LC-HRMS based on mixed-mode chromatography for the separation of teicoplanin and the unravelment of its composition

[Display omitted] •Mixed-mode chromatography is a suitable choice for the separation of teicoplanin.•All major components, including isomers, were resolved in less than 18 min.•Coupled to HRMS/MS, it allowed the novel characterization of minor components. Liquid chromatography – high resolution mass spectrometry (LC-HRMS) based on mixed-mode chromatography has proven to be a useful tool in pharmaceutical analysis for the characterization of very polar antibiotics such as teicoplanin. This compound is a semisynthetic glycopeptide antibiotic thought to be a mixture of five major compounds (named A2-1 through A2-5) and four minor ones (RS-1 to RS-4), all sharing the same glycopeptide core structure (dubbed A3-1) and differing only on the length of a hydrocarbon side chain. These nine compounds have been fully characterized in the past thirty years by means of HPLC coupled to UV and MS detectors. However, HPLC separations were all based on octadecylsilica columns (C18), which nowadays may not be the best choice for the chromatographic separation of such polar and charged compounds. In this work, several different chromatographic alternatives are tested for the separation of teicoplanin, including mixed-mode chemistries. It has been demonstrated that a C18-PFP mixed-mode column provides the best chromatographic resolution, furthermore, confirming the existence of several minor compounds in the composition of teicoplanin. Up to fourteen minor components were characterized by means of High-resolution MS/MS and confirmed by the analysis of a commercial teicoplanin product (Targocid®).