###### Abstract  
As a universal quorum sensing (QS) signal, autoinducer-2 (AI-2) is utilized by both Gram-negative and Gram-positive bacteria to coordinate several group behaviors, such as biofilm formation, virulence, and motility, when the bacterial cell density exceeds the thresholds. The determination of the AI-2 level is essential to understand the physiological and biochemical processes involved in bacterial communication. However, the current methods for AI-2 determination are complicated, time-consuming, and require costly equipment, such as a mass spectrometer (MS) or fluorescence detector (FLD). In this study, we present a new and easily applicable method for AI-2 determination. This method, based on the primary derivatization of AI-2 with 2,3-diamionnaphthalene (DAN), uses an affordable high-performance liquid chromatography (HPLC) instrument with a UV detector. Under optimized conditions, our method showed a good linearity (r\({}^{2}\) = 0.999) and demonstrated the effective detection of AI-2 levels in various environmental samples, as follows: 0.38 (\(\pm\)0.05) uM for _E. coli_ K12, 0.48 (\(\pm\)0.05) uM for _Aeromonas_ sp. YB-2, 0.32 (\(\pm\)0.06) uM for the _Enterobacter_ sp. YB-3, and 0.28 (\(\pm\)0.16) uM for activated sludge.  
autoinducer-2; (S)-4,5-dihydroxy-2,3-pentandione; HPLC-UV; quorum sensing; activated sludge +
Footnote †: journal: Applied Sciences  
17 September 2021  
Accepted: 28 September 2021  
Submitted: 30 September 2021  
The detection of AI-2, however, has proven to be more complicated, as it can form various equilibrium structures through spontaneous cyclization in the aqueous phase (Figure S1) [20].  
The current methods used to detect AI-2 can be classified into two categories (Table 1): (i) methods that require a derivative step (e.g., HPLC with fluorescence detector (HPLC-FLD) [21], LC-MS [22; 23], GC-MS [24], etc.) and (ii) methods that do not require derivatization (e.g., AI-2 bioassay [25; 26]).  
One example of the second type is the widely applied AI-2 bioassay method that uses the bioluminescent response of an AI-2 reporter strain (e.g., _Vibrio harveyi_ BB strains) to measure the intensity of the AI-2 signal. Although the AI-2 bioassay has a relatively low detection limit, it requires a long preparation (>12 h) and measurement (5-7 h) time, in addition to sophisticated analytical skills. Moreover, the reproducibility of the method is relatively poor and depends on the AI-2 reporter strain and the state of the sample. Conversely, the first type of methods (HPLC-FLD, LC-MS, and GC-MS), which require a derivatization procedure for the fixation of the AI-2 signal structures, have the disadvantages that the additional derivative step is relatively less sensitive, and that advanced and costly detectors, such as a mass spectrometer (MS) or an FLD, are required. Nevertheless, these methods have the advantages of a short measuring time (<30 min), high reproducibility, and ease of application.  
In view of the limitations of the current AI-2 determination methods, we developed a new method that uses a relatively accessible and low-cost HPLC-UV detector. Here, we demonstrate the validity of this new method for various environmental samples of pure and mixed bacterial cultures, such as _E. coli_ K12 [9; 27], _Aeronomas_ sp. YB-2 [9], _Enterobacter_ sp. YB-3 [9], and activated sludge.