Detection of Microbiological Contamination and Malfunctions in Technical Equipment Used in the Milk Processing Industry Using Acyl‐Homoserine Lactones With Liquid Chromatography/Triple Quadrupole Mass Spectrometry

RATIONALE: Bacterial contamination causes sensory changes and health risks and is a critical quality factor in the food industry. Plate-based microbiological methods have long incubation times, making rapid contamination control difficult. Homoserine lactones (HSLs) from Gram-negative bacteria mediate quorum sensing that drives biofilm and toxin formation; our approach uses four homoserine lactones to rapidly identify contamination. METHODS: A sample preparation and LC/MS/MS method were established and validated for the four HSLs of different polarity: N-butyryl-L-homoserine lactone, N-(3-hydroxy-butanoyl)-L-homoserine lactone, N-(3-oxooctanoyl)-L-homoserine lactone, and N-(3-oxododecanoyl)-L-homoserine lactone. This method uses positive electron spray ionization with a triple quadrupole mass analyzer and γ-decalactone as the internal standard. The validated method was then used to analyze HSLs extracted from technical cooling water by solid-phase extraction. RESULTS: The method provides same-day results without production shutdowns and baseline-separated chromatography. Applied to real cases, HSLs profiling identified contamination and malfunctions with six true-positive and six true-negative classifications. CONCLUSION: HSL-targeted LC/MS/MS offers a practical, rapid, and sensitive tool for detecting leaks, biofilms, and hidden microbial activity in complex industrial water matrices. The approach enables earlier risk management, reduced downtime, and routine monitoring in milk-processing facilities.