Quorum Sensing

7. The area under each of these chromatographic peaks is
   integrated. The concentration of the analyte A in the culture
   medium is given by the equation:

C
A
 2 =H

whereC¼concentration of internal standard;A¼area of the

HAQ peak; andH¼area of the internal standard peak. The

factor of 2 is to keep into account the dilution factor due to the

addition of the methanol containing the internal standard. For

HHQ and HQNO and the other members of these families

with different chain lengths, the internal standard used is

HHQ-d 4 , while for the congeners of the PQS family the inter-

nal standard used is PQS-d 4 (seeNote 6).

3.2 Quantification
of HAQs from a
Complex Matrix
in MRM Mode

1. HAQs can be analyzed from a sample of muscle tissue from a
   mouse infected withP. aeruginosa[9]. One hundred milligram
   of muscle tissues are put in a 2 ml microcentrifuge tube to
   which is added 500μl of methanol containing 0.2 mg/l of
   HHQ-d 4 and PQS-d 4. The tissues are homogenized with a
   Polytron and then centrifuged at 13,000
   gfor 15 min. The
   supernatant is then collected and 80μl injected in the HPLC
   under the same conditions as in Subheading3.1.


2. The source operating parameters are the same as in full-scan
   mode. In MRM mode the following transitions are monitored:
   for HHQ 244! 159; HHQ-d 4 248! 163; HQNO
   260 !159; PQS 260!175; and PQS-d 4264 !179. The
   pressure of the collision gas (argon) is set at 2
   10 ^3 mTorr
   and the collision energy at 30 V for all transitions.


3. The area of each chromatographic peak is integrated and
   the concentration of each compound is calculated as above
   (seeNote 7).

3.3 Direct
Quantification
of Targeted AHLs
from Culture Broth
in MRM Mode

1. The bacteria are cultivated as in Subheading3.1 (seeNote 8).
   To 480μl of culture is added 120μl of anhydrous acetonitrile
   containing 15 mg/l HHQ-d 4 and the mixture is vortexed and
   then centrifuged at 13,000
   gfor 10 min. A 500μl aliquot is
   collected in a HPLC vial and 15μl is injected (seeNote 9).


2. The solvent gradient is as follows: from 0 to 1 min 100%
   solvent A; from 1 to 5 min 50% solvent B; from 5 to 13 min
   100% solvent B; from 13 to 23 min 100% solvent B; from 23
   to 25 min 100% solvent A; and from 25 to 28 min 100%
   solvent A. Flow rate is set at 400μl/min split to 40μl/min
   by the splitter. Under these conditions the open form of each
   AHL always has a shorter retention time than the
   corresponding closed form.

54 Franc ̧ois Le ́pine et al.