title: Development and validation of a UHPLC-MS/MS procedure for quantification of the Pseudomonas Quinolone Signal in bacterial culture after acetylation for characterization of new quorum sensing inhibitors.
abstract: The appearance of antibiotic resistance requires novel therapeutic strategies. One approach is to selectively attenuate bacterial pathogenicity by interfering with bacterial cell-to-cell communication known as quorum sensing. The PQS quorum sensing system of Pseudomonas aeruginosa employs as signal molecule the Pseudomonas Quinolone Signal (PQS; 2-heptyl-3-hydroxy-4-(1H)-quinolone), a key contributor to virulence and biofilm formation. Thus, interference with PQS production is considered as promising approach for the development of novel anti-infectives. Therefore, in this study, we developed and validated an ultra-high performance liquid chromatographic-tandem mass spectrometric approach for reliable quantification of PQS in P. aeruginosa cultures for activity determination of new quorum sensing inhibitors. The poor chromatographic properties of PQS reported by others could be overcome by fast microwave-assisted acetylation. The validation procedure including matrix effects, recovery, process efficiency, selectivity, carry-over, accuracy and precision, stability of the processed sample, and limit of quantification demonstrated that the method fulfilled all requirements of common validation guidelines. Its applicability was successfully proven in routine testing. In addition, two-point calibration was shown to be applicable for fast and reliable PQS quantification saving time and resources. In summary, the described method provides a powerful tool for the discovery of new quorum sensing inhibitors as potential anti-infectives and illustrated the usefulness of chemical derivatization, acetylation, in liquid chromatography-mass spectrometry analysis.
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany. Electronic address: christine.maurer@helmholtz-hzi.de.
authors.forename: Christine K
authors.initials: CK
authors.lastname: Maurer
authors.forename: Anke
authors.initials: A
authors.lastname: Steinbach
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Hartmann
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000592:standards / Q000639:trends; D013058:Mass Spectrometry / Q000592:standards / Q000639:trends; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D015363:Quinolones / Q000032:analysis*; D053038:Quorum Sensing / Q000187:drug effects; D053719:Tandem Mass Spectrometry / Q000592:standards* / Q000639:trends
chemical_list: C407944:2-heptyl-3-hydroxy-4-quinolone; D000900:Anti-Bacterial Agents; D015363:Quinolones
keywords: Acetylation; Pseudomonas Quinolone Signal; Quorum sensing inhibitors; Two-point calibration; UHPLC–MS/MS