Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry


In recent years, use of supercritical-fluid chromatography (SFC) with CO2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC–HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 157

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Williams P. Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology. 2007;153(Pt 12):3923–38.

  2. 2.

    Fuqua WC, Winans SC, Greenberg EP. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994;176(2):269–75.

  3. 3.

    Williams P, Winzer K, Chan W, Cámara M. Look who's talking: communication and quorum sensing in the bacterial world. Philos Trans R Soc B. 2007;362(1483):1119–34.

  4. 4.

    von Bodman SB, Willey JM, Diggle SP. Cell-cell communication in bacteria: united we stand. J Bacteriol. 2008;190(13):4377–91.

  5. 5.

    González JF, Venturi VT. A novel widespread interkingdom signaling circuit. Trends Plant Sci. 2013;18(3):167–74.

  6. 6.

    Sperandio V. Novel approaches to bacterial infection therapy by interfering with bacteria-to-bacteria signaling. Expert Rev Anti-Infect Ther. 2007;5(2):271–6.

  7. 7.

    Càmara M, Daykin M, Chhabra SR. Detection, purification, and synthesis of N-acyl homoserine lactone quorum sensing signal molecules. Methods Microbiol. 1998;27:319–30.

  8. 8.

    Doberva M, Stien D, Sorres J, Hue N, Sanchez-Ferandin S, Eparvier V, et al. Large diversity and original structures of acyl-homoserine lactones in strain MOLA 401, a marine Rhodobacteraceae bacterium. Front Microbiol. 2017;8:1152.

  9. 9.

    Wang Y, Zhang X, Wang C, Fu L, Yi Y, Zhang Y. Identification and quantification of acylated homoserine lactones in Shewanella baltica, the specific spoilage organism of Pseudosciaena crocea, by ultrahigh-performance liquid chromatography coupled to triple quadrupole mass spectrometry. J Agric Food Chem. 2017;65(23):4804–10.

  10. 10.

    Patel NM, Moore JD, Blackwell HE, Amador-Noguez D. Identification of unanticipated and novel N-Acyl L-Homoserine Lactones (AHLs) using a sensitive non-targeted LC-MS/MS method. PLoS One. 2016;11(10):e0163469.

  11. 11.

    Steindler L, Venturi V. Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors. FEMS Microbiol Lett. 2007;266(1):1–9.

  12. 12.

    Cataldi TR, Bianco G, Frommberger M, Schmitt-Kopplin P. Direct analysis of selected N-acyl-L-homoserine lactones by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2004;18(12):1341–4.

  13. 13.

    Charlton TS, de Nys R, Netting A, Kumar N, Hentzer M, Givskov M, et al. A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol. 2000;2(5):530–41.

  14. 14.

    Frommberger M, Hertkorn N, Englmann M, Jakoby S, Hartmann A, Kettrup A, et al. Analysis of N-acylhomoserine lactones after alkaline hydrolysis and anion-exchange solid-phase extraction by capillary zone electrophoresis-mass spectrometry. Electrophoresis. 2005;26(7-8):1523–32.

  15. 15.

    Ortori CA, Dubern JF, Chhabra SR, Cámara M, Hardie K, Williams P, et al. Simultaneous quantitative profiling of N-acyl-L-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS. Anal Bioanal Chem. 2011;399(2):839–50.

  16. 16.

    Purohit AA, Johansen JA, Hansen H, Leiros HK, Kashulin A, Karlsen C, et al. Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family. J Appl Microbiol. 2013;115(3):835–47.

  17. 17.

    Leipert J, Treitz C, Leippe M, Tholey A. Identification and quantification of N-acyl homoserine lactones involved in bacterial communication by small-scale synthesis of internal standards and matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom. 2017;28(12):2538–47.

  18. 18.

    Laboureur L, Bonneau N, Champy P, Brunelle A, Touboul D. Structural characterisation of acetogenins from Annona muricata by supercritical fluid chromatography coupled to high-resolution tandem mass spectrometry. Phytochem Anal. 2017;28(6):512–20.

  19. 19.

    He PX, Zhang Y, Zhou Y, Li GH, Zhang JW, Feng XS, et al. Analyst. 2019.

  20. 20.

    West C. Current trends in supercritical fluid chromatography. Anal Bioanal Chem. 2018;410(25):6441–57.

  21. 21.

    Liu LX, Zhang Y, Zhou Y, Li GH, Yang GJ, Feng XS. The application of supercritical fluid chromatography in food quality and food safety: an overview. Crit Rev Anal Chem. 2019:1–25.

  22. 22.

    Barthélemy M, Elie N, Pellissier L, Wolfender JL, Stien D, Touboul D, et al. Structural identification of antibacterial lipids from Amazonian palm tree endophytes through the molecular network approach. Int J Mol Sci. 2019;20(8):E2006.

  23. 23.

    Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinf. 2010;11:395.

  24. 24.

    Myers OD, Sumner SJ, Li S, Barnes S, Du X. One step forward for reducing false positive and false negative compound identifications from mass spectrometry metabolomics data: new algorithms for constructing extracted ion chromatograms and detecting chromatographic peaks. Anal Chem. 2017;89(17):8696–703.

  25. 25.

    Olivon F, Elie N, Grelier G, Roussi F, Litaudon M, Touboul D. MetGem software for the generation of molecular networks based on the t-SNE algorithm. Anal Chem. 2018;90(23):13900–8.

  26. 26.

    MacDougall D, Amore FJ, Cox GV, Crosby DG, Estes FL, Freeman DH, et al. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal Chem. 1980;52:2242–9.

  27. 27.

    West C, Lesellier E. Orthogonal screening system of columns for supercritical fluid chromatography. J Chromatogr A. 2008;1203(1):105–13.

  28. 28.

    Riddell N, Bavel B, Jogsten IE, McCrindle R, McAlees A, Chittim B. Coupling supercritical fluid chromatography to positive ion atmospheric pressure ionization mass spectrometry: ionization optimization of halogenated environmental contaminants. Int J Mass Spectrom. 2017;421:156–63.

  29. 29.

    Sawana A, Adeolu M, Gupta RS. Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species. Front Genet. 2014;5:429.

  30. 30.

    Coenye T, Vandamme P. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ Microbiol. 2003;5(9):719–29.

  31. 31.

    Suárez-Moreno ZR, Caballero-Mellado J, Venturi V. The new group of non-pathogenic plant-associated nitrogen-fixing Burkholderia spp. shares a conserved quorum-sensing system, which is tightly regulated by the RsaL repressor. Microbiology. 2008;154(Pt 7):2048–59.

  32. 32.

    Coutinho BG, Mitter B, Talbi C, Sessitsch A, Bedmar EJ, Halliday N, et al. Regulon studies and in planta role of the BraI/R quorum-sensing system in the plant-beneficial Burkholderia cluster. Appl Environ Microbiol. 2013;79(14):4421–32.

  33. 33.

    Krick A, Kehraus S, Eberl L, Riedel K, Anke H, Kaesler I, et al. A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactone. Appl Environ Microbiol. 2007;73(11):3587–94.

  34. 34.

    DiMango E, Zar HJ, Bryan R, Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Investig. 1995;96(5):2204–10.

  35. 35.

    Smith RS, Kelly R, Iglewski BH, Phipps RP. The Pseudomonas autoinducer N-(3-oxododecanoyl) homoserine lactone induces cyclooxygenase-2 and prostaglandin E2 production in human lung fibroblasts: implications for inflammation. J Immunol. 2002;169(5):2636–42.

  36. 36.

    Tateda K, Ishii Y, Horikawa M, Matsumoto T, Miyairi S, Pechere JC, et al. The Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone accelerates apoptosis in macrophages and neutrophils. Infect Immun. 2003;71(10):5785–93.

  37. 37.

    Horikawa M, Tateda K, Tuzuki E, Ishii Y, Ueda C, Takabatake T, et al. Synthesis of Pseudomonas quorum-sensing autoinducer analogs and structural entities required for induction of apoptosis in macrophages. Bioorg Med Chem Lett. 2006;16(8):2130–3.

  38. 38.

    Le Balc’h E, Landman C, Tauziet E, Brot L, Quevrain E, Rainteau D et al.. 3-oxo-C12:2-HSL, a new N-acyl-homoserine lactone identified in gut ecosystem exerts an anti-inflammatory effect and does not modify paracellular permeability. The 12th Congress of ECCO – European Crohn’s and Colitis Organisation; 2017; Barcelona.

Download references


This work was supported by the Agence Nationale de la Recherche (grant ANR-16-CE29-0002-01 CAP-SFC-MS), an Investissement d’Avenir grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-01), a joint Agence Nationale de la Recherche and Swiss National Science Foundation (SNF) grant (SECIL, reference ANR-15-CE21-0016 and SNF no. 310030E-164289), and a grant from Région Ile-de-France (DIM Analytics).

Author information

Correspondence to David Touboul.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Published in the topical collection Current Progress in Lipidomics with guest editors Michal Holčapek, Gerhard Liebisch, and Kim Ekroos.

Electronic supplementary material


(PDF 1607 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hoang, T.P.T., Barthélemy, M., Lami, R. et al. Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry. Anal Bioanal Chem (2020).

Download citation


  • Supercritical-fluid chromatography
  • N-Acyl homoserine lactone
  • Quorum sensing
  • Quantification
  • Molecular networking