Preparation of fluorescent labeled gentamicin as biological tracer and its characterization by liquid chromatography and high resolution mass spectrometry

• Synthesis of fluorescent mono-labeled gentamicin.
• Purification of the single-labeled fluorescent product by semi-preparative HPLC.
• Application of high resolution mass spectrometry for product quality control.
• Structural characterization of this important biological probe as well as of gentamicin by HR TOF MS and MS/MS.

This work deals with the preparation of single-labeled bioconjugates of the antibiotic Gentamicin (GT) with the sulforhodamine-derived fluorescence dye Texas Red®-X (TR), its purification by high-performance liquid chromatography (HPLC) and its characterization by high-resolution mass spectrometry. Aminoglycosides such as GT are efficient antibiotics, but also problematic due to severe side effects such as nephro- and ototoxicity. Fluorescent labeled GT is used to visualize cellular uptake and distribution of the antibiotic to finally understand the mechanisms of serious adverse drug reactions. Pharmaceutically administered GT is a mixture of mainly four different components, which exhibit three (GT(C1)) or four (GT(C1a), GT(C2), GT(C2a)) primary amino functional groups which can be coupled with the labeling reagent TR. Thus, multiple labeling could be envisaged which was assumed to be detrimental for uptake studies by fluorescence imaging. The proposed synthesis aimed at preparation of single labeled product and together with the employed purification strategy indeed yielded single labeled GT as product. Analytical control of the reaction product was carried out by means of mass spectrometry (UHPLC-ESI-QTOF-MS/MS) to rule out over-labeling of GT, which would alter the physicochemical characteristics of GT and its cellular uptake significantly. Moreover, LC–MS/MS analysis gave valuable insights into structural diversity of single labeled products. Further, high-resolution MS and MS/MS spectra of underivatized GT are provided as well. The analytical information on preparation strategy and structure diversity is valuable for studies with a clinical focus on research of aminoglycoside toxicity. Furthermore, it is deemed to be useful for the development of LC–MS/MS assays for the determination of aminoglycosides or the fast screening of synthetic biology samples from biotechnological drug discovery.

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