Department of Pharmaceutical Sciences, University of Maryland Baltimore, School of Pharmacy, 20 N Pine Street, Baltimore, MD, 21201, USA.
Department of Microbiology, Immunology & Cell Biology, West Virginia University, School of Medicine, Morgantown, WV, 26506, USA.
Anal Bioanal Chem. 2020 Mar;412(7):1521-1534. doi: 10.1007/s00216-019-02374-0. Epub 2020 Jan 29.
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.
铜绿假单胞菌是一种机会性病原体，在感染过程中会产生许多外产物，帮助其逃避宿主免疫系统并从宿主环境中获取营养。这些产物包括一组分泌的 2-烷基-4(1H)-喹啉代谢物(AQ)，它们具有多种生物活性。在这里，我们描述了一种基于液相色谱-串联质谱(LC-MS/MS)的方法，用于定量复杂生物基质中多种 AQ 同系物的验证。该测定法经过选择性、灵敏度、线性、准确性、精密度、携带、稀释完整性、回收率、基质效应和各种稳定性(冻融、台式、长期储存和自动进样器/制备后)方面的验证。使用 6 种不同 AQ 同系物的真实标准品，我们报告了所有经过验证的 AQ 标准品在 25 至 1000 nmol/L 之间线性范围内的准确定量。该方法成功应用于定量铜绿假单胞菌细胞培养物和感染铜绿假单胞菌的小鼠肺部的 AQ 浓度。此外，我们还证实了细胞培养物中几种 AQ 同系物的不饱和形式的存在。
