二维超高效液相色谱-四极杆/飞行时间质谱法解析替考拉宁杂质

建立了二维超高效液相色谱-四极杆/飞行时间质谱法（2D-UPLC-Q/TOF-MS）对替考拉宁组分分离和杂质结构解析的分析方法，有效地解决了流动相中含不挥发性磷酸盐的色谱系统不适用于液相色谱-质谱快速鉴定替考拉宁杂质的难题。一维超高效液相色谱以Octadecyl silica （ODS） hypersil色谱柱（250 mm×4.6 mm， 5 μm）进行色谱分离，以3.0 g/L磷酸二氢钠溶液（pH 6.0）/乙腈=9/1 （v/v）为流动相A、3.0 g/L磷酸二氢钠溶液（pH 6.0）/乙腈=3/7 （v/v）为流动相B进行梯度洗脱；二维超高效液相色谱以Waters ACQUITY UPLC BEH C₁₈色谱柱（50 mm×2.1 mm， 1.7 μm）进行脱盐，以0.01 mol/L甲酸铵（pH 6.0）和乙腈为流动相进行梯度脱盐洗脱。质谱在电喷雾离子源、正离子模式下，采用全信息串联质谱（MS^E）模式采集质谱数据，锥孔气流速50 L/h，锥孔电压60 V，离子源温度120 ℃，雾化气流速900 L/h，雾化气温度500 ℃，毛细管电压2500 V，碰撞能量20~50 eV。根据杂质精确质量数及其二级质谱信息推导其结构，并对替考拉宁主要成分TA_2-2的裂解规律进行了推导，发现了2个母核特征离子；对《欧洲药典》10.0收录的10个组分及22个杂质组分进行二级质谱分析，发现了3个新杂质组分。采用该法既可以使用一维超高效液相色谱根据相对保留时间进行组分准确定位，也可以使用二维超高效液相色谱-四极杆/飞行时间质谱二级质谱信息快速、简便、灵敏地对杂质进行结构鉴定，为替考拉宁的质量控制和工艺优化提供了一种新思路。

Analysis of teicoplanin impurities by two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

A two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry （2D-UPLC-Q/TOF-MS） method was established for the separation and structural analysis of the components in teicoplanin. This method effectively solved the problems associated with chromatographic systems， such as liquid chromatography-mass spectrometry （LC-MS）， which used a non-volatile phosphate buffer as the mobile phase and were not suitable for the rapid identification of impurities. Moreover， this method circumvented the complexities associated with locating and identifying impurities using the original method by re-establishing a chromatographic system suitable for LC-MS. In this study， for one-dimensional （1D） chromatography， the chromatographic separation was performed on an Octadecyl silica （ODS） hypersil column （250 mm×4.6 mm， 5 μm） with gradient elution using 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=9/1 （v/v） as mobile phase A and 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=3/7 （v/v） as mobile phase B. The column temperature was maintained at 30 ℃ and an ultraviolet detector was used at 254 nm for analysis. For 2D chromatography， desalting was performed on a Waters ACQUITY UPLC BEH C₁₈ column （50 mm×2.1 mm， 1.7 μm） with gradient elution using ammonium formate buffer （pH 6.0） and acetonitrile as the mobile phases. The column temperature was maintained at 45 ℃. The MS data for the components and impurities were collected by positive ion electrospray ionization （ESI） using the full-information tandem MS mode （MS^E）. The cone and nebulizer gas flow rates were set at 50 and 900 L/h， respectively. The ion source and nebulizer gas temperatures were set at 120 ℃ and 500 ℃， respectively. The ESI and cone needle voltages were set at 2500 and 60 V， respectively. The collision energy was set at 20-50 eV. The molecular formulas of the components and impurities were determined using their exact masses and isotope distributions， and the structural components and impurities of teicoplanin were deduced from their fragment ions according to the fragmentation pathway of the TA_2-2 component. Moreover， the 10 components reported in the European Pharmacopoeia 10.0 were analyzed and 22 impurities of teicoplanin were identified by 2D-UPLC-Q/TOF-MS. Three new impurities and two characteristic fragment ions of the teicoplanin parent nucleus were detected， and the fragmentation pathway of TA_2-2 was deduced. Using this method， 1D-UPLC is applicable for the accurate qualification of components based on relative retention times， and 2D-UPLC-Q/TOF-MS is suitable for the rapid identification of the structure of components based on their fragment ions. The results indicate that 2D-UPLC-Q/TOF-MS may be used to analyze the structure of impurities in teicoplanin based on their exact masses， isotope distributions， and fragment ions. The method is rapid， simple， and sensitive， which provides a novel strategy for the quality control and process optimization of teicoplanin.