依替米星中杂质的分离纯化及结构鉴定.docxVIP

依替米星中杂质的分离纯化及结构鉴定 李正义 晏培培 殷晓伟 王鹏飞 殷乐 孙小强 常州大学石油化工学院 摘要： 目的:对依替米星及其杂质进行分离纯化，并对各杂质进行结构确定。方法:采用 高效液相色谱-蒸发光散射法，选用Ultimate LP 5色谱柱(250 mmX4.6 mm, 5um),以0.2 mol - L-1三氟乙酸溶液■甲醇(84： 16)为流动相，流速0. 5 m L-minl，漂移管温度65°C,进行HPLC-ELSD分析;采用Ultimate LP-5色谱柱 (250 mmX21. 2 mm, 5 m),以 0. 2 mol ? L三氟乙酸水溶液-甲醇(90 : 10)为 流动相，流速6 ni L ? min-1, ELSD为检测器，进行HPLC制备分离；以甲醇-水 (80 : 20)为流动相，流速0. 8 m L ? min-1,进行MS分析；以D2O为溶剂,釆用一 维和二维核磁共振谱，对制备得到的杂质进行结构测定。结果:制备分离出4种 微量杂质(W0.03%) , MS及NMR分析确定了它们的结构分别为1-N-乙基加洛糖 胺、1-N-乙基-3‘ , 4’ -二脱氧新霉胺、1-N-乙基-3 -N-脱甲基庆大霉素Cla, 1-N-乙基小诺霉素。结论:通过HPLC-ELSD及其HPLC制备分离方法的联用，能迅 速检测并分离纯化岀微量杂质；综合运用各种一维和二维核磁共振技术对依替米 星(ETM)及其杂质的结构进行对比分析，建立了一种快速、准确分析氨基糖昔 类抗生素结构的方法。 关键词： 氨基糖昔类抗生素;依替米星；1-N-乙基加洛糖胺、1-N-乙基-3’，4’ -二脫氧 新霉胺、—乙基-3 -7-脱甲基庆人霉素Cb, — 乙基小诺霉素;质谱;a 磁共振;蒸发光散射检测; 作者简介：孙小强 Tel: (0519)E-ma订：sunxiaoqiang@yahoo. com 作者简介：李正义 Tel: (0519)E-ma订：zyli@cczu. edu. cn 收稿日期：2016年12月11 H 基金：国家自然科学基金 Separation, purification and struetural identification of impurities in etimicin LI Zheng-yi YAN Pei-pei YIN Xiao-wei WANG Peng-fei YIN Yue SUN Xiao-qiang School of Petrochemical Engineering, Changzhou University; Changzhou Fangyuan Pharmaceutical Co., Ltd.; Abstract： Objective:Etimicin (ETM) and its various impurities were analyzed by an HPLC-ELSD method, and the impuri ties were further separated and purified for the struc tu ral ide ntification. Met hods: IIPLC-ELSD analysis was eipplied on the Ultimate LP Ci8 column (250 mmX4. 6 mm, 5 u m) , 0. 2 mol ? L 1 trifluoroacetic acid aqueous solution-methanol (84 : 16) as the mobile phase with the flow rate of 6 m L ? min and the drift tube temperature of 65°C. Preparative HPLC was applied, using methanol-water (80 : 20) as the mobile phase with the flow rate of0 8 m L ? mirf for MS analysis. The st rue tu res of prepared impurities in eti mincin were determined by ID and 2D XMR analysis with D20 as solvent. Results:Four trace imp urities (WO. 03%) were separated and purified. And their structures were 1-N-ethyl garamine, l-N-