Robust HPLC-MS/MS quantification of N-acyl homoserine lactones (AHLs) in biomass-associated solid matrices reveals...

N-acyl homoserine lactones (AHLs) are quorum-sensing signals that regulate biofilm formation, extracellular polymeric substance production, and other collective microbial behaviors in biological wastewater treatment systems. However, reliable AHL quantification in biomass-associated solid matrices remains analytically challenging because sludge and biofilm extracts contain co-extracted constituents that cause severe matrix effects and chain-length-dependent recovery losses. Here, an ultrasonic extraction-liquid-liquid extraction (UE-LLE) workflow coupled with HPLC-MS/MS was developed and validated for ten AHLs in three representative solid matrices: suspended sludge and carrier biofilm from an integrated fixed-film activated sludge (IFAS) system, and carrier biofilm from a moving-bed biofilm reactor (MBBR). Ethyl acetate outperformed dichloromethane and methanol during ultrasonic extraction, and post-extraction LLE clean-up reduced co-extracted interferences more effectively than HLB solid-phase extraction. A grouped multi-internal-standard strategy using non-native C7-, C9-, and C11-HSL corrected residual chain-length-dependent bias without requiring matrix-matched calibration. The optimized method achieved relative recoveries of 70-121%, relative matrix effects of 78-115%, and limits of detection below 0.26 ng/g MLVSS across all three matrices. When combined with an established aqueous-phase workflow and applied to the paired samples from the tested IFAS, MBBR, aerobic granular sludge, and activated sludge systems, aqueous-phase measurements accounted for only 20-48% of the total measured target AHL concentration on a common reactor-volume basis. Thus, for these analyzed samples, aqueous-only quantification would have underestimated the measured target AHL pool by approximately 2.1-5.0-fold and could bias cross-system comparisons. Overall, this workflow provides a robust solid-matrix analytical approach that complements aqueous-phase analysis and strengthens the quantitative basis for AHL occurrence assessment and quorum-sensing interpretation in wastewater treatment systems.