高效液相色谱-电喷雾-离子阱质谱法快速推定硫酸奈替米星中有关物质的结构

 目的 应用高效液相色谱-电喷雾-离子阱质谱（HPLC-ESI-IT-MSn）法及TFAfix技术,快速鉴定硫酸奈替米星样品中有关物质的结构。方法 Agilent SB-C₁₈(4.6 mm×150 mm , 3.5 μm)色谱柱,水-三氟乙酸-甲醇(84∶1∶15)为流动相,流速0.5 mL·min^-1;离子肼质谱仪正离子检测,柱后分流（3∶2）进样,电喷雾离子源,离子源温度350 ℃,雾化室压力275.8 kPa,干燥气流速9 L·min^-1,采用TFAfix技术,即在柱后添加丙酸-异丙醇（20∶80）溶液,改善流动相中三氟乙酸对电喷雾离子化的抑制作用;对有标准品的有关物质,其结构通过与对照品的色谱质谱行为来确定;对无对照品的有关物质,主要以奈替米星和西索米星质谱行为为模板,根据它们多级质谱信息来推定。结果 硫酸奈替米星原料中检出9个有关物质,推定出其中7个物质的结构,分别为西索米星、去甲基西索米星、1-N-乙基-加洛糖胺、5-O-乙基-奈替米星、2′-N-乙基-奈替米星、3″-N-乙基-奈替米星、3-N-丙基-依替米星,解析了另两物质的部分结构。结论 建立的方法可以用于硫酸奈替米星原料中有关物质结构的快速推定,为奈替米星质量控制和工艺优化研究提供了可靠快速的分析手段。

Rapid Characterization of the Related Substances in Netilmicin Sulfate Sample by HPLC-ESI-IT-MSn

OBJECTIVE To characterize rapidly the related substances in netilmicinsulfate sample by a HPLC-ESI-IT-MSⁿ method with a TFAfix technology. METHODS The method was carried out using an agilent SB-C₁₈ (4.6 mm×150 mm , 3.5 μm) column and a mobile phase containing a mixture of water, trifluoroacetic acid and methanol（84∶1∶15） at a flow rate of 0.5 mL·min^-1. Two fifth of the column eluent was sent into the MS for peak characterization by splitting mode. The identification of related substances in the netilmicinsulfate bulk sample was performed with a ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode, the temperature of ESI ion source is 350 ℃, nebulizing pressure is 275.8 kPa, and dry gas flow is 9 L·min^-1, a solution cotaining propionic acid and isopropanol（80∶20） was infused into the eluent through a T-piece to decrease the MS signal suppression of trifluoroacetic acid . Related substanes which their reference substances can be obtained were identified by comparing their chromatographic and MS behaviors with those of corresponding reference substances, the other unknown related substances were deduced using the collision-induced dissociation (CID) spectra of as templates. RESULTS A total of nine compounds were were characterized in commercial samples, among which seven impurities were identified, they are sisomicin, de-N-methyl-sisomicin, 1-N-ethylgaramine, 5-O-ethyl-netilmicin, 2′-N- ethyl-netilmicin, 3″-N- ethyl-netilmicin and 3-N-propyl-etimicin,respectively, and the other two compounds were deduced partially, too. CONCLUSION The established method is suitable for the rapid identification of related substances in netilmicinsulfate samples and quality control.