Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide.

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.