Zhejiang University of Technology, Hangzhou 310014, China; Zhejiang Institute for Food and Drug Control, Hangzhou 310052, China.
Zhejiang University of Technology, Hangzhou 310014, China.
Anal Chim Acta. 2017 Nov 1;992:42-54. doi: 10.1016/j.aca.2017.08.028. Epub 2017 Sep 14.
High-resolution mass spectrometry had been routinely used for structure identification of impurity. However, all LC-MS methods were based on a volatile mobile phase, and a non-volatile system is used in the official analytical method of United States Pharmacopoeia for cefpiramide which limited the use of mass spectrometry for structure characterization of the impurities. Here we presented the utilization of a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) with positive and negative modes of electrospray ionization for characterization of eight impurities in cefpiramide. Trap-free two-dimensional liquid chromatography and online desalting technique made it possible to characterize the impurity in cefpiramide in the condition of official standard, and the TIC chromatogram of LC-MS was in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities, which could further improve the method of official monographs in pharmacopoeias. Each peak separated by the non-volatile mobile phase was trapped by a 20 μL quantitative loop then transferred into a system with a volatile mobile phase connected to a MS detector. In the first dimension, the column was Kromasil C analytical column (250 mm × 4.6 mm, 5 μm) with a non-volatile salt mobile phase at the flow rate of 0.8 mL min. In the second dimension, the column was Shimadzu Shim-pack GISS C (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase at the flow rate of 0.3 mL min. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely. The fragmentation behavior of cefpiramide and its eight impurities were studied. The structures of eight impurities in cefpiramide drug substance were deduced based on the HPLC-MS data, in which seven impurities were novel impurities. The forming mechanisms of degradation products in cefpiramide were also studied.
高分辨率质谱已被常规用于杂质的结构鉴定。然而，所有 LC-MS 方法都基于挥发性流动相，而美国药典中头孢吡肟的官方分析方法则使用非挥发性系统，这限制了质谱在杂质结构特征化方面的应用。在这里，我们介绍了使用无阱二维液相色谱与高分辨离子阱/飞行时间质谱（2D LC-IT-TOF MS）联用，采用正、负离子模式电喷雾电离，对头孢吡肟中的 8 种杂质进行了表征。无阱二维液相色谱和在线脱盐技术使得在官方标准条件下对头孢吡肟中的杂质进行特征化成为可能，LC-MS 的 TIC 色谱图与官方分析方法的 LC 色谱图在杂质的峰序上一致，这可以进一步改进药典中的官方专论方法。通过非挥发性流动相分离的每个峰都被 20μL 定量环捕获，然后转移到与 MS 检测器相连的具有挥发性流动相的系统中。在第一维中，色谱柱为 Kromasil C 分析柱（250mm×4.6mm，5μm），非挥发性盐流动相的流速为 0.8mL min。在第二维中，色谱柱为 Shimadzu Shim-pack GISS C（50mm×2.1mm，1.9μm），挥发性盐流动相的流速为 0.3mL min。通过多次中心切割 2D-LC 方法和在线脱盐技术，完全解决了非挥发性盐流动相与质谱不兼容的问题。研究了头孢吡肟及其 8 种杂质的碎裂行为。根据 HPLC-MS 数据推断出头孢吡肟原料药中 8 种杂质的结构，其中 7 种杂质为新杂质。还研究了头孢吡肟中降解产物的形成机制。
