The quorum sensing is a chemical language allowing bacteria to interact [1], through the excretion of a set of molecules called auto-inducers. From a threshold population density, and thus a threshold concentration of auto-inducing molecules, their detection by an intracellular receptor regulates bacterial gene expression, including resistance or virulence genes. Our study focuses on the auto-inducing N-acyl-homoserine lactones (AHL) [2] of Gram-negative bacteria from the Burkholderia and Paraburkholderia genus as some of these bacteria species are described as opportunistic pathogens for humans [2]. The AHL differ in their acyl-chain length (generally from C4 to C18) and may be modified by a 3-oxo or 3-hydroxy substituent, a terminal methyl branch, or various degrees of unsaturations [3]. As the bacterial signal specificity depends on all of those, including the instauration’s position on the acyl chain, it is essential to obtain a complete structural charaterization of AHL to better understand the population regulation and virulence phenomenon. Although several mass spectrometry (MS) methods have been developed to study AHL [4], only NMR analyses requiring the purification of a large amount of product enable the localization of the double bonds on the acyl chain. Thus, this study aims to develop a new method that will significantly reduce the amount of biological material needed. This method is based on LC-MS/MS or/and SFC-MS/MS analyses of extracts derived by a Paternò-Büchi reaction in solution or post-column [5]. In parallel, in situ characterization will be performed by the deposition of a photoreactive matrix on the bacterial cultures followed by imaging MS analyses. References 1. J. Baltenneck, S. Reverchon, F. Hommais, Microorganisms, 9, 239 (2021) 2. L. Eberl, Int. J. Med. Microbiol., 296, 103-110 (2006) 3. M. Churchill, L. Chen, Chem. Rev., 111, 68-85 (2011) 4. N. Patel, J. Moore, H. Blackwell, D. Amador-Noguez, PloS One, 11, e0163469 (2020) 5. T. Xu, Z. Pi, F. Song, S. Liu, Z. Liu, Anal. Chim. Acta, 1028, 32-44 (2018)