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The reversed phase separation was achieved on a BEH C18 packing with 1.7?��m particle http://www.selleckchem.com/products/pf-562271.html diameter and column dimensions of 100 �� 2.1?mm (Waters). The thermostat of the column was set to 60?��C and the autosampler to 27?��C. The flow rate was 0.9?mL?min?1 and the injection volume was 20?��L via full loop. For the separation of the AHLs, a linear solvent strength gradient was applied starting with water containing 10%?v/v acetonitrile increasing to 100% acetonitrile in 1?min. The serines were determined using a separate method with the mobile phase of 10?mM ammonium formate at pH 4.25 containing 10%?v/v acetonitrile for 0.2?min following solvent gradient to 100% acetonitrile within half a minute. The detection wavelength was set to 197?nm (1.2?nm http://www.selleck.cn/products/azd6738.html width) with the scan rate of 20?Hz. The peak areas were integrated using empower 2 (Waters). Positive FT-ICR spectra for qualitative analysis of AHLs were acquired on a Bruker Daltonics (Bremen, Germany) Apex Qe 12T system equipped with an APOLLO II source and microspray infusion of 120?��L?h?1 with a scan number of 256. Spectra were acquired in the broadband mode and were calibrated externally on clusters of arginine. Peaks exceeding a threshold signal-to-noise ratio of 3 were exported to peak lists. The resulting text files containing >2300 mass�Cintensity pairs were scanned using a software tool written in python (http://www.python.org) for the presence of peaks from a reference file with theoretically possible protonated and sodium-cationized homoserine lactone masses with an error http://www.selleckchem.com/products/VX-765.html Only the m/z of those AHLs were considered for which the 13C isotope peak was detectable. The fitting of the mathematical model to the experimental data was performed using a minimization procedure (least square method) of the software tool matlab, version 7.5.0.338 (R2007b, The Mathworks Inc., Natick, MA). For studying the AHL production and kinetics of P. putida IsoF, two different set-ups were used: (1) a 5-L beaker from which 200?mL was taken at each time point and (2) a set of 500-mL baffled flasks each containing sample volumes of 200?mL to allow the comparison of production rates under different culture conditions. The cultures were incubated for 34?h in a well-buffered medium (pH 6.8�C7.2) and grew exponentially from 6 to 13?h, followed by the late exponential phase from 13 to 19?h after inoculation (Fig. 1a). Table 1 summarizes the detected putative AHLs extracted from the supernatants. The highest m/z was found for 3-oxo-C10-HSL (1.7 �� 109), followed by 3-oxo-C12-HSL (1.7 �� 108) and 3-oxo-C8-HSL (1.4 �� 107) as can be seen in Fig. 2b, as well. The other possible AHLs with lower intensity differed mostly in the saturation and/or the keto- or hydroxyl substitution of the side chain and not so much in its length. To confirm the identification, the same sample was separated by UPLC and detected by a photo diode array (Fekete et al., 2007).