Detection and Quantitation of Bacterial Acylhomoserine Lactone Quorum Sensing Molecules via Liquid Chromatography–Isotope Dilution Tandem Mass Spectrometry

A range of acylhomoserine lactones (AHLs) are used as intraspecies quorum sensing signals by Gram-negative bacteria, and the detection and quantitation of these molecules is of interest. This manuscript reports a liquid chromatographic–isotope dilution tandem mass spectrometric method for the quantitation of these molecules. A divergent solid-phase synthesis of stable-isotope-labeled AHLs suitable for use as an internal standard is reported. This route relies on the biomimetic conversion of a dideuterated methionine equivalent, <i>N</i>-Fmoc-(4,4-²H₂)­methionine, to the desired labeled AHL, and a representative series of eight of these molecules was produced in >95% purity and yields up to ∼50%. The representative AHL internal standards were then used to develop an optimized liquid chromatographytandem mass spectrometric (LC–MS/MS) separation and detection protocol for these molecules, which relies on a high-efficiency C18 core–shell column to minimize the time necessary for separation. The addition of internal standards at different steps during sampling was also found to affect the analysis for hydrophobic AHLs with addition prior to cell removal giving the most accurate results. Taken together, the use of the internal standards and separation method reported herein provides a rapid and quantitative method for the study of AHL production in bacteria