| Objective 1)To establish an HPLC method to determine the related-substance of glycididazole sodium;2)Isolation and preparation of an unknown impurity separation of glycididazole sodium;3)Structural identification of the unknown photolysis impurities in glycididazole sodium;4)To establish an HPLC method to determine and analysis Photolysis product in glycididazole sodium.Methods 1)The assay was conducted on a shiseido CAPCELL ODS column(250mm×4.6 mm,5 μm),with 0.05 mol/L solution of ammonium acetate buffer(adjust pH to 7.1 with sodium hydroxide)-acetonitrile(80∶20)as mobile phase at the flow rate of 1.0 mL/min,the detection wavelength was set at 316 nm.Temperature was 35℃,and the injection volume was 20 μL;2)The assay was conducted on a C18HCE column(10μm,DAC50mm,300g),with A phase 0.1%formic acid as aqueous solution,B phase acetonitrile as mobile phase,at the flow rate of 1.0ml/min,the detection wavelength was set at 316 nm.Temperature was 35℃,and the injection volume was 60mL/110mL;3)Determination of molecular and planar structure of the unknown photolysis impurities in glycididazole sodium by mass spectrometry and nuclear magnetic resonance spectroscopy.Wight the unknown photolysis impurities in glycididazole sodium about 5mg,dissolved in methanol solution of 1mL,Diluted with acetonitrile-water solution to 5μg/mL,using infusion mode,the positive ion scan mode was selected,and the first and two stage mass spectrometry analysis was carried out.Wight the unknown photolysis impurities in glycididazole sodium about 10mg,Diluted with methyl sulfoxide of 1mL,to avoid light,cold storage to the nuclear magnetic tube,III HD AVANCE 500 liquid 500 MB(high resolution)superconducting nuclear magnetic resonance spectrometer(including one dimensional spectrum 1H and 13C NMR,as well as the two-dimensional spectrum 1H-1H-COSY,DEPT,HSQC and HMBC);4)The assay was conducted on a Hypersil column(C18,250*2.1 mm 4.6 μm)with 0.05 mol/L solution of ammonium acetate buffer(adjust to pH 7.1 with potassium hydroxide)Conclusion 1)Glycididazole sodium was completely separated from impurities.The linear concentration range of glycididazole sodium was 0～18.42 μg·mL-1 with the correlation coefficient of 0.9998.And the linear concentration range of metronidazole was 0～20.12 μg/mL with the correlation coefficient of 1.000,the average recovery(n=3)was 98.8%(RSD%=0.9).The solution was stable within 2.5 hours under room temperature(RSD%=2.28).The method is sensitive,accurate and specific.It can be used to determination related-substance in glycididazole sodium;2)According to pre-experiment,8.874min,14.057min and 17.456min were collected respectively.In turn,the former impurity,the glycididazole sodium,the unknown photolysis product.Its purity was 99.08%,100%,100%,respectively;3)A mass spectrometry analysis was obtained for the accurate nuclear 1][M of sodium bis,metronidazole and the unknown photolysis products,520.1380,170.0711 and440.1511,respectively.According to HR-EIS-MS given a quasi ion front,determine its molecular formula is C16H2108N7 and The planar structure is determined by resonance NMR4)The front(about 11.2min),without interference,the detection limit is 0.5ng,the limit of quantitation was 2.5ng,with good linearity in the range of 5-100ug/mL,the recovery rate is between 101.1%and 102.9%,in group RSD were less than 0.9%,inter batch precision was less than 1.0%.photolysis products of mobile phase sample plate for 24 hours stable photoproducts saline for 6 hours.All the indexes were in accordance with the provisions or requirements of the analytical method,so this method is suitable for the analysis of the content of photolysis products in the sample. |
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