Applied Microbiology and Biotechnology

, Volume 99, Issue 21, pp 9177–9187 | Cite as

Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantitative determination of rhamnolipid congeners

  • Michelle Rudden
  • Konstantina Tsauosi
  • Roger Marchant
  • Ibrahim M. Banat
  • Thomas J. Smyth
Methods and protocols


Rhamnolipids (RLs) are synthesised as a complex mixture of congeners comprising either one or two molecules of rhamnose glycosidically linked to a dimer of 3-hydroxy fatty acids varying in chain length and degree of saturation. Currently, HPLC-MS/MS is the most precise and accurate method for RL determination, while accurate quantification is limited. In this study, a rapid ultra pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the rapid and quantification of individual RL congeners. Increased RLs specificity was achieved using tandem mass spectrometry in multiple reaction monitoring (MRM) mode which was used to quantify RL isomer pairs such as Rha-Rha-C8-C10/Rha-Rha-C10-C8 which are difficult to resolve chromatographically. UPLC showed an 18-fold reduction in retention time for Rha-Rha-C10-C10 (1.07 min) and a 17-fold reduction for Rha-C10-C10 (1.36), the major rhamnolipids present, compared to HPLC, with a total run time less than 2.2 min. The results show that the linear range for the main RL congeners (Rha-C10-C10 and Rha-Rha-C10-C10) is 0.1 to 100 μg/mL. The LOD and LOQ for Rha-C10-C10 is 0.05 and 0.1 μg/mL and for Rha-Rha-C10-C10 is 0.1 and 0.5 μg/mL, respectively. The method was validated for linearity, intra- and inter-day precision and accuracy in accordance with FDA guidelines. The method was applied for the quantification of 14 individual RL congeners produced by Pseudomonas aeruginosa ST5 and comparison of RLs composition on four different carbon sources. Quantification of the individual congeners showed a conserved congener distribution irrespective of carbon source with a preferential selection for C10 β-hydroxyacids as the lipid component of RLs. The only statistically significant differences detected were between actual RL yields on the various carbon sources.


Rhamnolipids UPLC-MS/MS Quantification Congeners Triple quadrupole 



IB, RM and KT acknowledge European Commission within the FP-7 PROJECT under Grant Agreement No. 312139 ‘Integrated Biotechnological Solutions for Combating marine Oil Spills’—KILL•SPILL.

Authors also acknowledge the Institute of Biomedical Sciences, Ulster University, Coleraine, UK.

Conflict of interest

Authors declare that they have no competing interests.

Supplementary material

253_2015_6837_MOESM1_ESM.pdf (413 kb)
ESM 1 (PDF 412 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Michelle Rudden
    • 1
  • Konstantina Tsauosi
    • 1
  • Roger Marchant
    • 1
  • Ibrahim M. Banat
    • 1
  • Thomas J. Smyth
    • 2
    • 3
  1. 1.School of Biomedical ScienceUlster UniversityColeraineUK
  2. 2.Department of Life SciencesInstitute of Technology Sligo, Co.SligoRepublic of Ireland
  3. 3.Teagasc Food Research Centre AshtownDublinIreland

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