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Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction

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Abstract

Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.

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Acknowledgments

The authors are grateful to Benjamin Küpper and Rolf Wichmann from TU Dortmund, Laboratory of Biochemical Engineering for providing fermentation broth as sample material. TT and LMB gratefully acknowledge financial support by the Deutsche Bundesstiftung Umwelt (DBU).

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  1. Jeannine Engelen

    Present address: A&M Stabtest GmbH, Kopernikusstr. 25, 50126, Bergheim, Germany

Authors and Affiliations

  1. Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149, Münster, Germany

    Beate Behrens & Heiko Hayen

  2. Department of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaussstr. 20, 42119, Wuppertal, Germany

    Jeannine Engelen

  3. iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Till Tiso & Lars Mathias Blank

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  1. Beate Behrens
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Correspondence to Heiko Hayen.

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Behrens, B., Engelen, J., Tiso, T. et al. Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction. Anal Bioanal Chem 408, 2505–2514 (2016). https://doi.org/10.1007/s00216-016-9353-y

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