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.