- Using a Pasteur pipette, the organic phase (top when ethyl
acetate is used) is transferred to a HPLC vial, being very careful
not to carry over any emulsion from the interface. - Especially if one wants to precisely quantify the concentration
of AHL present, steps 2– 4 should be repeated twice. The
organic phases are pooled together. - The solvent is then evaporated under a gentle stream of nitro-
gen gas, and finally the dry residue is kept in a tightly closed vial
at 20 C. - At the time of injection, the extract is solubilized in 500μl
anhydrous acetonitrile (seeNote 10). Vortex for 30 s to fully
dissolve the residue (seeNote 11). - Injection and quantification are performed as described above.
4 Notes
- HAQs have been detected in mostP. aeruginosawild-type
strains we have tested, except PAK and PA7. - The cultivation can also be performed in larger volumes, such
as 50 ml of TSB in 250 ml flasks. However, we found that upon
scaling up, the concentration of AHLs and HAQs tends to
decrease significantly with larger culture volumes for the same
final cell density. - HAQs such as PQS or HQNO accumulate up to concentra-
tions in excess of 15 mg/l in the culture medium under the
described culture conditions and are thus easily detected in
Table 1
Mass of various AHLs [11]AHLs Mass [M + H]+
C 4 -HSL 172
C 6 -HSL 200
3-oxo-C 6 -HSL 214
C 8 -HSL 228
3-oxo-C 8 -HSL 242
C 10 -HSL 256
3-OH-C 10 -HSL 272
3-oxo-C 10 -HSL 270
C 12 -HSL 284
3-oxo-C 12 -HSL 29856 Franc ̧ois Le ́pine et al.