| Objective:In this paper,the residue of related substances and organic solvents in the optimized2,5’-dibromo-4,5,2’-trihydroxybenzophenone which a new chemical drug being developed by the research group and was abbreviated LF1-API was examined to determine the limit of impurity and organic soluble dose to control the quality of the new drug completely.The stability of the test indexes was studied by the crystal form examination and quality standard to determine the validity period of the API and to provide scientific basis for its subsequent packing,storage and transportation conditions.Through the cytotoxicity test of the detected related substances,the test foundation is laid for the safety prediction of API and the determination of the inspection limit of related substances.Methods:The related substances of the starting materials including to phthalate and coller-methoxybenzoyl chloride that were abbreviated LF1-SM01,LF1-SM02 respectively,intermediates that were abbreviated LF1-01,LF1-02 respectively,and the generated crude drugs that was abbreviated LF1-API were examined by HPLC.LF1-SM02 cannot be directly determined by HPLC because it was easily reacted with water to generate acid.Therefore,it was first completely methylated and then converted into o-methoxybenzene methyl ester whose structure has been confirmed by hydrogen spectrum.The limit of the LF1-SM02 was checked by measuring the content of its methyl esterification product.HPLC chromatographic conditions were Agilent C18 column,and mobile phase A was an aqueous solution adjusted with phosphoric acid to pH 2.0,mobile phase B was acetonitrile.The column temperature was 30℃,detection wavelength was 230 nm,flow rate was 1.0ml/min,and injection volume was 10 μl.HPLC-MS was used to confirm the structure of the LF1-API and its impurities.The mass spectrometry conditions were electrospray ion source(ESI),positive ion MRM mode scanning,ionizing spray voltage 1000 V,vertebral foramen voltage 100 V,atomizing gas was nitrogen and the gas pressure was 10 psi,thetemperature of the dry gas was 139℃,the speed of the dry gas was 1 L/h,and the mass scanning mass range was 100-1000.Residual organic solvents including to ethanol,n-hexane,dichloromethane,tetrahydrofuran used in the LF1-API synthesis process were detected by gas chromatography.The chromatographic conditions were 6%cyanopropylphenyl-94% dimethyl based on a capillary column with a fixed siloxane as the fixed solution.The initial temperature was 60℃,maintained for 5 minutes,heated to 80 ℃at a rate of 5 ℃/min,maintained for 5 minutes,and then at 30 ℃/min.The temperature was raised to 200℃ for 3 minutes,the inlet temperature was 200℃,the hydrogen flame ion detector,the detector temperature was 250℃,nitrogen was the carrier gas,the split ratio was 10:1,and the headspace bottle equilibrium temperature was 90℃.The time was30 minutes and the headspace injection was performed.Influencing factor test,accelerated test and long-term test were carried out.To test the stability of the LF1-API by testing its properties,melting point,crystal form inspection,related substance,and content.The cytotoxicity of 2,5′-dibromo-4,5,2′-trihydroxybenzophenone which was abbreviated LF1-API-imC on EA.hy 926 vascular endothelial cells was investigated by the MTT.Results:The HPLC methods for related substances of LF1-API have been established.LF1-SM01-imA is not detected in the starting material LF1-SM01 test article,but LF1-SM01-imB can be detected with the detection amount below 0.1%.LF1-SM02-imB was not detected in the test sample of LF1-SM02,LF1-SM02-imA can be detected and it was checked in the test sample of LF1-01 continuously.LF1-01-imA,LF1-SM01,LF1-SM02-imA and LF1-SM01-imB were not detected in the test samples of LF1-01,but a king of other unknown single impurities was detected with the detection amount below0.1%.LF1-02-imA and LF1-01 were not detected in the test samples of LF1-02,but LF1-02-imB could be detected with the detection amount below 0.5%.LF1-API-imA,LF1-API-imB and LF1-02 were not detected in the three batches test samples of LF1-API,and LF1-API-imC could be detected with the detection amount below 0.1%.The structure of LF1-API and its three impurities were confirmed by comparing with the mass spectrum of the reference substance.A headspace capillary column gas chromatography method was established for the determination of residual organic solvents including to ethanol,n-hexane,methylene chloride,tetrahydrofuran in LF1-API.Ethanol can be detected in allthree batches of bulk drug test samples.The residual ethanol was below the control limit of0.5%,while the other three solvents were not detected.The stability test showed that compared with the test results of 0-day,the test indexes of the influencing factor test,accelerated test and long-term test had not changed significantly,and the time and intensity of the characteristic peak detected by X-ray diffraction method had no obvious difference.Cytotoxicity test showed that when LF1-API-imC contained in LF1-API was in the range of 0.01~0.5 mol/L,the cell survival rate were greater than 98.0%,and there was no significant change in cell survival rate at various concentrations.Conclusion:The inspection method of related substances of LF1-API meet the requirements of methodological verification and could effectively detect related substances in LF1-API.The single impurity in LF1-API not exceed 0.1%,and the total impurities not exceed1.0%.The inspection method of organic solvent residues of LF1-API meet the requirements of methodological verification,and could effectively detect four residual solvents.The organic solvent residues of the LF1-API produced by the current process meet the requirements.The packaging material of LF1-API did not need to be protected from light.LF1-API was stable under the conditions of 40℃/RH75%.LF1-API-imC in the bulk drug produced by the current process had no cytotoxicity to vascular endothelial cells EA.hy926 at the planned clinical dosage. |
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