Quorum Sensing

or other metabolites directly in culture broth. In multiple reaction

monitoring (MRM) mode, one takes advantages of the ability of

the instrument to select and fragment specific pseudomolecular

ions, and monitor the intensity of one specific resulting fragment

ion. This scanning mode is limited to a preset series of fragmenta-

tion reactions (called transitions), but due to the specificity of the

fragmentation, it provides a much better signal-to-noise ratio than

the full-scan mode, thus increasing the sensitivity of the analysis.

The MRM mode is especially interesting for the analysis of AHLs,

or for low concentrations of HAQs produced in complex matrices,

such as from infected animal tissue samples.

3.1 Direct
Quantification of HAQs
from Culture Broth
in Full-Scan Mode

1.P. aeruginosaPA14 cultivated in TSB medium at 37C and

200 rpm overnight (seeNote 1) is used to inoculate 3 ml of

fresh TSB medium at a starting OD 600 of 0.05 (seeNote 2).

The cultures are then incubated under the same conditions,

typically until they reach an OD 600 of 3.0 (seeNote 3). A 300μl

culture sample is then transferred to a microcentrifuge tube and

300 μl methanol containing the internal standards is added (see

Note 4). Vortex briefly.

2. The tube is centrifuged at 13,000
   gfor 15 min to pellet the
   cells and debris, and then 500μl of the supernatant is pipetted
   in a borosilicate HPLC vial, from which 20μl is injected in the
   HPLC.


3. The solvent gradient for the chromatographic run is as follows:
   from 0 to 1 min 70% solvent A; from 1 to 13 min 100% solvent
   B; from 13 to 23 min 100% solvent B; from 23 to 25 min 70%
   solvent A; and from 25 to 28 min 70% solvent A (seeNote 5).
   Flow rate is set at 400μl/min split to 40μl/min by the T
   splitter.


4. The MS parameters are positive mode; needle voltage 3.0 kV;
   cone 30 V; block temperature 120C and drying gas 150C;
   nebulizing gas 20 l/min; and drying gas 200 l/min.


5. In full-scan mode, the scanning range is set tom/z100–400.
   The chromatogram of all the ions monitored (total ion chro-
   matogram or TIC) is presented in Fig.2.


6. Figure3 shows the chromatogram of the pseudomolecular ions
   (M + H)+of HHQ, PQS, and HQNO at 244, 260, and 260,
   respectively, and those of the internal standards HHQ-d 4 and
   PQS-d 4 at 248 and 264, respectively. With this column the
   retention time of PQS is 21.8 min while the one of the isobaric
   (an ion with the same nominalm/zvalue) HQNO is 19.9 min.
   All members of the HQNO family have a retention time
   shorter than those of the corresponding alkyl chain length in
   the PQS family.

52 Franc ̧ois Le ́pine et al.