Quantitative LC–MS/MS Analysis of Endogenous <i>Pseudomonas aeruginosa</i> Isomeric Metabolites Biliverdin IX Alpha, Beta, and Delta in Cell Culture Supernatant, Cell Pellet, and Lung Tissue

<i>Pseudomonas aeruginosa</i> (<i>Pa</i>) utilizes heme as an iron source from the host during infection. Biliverdin beta and delta (BVIXβ and BVIXδ) are generated by HemO, specific to <i>Pa</i>, while biliverdin alpha is generated from the bacterial BphO system and by mammalian heme oxygenases. Here, we have developed and characterized a quantitative LC-MS/MS assay for the separation of three endogenous isomers, BVIXα, BVIXβ, and BVIXδ. The assay was validated for accuracy, precision, linearity, extraction recovery, solution stability, freeze-thaw stability, benchtop stability, postextraction stability, and nonspecific oxidation of BVIX. The addition of an antioxidant, butylated hydroxytoluene, during sample preparation is needed in order to prevent coupled oxidation from inflating quantitative values of BVIX. The assay development included optimization of a liquid-liquid extraction for bacterial culture supernatants and sample preparation procedures for cell pellets and tissue homogenate to reduce sample demand and automate the extraction procedure in a 96-well format, to enhance extraction throughput. This method was applied to analyze isomer distribution in <i>Pa</i> supernatant, bacterial pellet, and infected lung tissue from <i>Pa</i>-challenged mice. This method can be used in the future for low-volume culture samples, as well as tissue samples, to understand the mechanisms of virulence and inform future drug development.