Since　highly　sensitive　on-line　coupling　of　UPLC　with　FTICR-MS　is　technically　infeasible　due　to　their　different　scan　rates,　at-line　coupling　of　these　techniques　was　developed　for　rapid　analysis.　To　enable　cutting　of　one　peak　of　the　chromatogram　into　one　fraction,　several　conditions　and　relationships　were　investigated,　e.g.　the　optimum　volume　of　the　inserted　delay　loop,　the　relationship　between　retention　time,　loop　outlet　drop　speed,　individual　drop　volume　versus　mobile　phase　composition　under　constant　speed,　and　linear　solvent　strength　gradient　elution　modes.　Good　and　reproducible　results　were　achieved　applying　UPLC　as　an　efficient　separation　and　fast　fractionation　tool　before　the　FTICR-MS　measurements.　A　chip-based　nanoelectrospray　ionization　system　was　employed　which　was　perfectly　suited　to　handling　the　small-volume　fractions　and　was　thus　chosen　for　the　at-line　coupling.　The　method　was　initially　applied　to　spiked　extracts　of　cell-free　bacterial　culture　supernatants　in　which　bacterial　signalling　compounds,　namely　N-acyl　homoserine　lactones　(AHL),　were　detected.　Good　reproducibility　and　high　recovery　was　observed.　Afterwards,　a　culture　supernatant　of　Erwinia　sp.　JX3.2,　a　putative　AHL　producer,　was　investigated　and　N-hexanoyl-homoserine　lactone　was　determined　as　a　possible　signalling　molecule.　More　reliable　assignments　were　achieved　by　use　of　at-line　coupling　of　UPLC　and　FTICR-MS　compared　with　off-line　measurements.　©　2007　Springer-Verlag.