Evidence for an imidazoline by-product from glycans using tandem mass spectrometry

•Separation and identification of unknown imidazoline by-product of labeling procedure.•Imidazoline substructure was established through High Resolution Mass Spectrometry.•Experiments showing chemical susceptibility and isotopically labeled tracers.•First study to identify this minor species from biologic.•Impacts all N-acetylglucosamine-type N-linked glycan profiling.

Herein is reported the separation and identification of a previously unknown imidazoline by-product originating from the fluorescent labeling procedure when applied to enzymatically released N-linked glycans of a human IgG1. The imidazoline by-product was generated via the reductive amination procedure with either sodium cyanoborohydride or 2-picoline borane. Using ultra performance liquid chromatography (UPLC) in conjunction with hydrophilic interaction-based chromatography (HILIC), the 2-aminobenzoic acid (2-AA)-labeled glycans were well-resolved from imidazoline by-products to facilitate direct identification utilizing electrospray ionization mass spectrometry (ESI-MS) with fragmentation. It was found that this minor species (∼2%) was 18.0105 u less than the neighboring peak GlcNAc2Man3GlcNAc2Fuc peak, abbreviated as A2G0F at 1582.5899 u. While this mass loss corresponds to the mass of a water molecule, the molecular location of loss of water was not straightforward in consideration of the biantennary A2G0F structure.Model studies were carried out using A2G0F standard and N-acetyllactosamine to identify the impurity as an imidazoline ring structure located at the reducing end of the glycan as confirmed by high resolution mass fragment ions. Imidazoline content decreased when the reductant concentration was increased. To conclude, evidence for the imidazoline structure was accomplished through high resolution, high accuracy mass spectrometry (HRAM), and experiments showing chemical susceptibility and isotopically labeled tracers. This study is the first to identify these minor species which likely impact all N-acetylglucosamine-type N-linked glycans from biologics.