normal scanning mode. However, HHQ and the other mem-
bers of the same family of compounds are the precursors of
PQS and its congeners and thus show a decrease in their
concentration following an initial increase. It is thus important
to select the proper cultivation time if HHQ and its congeners
are to be measured.
- HAQs such as PQS and HHQ have a limited solubility in water.
For example the solubility of PQS is<5 mg/l in TSB. How-
ever, concentrations in excess of 15 mg/l have often been
measured in whole cultures. This is an indication that these
compounds are either pseudosolubilized by bacterial exopro-
ducts or adsorbed on the cell surface. As bacteria must be
removed from the medium prior to analysis, in order to avoid
plugging the HPLC column, this can have an important effect
on the concentration of HAQs detected in the samples. To
dissociate HAQs from the bacteria cell surface, methanol is
added to the culture at a 50% (v/v) concentration prior to
removing the cells by centrifugation [8]. In fact, we measure
typically 50% less HAQs if the cells are centrifuged prior to
adding methanol. Thus addition of methanol (which contains
the internal standards) to the culture samples serves two pur-
poses: to release bound HAQs and to act as carrier for the
internal standards for quantification purposes.
- Because of occasional uncertainties in the supply of acetonitrile,
an alternative gradient method can be used with 2-propanol as
solvent according to the following steps: at the moment of
injection 90% solvent A; from 0 to 3 min 40% solvent B (2-
propanol containing 1% acetic acid); from 3 to 10 min 65%
solvent B; from 10 to 21 min 70% solvent B; from 21 to 23 min
100% solvent B; from 23 to 25 min 100% solvent B; from 25 to
26 min 90% solvent A; and from 26 to 30 min 90% solvent A.
- One problem with PQS quantification is that when the HPLC
column ages, the shape of the PQS peaks tends to present
tailing and the area of the corresponding peak often varies
considerably from one injection to another, which makes the
quantification more difficult without using an appropriate
deuterated internal standard. The PQS-d 4 internal standard
fluctuates in the same manner, thus correcting for these
variations.
- The most abundant members of each families of compound can
be quantified using their respective internal standard (PQS-d 4
for the PQS family and HHQ-d 4 for all the other HAQs) by
adding (or subtracting) 28 Da to the weight of the
corresponding pseudomolecular ion of the internal standard
while monitoring the same fragment ion. Under these condi-
tions, fragmentation of all HAQs produces a fragment ion
common to all the congeners of a given family [10].
HAQ and AHL Analysis Using LC/MS 57
