Separation and characterization of allergenic polymerized impurities from cephalosporin for injection by trap free two-dimensional high performance size exclusion chromatography × reversed phase liquid chromatography coupled with ion trap time-of-flight mass spectrometry.

As requested by regulatory authorities, polymerized impurities are an important issue of quality control. In this study, we presented the utilization of a trap-free two-dimensional chromatography, which was consisted by a high performance size exclusion chromatography (HPSEC) and a reversed-phase liquid chromatography (RP-LC) coupled to ion trap time-of-flight mass spectrometry with positive mode of electrospray ionization, to separate and characterize ten allergenic impurities in ceftazidime for injection, cefazolin sodium for injection, cefoperazone sodium and sulbactam sodium for injection and cefamandole nafate for injection. An effective method for characterizing the polymerized impurities in β-lactam antibiotics was established on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension, the column was the Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5 μm) and the flow rate was 1.0 mL min with gradient elution using 0.005 mol L phosphate buffer solution at pH 7.0 and acetonitrile as mobile phase. In the second dimension, the analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) using ammonium formate solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase at a flow rate of 0.4 mL min. Eluent associate with each peak separated in the first dimension was trapped by a 20 μL quantitative loop and then transferred (via a six-port valve) into the second dimension system with volatile mobile phase. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile mobile phase and mass spectrometry was solved completely. The fragmentation behaviors of ten allergenic impurities were studied. The structures of ten allergenic impurities in cephalosporin drug substance were deduced based on the HPLC-MS data, in which four impurities were polymerized impurities. The forming factors of polymerized impurity in cephalosporins were also studied.