Abstract:Quorum sensing, the communication between microorganisms, is mediated by specific diffusible signal molecules. Gram-negative bacteria generally use N-acyl-homoserine lactones (AHLs) as their command language in coordinating their intra- and intercellular population behaviors. AHLs play an important role in controlling diverse physiological processes, including generation of extracellular toxin and enzymes by pathogenic bacteria, formation of biofilm, bioluminescence, biosynthesis of antibiotics, bacteria motility, etc. The authors have found in their recent studies that communication of microbial signals is closely related to microbial degradation of organic pollutant. Therefore, the detection and quantification of AHLs are essential for investigation of quorum sensing in gram-negative bacteria. Currently, there are several AHLs detection methods, including mainly enzyme linked immunosorbent assay (ELISA), biosensor, thin layer chromatography (TLC) and chromatography. In this study, a fast, simple selective method was established for detecting N-acyl-homoserine lactones (AHLs) in soil solution by the GC-MS (gas chromatography-mass spectrometry). The novel method can detect AHLs qualitatively and quantitatively at the same time. Using the full scan mode, seven kinds of AHLs (C4, C6, C7, C8, C10, C12 and C14-HSL) standards were analyzed by GC-MS. The best results in terms of selectivity and time consumption of the analysis were obtained. Sample injection was done in the split mode (5:1) and pure helium (99.999%) was used as carrier gas at a flow rate of 1 ml min-1. The GC injector temperature was optimized to 290 ℃. The oven temperature program was optimized to start from 100 ℃ to 150 ℃ at a rate of 35 ℃ min-1 and then to 280 ℃ at a rate of 25 ℃ and maintained for 6 min. Under such optimized conditions, all the 7 kinds of AHLs were well separated within 12 minutes. Furthermore, the mass spectrum of the 7 AHLs exhibited a molecular ion peak [M] +that was unique of each compound, i.e. C4-HSL (m/z 171),C6-HSL(m/z 199),C7-HSL(m/z 213),C8-HSL(m/z 227),C10-HSL(m/z 255),C12-HSL(m/z 283) and C14-HSL(m/z 311). Probably as a result of the McLafferty rearrangement, they generated a common fragment ion m/z 143. As the 7 AHLs all contained abundant m/z 143 ions, the ions could be used as a marker fragment to detect AHLs in unknown samples on mass detector operating in the SIM mode. The detection limits of the seven AHLs (C4, C6, C7, C8, C10, C12 and C14-HSL) standards were 1.50, 2.00, 1.50, 2.00, 2.00, 2.50, and 2.50 µg l-1, respectively. The standard curves of all the 7 AHLs displayed nice linear relationships up to 2.0 mg l-1 and their coefficients of determination were all higher than 0.997. The recovery experiment using ethyl acetate as extracting agent shows that in the solutions with spiking concentrations being 0.5 mg l-1and 2.0 mg l-1, recoveries of 6 AHLs (C6, C7, C8, C10, C12 and C14-HSL) varied in the ranges of 76%~85% and 74%~97%, respectively, whereas the recovery of C4-HSL was relatively low, being about 55% for both spiking concentrations. In 3 soil solutions of Latosolic red soil and Yellow brown soil, different in soil/water ratio, the recoveries of C4-HSL ranged from 56% to 71% for both spiking concentrations, while the recoveries of the other 6 AHLs were all relatively higher, being in the ranges of 71%~108% and 68%~104% for spiking concentration of 0.5 mg l-1 and 2.0 mg l-1, respectively. No significant difference was observed in recovery of each AHL relative to soil solution, indicating that the organic and inorganic compounds in the soil solutions have no influence on extraction of AHLs in soil solutions using ethyl acetate. In addition, significant relationships were observed between the recovery of AHLs in different media and their logP (P stands for partion between normal butanol and water) as well as logS (solubility in water), but no relationship was between the recovery of AHLs and their polarity, showing that the recovery of AHLs were mainly related to their logP values and solubility in water. Therefore, this extracting method coupled with GC-MS can be effectively applied to analysis of AHLs in different soil solutions. Thereafter AHLs concentrations in the soil solutions were determined. It was found that the concentrations of the 7 AHLs varied in the range of 3.8~8.7 µg l-1and 4.2~9.8 µg l-1 in the solutions of Latosolic red soil and yellow brown soil, respectively. It could be concluded that AHLs in both aquatic solution and complex media, such as soil solution, can be analyzed using GC-MS coupled with ethyl acetate extraction。