 0,polyethylene glycol as a fixed liquid,the silica capillary column was DB-WAX(30m×0.45mm×0.85?m),with a FID detector,the initial column temperature was 50?for 10 minutes,then increased at the rate of 30?/min to 200?(hold 10min),The temperature of the detector was250?and the inlet temperature is 220?,Carrier gas was nitrogen,the pressure is 4.0psi,the split ratio is 20:1,the number of theoretical plates should be more than 5000according to the peak of ethanol.Headspace conditions:the balance temperature,quantitative ring temperature,transmission line temperature were controlled at 80?,90?,100?,the headspace bottle heating time was set at 40min.Under the above chromatographic conditions,the sensitivity,precision,linear range,repeatability were good,ethanol were within the scale of limitation.The method of determination of the related substances of AOH and AOH freeze-dried powder was self-control method of the principal component without the correction factor of the related substance.High performance liquid chromatography(HPLC)was adopted to determine the related substances of AOH freeze-dried powder,and the specificity of the method was investigated through strong acid,strong base,strong oxidation,high temperature,high humidity and light damage test.The test results showed that there was no obvious related substances produced under 5mol/L sodium hydroxide,3mol/L hydrochloric acid and heated for 8 hours at 90 degrees in30%H2O2 solution,or place 5 days under high humidity(RH 92.5%)and light(4500Lx+500Lx)conditions.The experimental result indicated that the AOH freeze-dried powder was stable under the conditions of light,high temperature,high humidity,strong oxidation,strong base and strong acid.Under heated for 8 hours at90 degrees in 5mol/L hydrochloric acid solution and heated for 8 hours at 120centigrade,AOH freeze-dried powder was destructed with many related substances.Meanwhile,the established related substances method could well separate AOH from its related substances and the auxiliary materials have no interference to the detection.The AOH freeze-dried powder was stable under the circumstances of strong light illumination(4500 Lx+500Lx),high temperature(60?)and high humidity(25?,RH90%±5%).Current evidence from 6 month acceleration experiment(40?±2?,RH75%±5%),and 9 month long-term experiment(25?±2?,RH 60%±10%)proved AOH freeze-dried powder to be stable under those circumstances.Long-term experiment is undergoing.Conclusion:This thesis conducted the internal control standard researches of AOH,including test of physicochemical constants and the inorganic experiment.We also set up a quality determination method for AOH,s product and related substance assaying.Through the research of methodology,proving that the method that we set up for determining the content of AOH and its related substances met the requirements.A method for the determination of residual organic solvents in AOH was established.On this basis,this thesis designed a simple preparation process of AOH freeze-dried powder,and conducted quality standard and stability researches of the AOH freeze-dried powder,including the identification and inspection of AOH freeze-dried powder,set up a quality determination method for AOH freeze-dried powder,s product and related substance assaying.Through the research of methodology,proving that the method that we set up for determining the content of AOH freeze-dried powder and its related substances met the requirements.In summary,our research has laid down the foundation for preclinical study of AOH freeze dry powder and provides a scientific basis for the design of good production,package,storage and transportation conditions of the new drug.