离子交换树脂混用技术分离纯化妥布霉素
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摘要
目的开发利用离子交换树脂混用技术分离纯化妥布霉素的工艺。方法建立2,4-二硝基氟苯(DNFB)HPLC柱前衍生化检测妥布霉素及相关物质,以树脂吸附量和解吸量以及妥布霉素纯度为指标,通过静态吸附和动态吸附实验,对妥布霉素在树脂上吸附和解吸条件进行优化。结果从10种不同类型的树脂筛选出HZ-3C和JK110分离纯化妥布霉素,确定最优工艺条件如下:确定两种树脂最佳比例为3:1,上样p H值为9,上样流速为3BV/h,使用浓度0.20%和0.35%氨水溶液进行梯度洗脱。采用优化后的条件,妥布霉素纯度从32.85%提高到94.82%,回收率为88.26%。结论建立了一种新的妥布霉素分离纯化方法,优化后的工艺妥布霉素得率高、工艺简单易行,适合工业化生产。
Objective To develop the separation and purification processes of tobramycin with mixed resins.Methods An HPLC method is established for the determination of tobramycin and its related substances by precolumn derivatization with 2,4-dinitrofluorobenzene.Based on adsorption capacity and desorption capacity of resin and the purity of tobramycin,optimal conditions for adsorption and elution of tobramycin were performed with static and dynamic experiments.Results HZ-3 C and JK110 were selected from ten types of resin as the effective adsorbent for separation and purification of tobramycin.The purification conditions were specified as the following:The ratio of HZ-3 C and JK110 was 3:1,the p H value was 9,the flow rate was 3 BV/h.The optimum desorption conditions was obtained by gradient elution with 0.3% and 0.7% ammonia water,under these conditions,tobramycin content were increased from 32.85% to 94.82%,with a tobramycin recovery rate of 88.26%.Conclusion The method for separation and purification of tobramycin was established,it was simple and easy,and suitable for industrial production with high purity and desorption rate.
Objective To develop the separation and purification processes of tobramycin with mixed resins.Methods An HPLC method is established for the determination of tobramycin and its related substances by precolumn derivatization with 2,4-dinitrofluorobenzene.Based on adsorption capacity and desorption capacity of resin and the purity of tobramycin,optimal conditions for adsorption and elution of tobramycin were performed with static and dynamic experiments.Results HZ-3 C and JK110 were selected from ten types of resin as the effective adsorbent for separation and purification of tobramycin.The purification conditions were specified as the following:The ratio of HZ-3 C and JK110 was 3:1,the p H value was 9,the flow rate was 3 BV/h.The optimum desorption conditions was obtained by gradient elution with 0.3% and 0.7% ammonia water,under these conditions,tobramycin content were increased from 32.85% to 94.82%,with a tobramycin recovery rate of 88.26%.Conclusion The method for separation and purification of tobramycin was established,it was simple and easy,and suitable for industrial production with high purity and desorption rate.
引文
[1]肖剑萍,温淑平,洪文荣.单组分妥布霉素工程菌的构建[J].中国抗生素杂志,2014,39(12):891-895.
[2]Elzaher A A,Mahrouse M A.Utility of experimental design in pre-column derivatization for the analysis of tobramycin by HPLC-fluorescence detection:Application to ophthalmic solution and human plasma[J].Anal Chem Insights,2013,8(8):9-20.
[3]Santos H S,Fran?a G M D,Romani E C,et al.Selective determination of tobramycin in the presence of streptomycin through the visible light effect on surface plasmon resonance of gold nanoparticles[J].Microchem J,2014,116(20):6-15.
[4]Borodina I,Sch?ller C,Eliasson A,et al.Metabolic network analysis of Streptomyces tenebrarius,a Streptomyces species with an active entner-doudoroff pathway[J].Appl Environ Microbiol,2005,71(5):2294-2302.
[5]郭养浩,陈剑锋,陈浩.氨基糖苷类抗生素的离子交换提纯方法:CN,101012246A[P].2007-08-08.
[6]陈剑锋.氨基糖苷类抗生素的大孔吸附树脂富集纯化方法:CN,1974586A[P].2007-06-06.
[7]王凯晨,宁方红,刘坐镇,等.大孔弱酸树脂对妥布霉素纯化的工艺研究[J].离子交换与吸附,2015,31(6):551-561.
[8]Russ H,Mccleary D,Katimy R,et al.Development and validation of a stability-indicating HPLC method for the determination of tobramycin and its related substances in an ophthalmic suspension[J].J Liq Chromatogr R T,1998,21(14):2165-2181.
[9]杨茂俊,吴章秀.高效液相色谱法分析国产妥布霉素及有关物质[J].中国抗生素杂志,1996,21(3):72-76.
[10]张星,阮奇,黄诗煌,等.大孔弱酸树脂提取西索米星的研究[J].中国抗生素杂志,2003,28(6):338-340.
[11]俞俊棠,唐孝宣.生物工艺学.上册[M].广州:华东理工大学出版社,2003.
[2]Elzaher A A,Mahrouse M A.Utility of experimental design in pre-column derivatization for the analysis of tobramycin by HPLC-fluorescence detection:Application to ophthalmic solution and human plasma[J].Anal Chem Insights,2013,8(8):9-20.
[3]Santos H S,Fran?a G M D,Romani E C,et al.Selective determination of tobramycin in the presence of streptomycin through the visible light effect on surface plasmon resonance of gold nanoparticles[J].Microchem J,2014,116(20):6-15.
[4]Borodina I,Sch?ller C,Eliasson A,et al.Metabolic network analysis of Streptomyces tenebrarius,a Streptomyces species with an active entner-doudoroff pathway[J].Appl Environ Microbiol,2005,71(5):2294-2302.
[5]郭养浩,陈剑锋,陈浩.氨基糖苷类抗生素的离子交换提纯方法:CN,101012246A[P].2007-08-08.
[6]陈剑锋.氨基糖苷类抗生素的大孔吸附树脂富集纯化方法:CN,1974586A[P].2007-06-06.
[7]王凯晨,宁方红,刘坐镇,等.大孔弱酸树脂对妥布霉素纯化的工艺研究[J].离子交换与吸附,2015,31(6):551-561.
[8]Russ H,Mccleary D,Katimy R,et al.Development and validation of a stability-indicating HPLC method for the determination of tobramycin and its related substances in an ophthalmic suspension[J].J Liq Chromatogr R T,1998,21(14):2165-2181.
[9]杨茂俊,吴章秀.高效液相色谱法分析国产妥布霉素及有关物质[J].中国抗生素杂志,1996,21(3):72-76.
[10]张星,阮奇,黄诗煌,等.大孔弱酸树脂提取西索米星的研究[J].中国抗生素杂志,2003,28(6):338-340.
[11]俞俊棠,唐孝宣.生物工艺学.上册[M].广州:华东理工大学出版社,2003.