Development and bioanalytical method validation of an LC-MS/MS assay for simultaneous quantitation of 2-alkyl-4(1H)-quinolones for application in bacterial cell culture and lung tissue.

Pseudomonas aeruginosa is an opportunistic pathogen that produces numerous exoproducts during infection that help it evade the host immune system and procure nutrients from the host environment. Among these products are a family of secreted 2-alkyl-4(1H)-quinolone metabolites (AQs), which exhibit a range of biological activities. Here, we describe the validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for quantifying multiple AQ congeners in complex biological matrices. The assay was validated for selectivity, sensitivity, linearity, accuracy, precision, carryover, dilution integrity, recovery, matrix effects, and various aspects of stability (freeze-thaw, bench-top, long-term storage, and autosampler/post-preparative). Using authentic standards for 6 distinct AQ congeners, we report accurate quantitation within a linear range between 25 and 1000 nmol/L for all of the validated AQ standards. This method was successfully applied to quantify AQ concentrations in P. aeruginosa cell culture and in the lungs of mice infected with P. aeruginosa. Further, we confirmed the presence of unsaturated forms of several AQ congeners in cell culture. Graphical abstract.