- The following transitions are monitored: C 4 -HSL 172!102;
o-C 4 -HSL 190!120; HHQ-d 4248 !163; 3-oxo-C 12 -HSL
298 !102; and o-3-oxo-C 12 -HSL 316!120. The pressure
of the collision gas is set at 2 10 ^3 mTorr and the collision
energy is 15 V for the AHLs and 33 V for HHQ-d 4. - A calibration curve is performed to obtain the response factor
of C 4 -HSL and 3-oxo-C 12 -HSL relative to HHQ-d 4. To do so,
a series of solutions with increasing concentration of the AHL
from 0 to 15 mg/l are made with a constant concentration of
10 mg/l of HHQ-d 4. The same response factor is used for the
open and closed forms of each AHL. - The concentration of the AHLs in the solution is calculated as
follows:
RCA 2 =H
whereR ¼response factor,C¼concentration of internal
standard (HHQ-d 4 ),A¼area of the AHL peak, andH¼area
of the internal standard (HHQ-d 4 ) peak.3.4 Detection
of Non-targeted AHLs
from Culture Broth
in Parent Mode
Knowing that the majority of AHLs generate a 102 Da fragment
(e.g.,seeSubheading3.3,step 4), it is possible to detect and
quantify non-targeted AHLs by looking for the parent ion of
daughter ion 102 using the Parent mode.- The samples are prepared as in Subheading3.3.
- The source operating parameters are the same as in full-scan
mode (seeSubheading3.1). In Parent mode, the internal stan-
dard is Parent of 163!248 (HHQ-d 4 ). The pressure of the
collision gas (argon) is set at 3 10 ^3 mBar (collision gas flow
of 0.35 (ml/min) and a collision energy of 15 V are used). - Identify the AHLs corresponding to the parent ions of 102 in
the list of Table1.
3.5 Quantification
of Low Concentrations
of HAQs or AHLs
by Solvent Extraction
To obtain a better sensitivity or to quantify molecules present in
minute concentrations, it is optimal to extract the AHLs from the
cultures and concentrate.- Internal standard (HHQ-d 4 ) is added to a 10 ml culture sample
to get a final concentration of 0.2–0.5 mg/l. The sample is
then transferred to a 15 ml glass tube closed with a PTFE-lined
twist cap. - Two milliliter ethyl acetate (or other appropriate solvent) is
added and the mixture is strongly vortexed for 30 s. If the
emulsion is stable (the phases do not separate), the tubes can
be centrifuged to separate the phases.
HAQ and AHL Analysis Using LC/MS 55