ABSTRACT
A two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry ï¼2D-UPLC-Q/TOF-MSï¼ method was established for the separation and structural analysis of the components in teicoplanin. This method effectively solved the problems associated with chromatographic systemsï¼ such as liquid chromatography-mass spectrometry ï¼LC-MSï¼ï¼ which used a non-volatile phosphate buffer as the mobile phase and were not suitable for the rapid identification of impurities. Moreoverï¼ 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 studyï¼ for one-dimensional ï¼1Dï¼ chromatographyï¼ the chromatographic separation was performed on an Octadecyl silica ï¼ODSï¼ hypersil column ï¼250 mm×4.6 mmï¼ 5 µmï¼ with gradient elution using 3.0 g/L sodium dihydrogen phosphate buffer ï¼pH 6.0ï¼/acetonitrile=9/1 ï¼v/vï¼ as mobile phase A and 3.0 g/L sodium dihydrogen phosphate buffer ï¼pH 6.0ï¼/acetonitrile=3/7 ï¼v/vï¼ as mobile phase B. The column temperature was maintained at 30 â and an ultraviolet detector was used at 254 nm for analysis. For 2D chromatographyï¼ desalting was performed on a Waters ACQUITY UPLC BEH C18 column ï¼50 mm×2.1 mmï¼ 1.7 µmï¼ with gradient elution using ammonium formate buffer ï¼pH 6.0ï¼ and acetonitrile as the mobile phases. The column temperature was maintained at 45 â. The MS data for the components and impurities were collected by positive ion electrospray ionization ï¼ESIï¼ using the full-information tandem MS mode ï¼MSEï¼. The cone and nebulizer gas flow rates were set at 50 and 900 L/hï¼ respectively. The ion source and nebulizer gas temperatures were set at 120 â and 500 âï¼ respectively. The ESI and cone needle voltages were set at 2500 and 60 Vï¼ 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 distributionsï¼ and the structural components and impurities of teicoplanin were deduced from their fragment ions according to the fragmentation pathway of the TA2-2 component. Moreoverï¼ 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 detectedï¼ and the fragmentation pathway of TA2-2 was deduced. Using this methodï¼ 1D-UPLC is applicable for the accurate qualification of components based on relative retention timesï¼ 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 massesï¼ isotope distributionsï¼ and fragment ions. The method is rapidï¼ simpleï¼ and sensitiveï¼ which provides a novel strategy for the quality control and process optimization of teicoplanin.