Fabrication of an allyl-β-cyclodextrin based monolithic column with triallyl isocyanurate as co-crosslinker and its application in separation of lipopeptide antibiotics by HPLC

Abstract

A novel microporous structure monolithic polymer was prepared via free radical polymerization within a stainless steel column (4.6 mm [i.d.] × 50 mm) for separation of lipopeptide antibiotics. The monolithic column was synthesized using allyl-β-cyclodextrin and methyl methacrylate as co-monomers, triallyl isocyanurate and ethylene glycol dimethacrylate as co-crosslinkers, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were evaluated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The homemade monolithic columns not only had a relatively uniform porosity structure, low back pressure, and good permeability, but also show good separation performance. For low molecular weight substances, six aromatic compounds were separated in 6 min and the column efficiency of naphthalene achieved 35, 660 plates m−1. The enantiomers of ibuprofen and ephedrine, which were used as standard samples, were separated by the homemade monolithic column. The application potential of the monolithic column for the separation of Chinese medicine bioactive substances was illustrated by the ephedrine and pseudoephedrine, which were successfully detected in a crude extract of ephedra. Based on the excellent separation performance of the column for the aforementioned low molecular weight substances, we assumed that there are many forces, such as hydrogen bonds, π-π bonds, and dipole-dipole interactions, that are involved between enantiomers and allyl-β-cyclodextrin. Thus, natural lipopeptide antibiotics in the fermentation broth were used as samples to verify the assumption. Eighteen peaks were precisely separated from the fermentation broth. Among the 18 peaks, 4 lipopeptide antibiotics were identified by mass spectrometry. Methodologic validation was confirmed using two enantiomers of ibuprofen, and all parameters meet the testing requirements.