The hydrolysis of unsubstituted N-acylhomoserine lactones to their homoserine metabolites. Analytical approaches using ultra performance liquid chromatography.

Derivatives of N-acylhomoserine lactones (HSLs) with different alkanoyl side chains occur as quorum or diffusion sensing molecules in gram-negative bacteria and their quantitative chemical analysis became important as a possible way to follow regulation processes of their pathogenicity towards plants and animals. The lactone-ring of HSLs is chemically and biologically not stable: the corresponding serines can be formed in alkaline conditions and these may presumably behave inactive for the biological system. A fast and MS compatible liquid chromatographic method applying high pressure (ultra performance liquid chromatography) with diode array detection was optimized for the rapid quantitative determination of HSLs and their corresponding hydrolysis products. The technique was used to follow and model the hydrolysis reactions of HSLs as function of pH under controlled conditions. Moreover, the method could be triggered to allow a confirmation in the assignment of the potential HSLs in real samples by analysis of the real samples before and after hydrolysis. Quantitative performance characteristics and the character of the hydrolysis reaction were studied as well. The optimized method was successfully applied to a bacterial culture supernatant real sample containing HSLs.

Development and application of a method for the analysis of N-acylhomoserine lactones by solid-phase extraction and ultra high pressure liquid chromatography.

A robust method based on solid-phase extraction (SPE) followed by ultra high pressure liquid chromatography (with trade name of Ultra Performance Liquid Chromatography: UPLC; Waters, Milford, MA, USA) is proposed for the determination of five derivatives of N-acylhomoserine lactones (AHLs) that play a biological role as signal molecules of several gram-negative bacteria. Different commercial SPE cartridges were tested for sample extraction, clean-up and preconcentration. Since the sample matrix was a complex growth media, careful optimization of the SPE with respect to washing procedure, elution solvent and sample solvent was necessary. No sample loss was observed when up to 100 mL spiked full media was added onto the cartridge. Applying UPLC for the determination of AHLs, the performance characteristics of the method showed good separation efficiency and high speed. In order to demonstrate the applicability of the method, supernatants with the known AHL producer Burkholderia cepacia LA3 grown in different media were investigated. Additionally, the method was successfully used for the degradation/uptake study of AHLs from a liquid matrix in which barley was grown under controlled condition.

Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors.

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the "quorum sensing" response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing beta-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography-electrospray ionization ion trap mass spectrometry (nano-LC-MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC-MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.