Development of an extraction method and LC-MS analysis for N-acylated-l-homoserine lactones (AHLs) in wastewater treatment biofilms

•Highest AHLs recoveries from liquids samples obtained by HLB columns.•UE (ultrasonic extraction) recovers the highest AHLs from biofilm samples.•HLB and UE combined with UPLC-MS/MS could successfully detect AHLs in biofilm samples.•AHLs in biofilms samples has been determined with a range of 1.33-337.59 ng/g.

N-Acylated-l-homoserine lactones (AHLs) play a vital role in Gram-negative bacteria communication by promoting the formation of extracellular polymeric substances (EPS) and biofilms. However, the low concentration of these AHL signals makes the process difficult to understand. A robust and sensitive pretreatment method for AHL detection was developed in this work. Compared with eight different solid-phase extraction (SPE) columns and three various solid extraction method, we found that the UE (ultrasonic extraction) and an Oasis hydrophilic-lipophilic-balanced (HLB) sorbent in column format combined with ultra-performance liquid chromatography linked to tandem mass spectrometry (UPLC-MS/MS) can be successfully used for systematic pretreating moving bed biofilm reactor (MBBR) biological samples to extract AHLs and determine concentration of AHLs in wastewater treatment biofilms. This easy-to-follow protocol makes it ideal for quantitative analyses of AHLs in wastewater treatment biofilms.