色谱-质谱联用技术鉴定环孢素A的有关物质

Identification of the related substances of cyclosporin A by LC-MS techniques

采用Hypersil BDS C 18(100 mm×4.6 mm,2.4μm)色谱柱,以乙腈-水-甲基叔丁基醚-甲酸(430∶520∶50∶1)为流动相进行等度洗脱,对环孢素A及其有关物质进行分离。在所建立的条件下,环孢素A及其有关物质分离良好,检测并鉴定出29个主要有关物质(9个工艺杂质,20个降解产物),其中13个为欧洲药典中列出的已知杂质或其光学异构体,其余16个均为未知结构的有关物质。采用电喷雾正离子化-飞行时间质谱法(ESI-TOF/MS)测定各有关物质的准确质量和元素组成,三重四极杆串联质谱(ESI-MS/MS)测定其碎片离子特征,通过质谱解析鉴定了29个有关物质的结构。建立的色谱-质谱联用分析方法能有效地分离鉴定环孢素A中的有关物质,可用于其发酵过程的质量控制。

To identify the related substances of cyclosporin A by LC-MS techniques,the separation of cyclosporin A and its related substances was carried out on a Hypersil BDS C 18(100 mm×4.6 mm,2.4μm)column with isocratic elution by a mixture of acetonitrile-water-MTBE and formic acid(430∶520∶50∶1)as the mobile phase.Cyclosporin A and its 29 related substances(9 process related and 20 degradants)were well separated under the established conditions.Among them,13 were listed in EP and the rest 16 were unknown products having not been reported before.Electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of parent ions of all the components,and triple quadrupoles tandem mass was employed for the product mass spectra determination.Thence,the structures of all the 29 detected substances were successfully characterized through spectra elucidation and the fragmentation pathways analysis.The established LC-MS method was successfully employed for the separation and identification of the related substances of cyclosporin A and it is useful for its fermentation processes and quality control.