Quorum Sensing

5. Evaporate ethyl acetate of the extracts with a rotary evaporator
   or centrifugal evaporator (maximum temperature 40C) (see
   Note 7).


6. Add 120μl of methanol (HPLC grade) to dissolve the dried
   extract and mix by vortexing for 10 s.


7. Centrifuge the crude extract at 13,800gfor 15 min at 4C
   to remove particles.


8. Transfer the supernatant (approximately 100μl) into a chro-
   matography vial fitted with a flat-bottom insert and cap the vial.

3.3 UPLC/MS
Analysis

1. Maintain the column temperature at 30C and keep the flow
   rate at 0.4 ml/min.


2. Balance the column for 15 min or longer until a stable baseline.


3. Inject 5μl of DSF or BDSF standard solutions, or 5μl of the
   extract from Subheading3.2,step 8, to a 1290 Infinity UPLC
   equipped with a Zorbax Eclipse XDB-C18 column.


4. Detect DSF family signals in a diode array detector under the
   detection wavelength of 220 nm with a bandwidth of4 nm.


5. The UPLC elutes are further introduced into an accurate mass
   TOF-MS equipped with a Jet Stream (JS) electrospray ioniza-
   tion (ESI) source in the negative ionization mode.


6. MS source operating parameters are as follows: (1) gas temper-
   ature: 325C; (2) drying gas: 8 l/min; (3) nebulizer: 35 psig;
   (4) sheath gas temperature: 350C; (5) sheath gas flow: 11 l/
   min; (6) capillary voltage (Vcap): 3500 V; (7) nozzle voltage:
   200 V; and (8) mass range: m/z 100–1700.

3.4 Quantification
of DSF Family Signals

1. Use the MassHunter Workstation Data Acquisition Software
   (revision B.04) to acquire data in the centroid mode. Typical
   mass spectra of DSF, BDSF, CDSF, and IDSF are shown in
   Figs.2–4.
   (a) To detect [BDSF-H]–, set the monoisotopic exact value
   (m/z) as 197.1547 (Fig.2).
   (b) To detect [CDSF-H]–, set the monoisotopic exact value
   (m/z) as 209.1547 (Fig.3).
   (c) To detect [DSF-H]–and [IDSF-H]–, set the monoisoto-
   pic exact value (m/z) as 211.1704 (Fig.4).


2. Integrate and quantify the area under each of the chro-
   matographic peaks.


3. Create a standard curve for DSF or BDSF by plotting the peak
   area vs. the known concentrations. Example standard curves
   are shown in Fig.5, in which the DSF or BDSF standards at the
   concentrations of 1μM, 5μM, 10μM, and 50μM were used
   (seeNote 8).

UPLC/MS Analysis for DSF-Family Signals 101