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
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
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DOI：
10.1007/s00216-019-02374-0
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发表时间：
2020-03-01
影响因子：
4.3
通讯作者：
Kane, Maureen A.
中科院分区：
文献类型：
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作者：
Brewer, Luke K.;Jones, Jace W.;Kane, Maureen A.
铜绿假单胞菌是一种机会性病原体，在感染过程中产生大量的外产物，帮助其逃避宿主免疫系统并从宿主环境中获取营养。这些产品中有一系列分泌型 2-烷基-4(1H)-喹诺酮代谢物 (AQ)，具有一系列生物活性。在这里，我们描述了基于液相色谱-串联质谱 (LC-MS/MS) 的方法的验证，用于定量复杂生物基质中的多种 AQ 同系物。该测定的选择性、灵敏度、线性、准确性、精密度、残留、稀释完整性、回收率、基质效应和稳定性的各个方面（冻融、台式、长期储存和自动进样器/后制备）均经过验证。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 之间的线性范围内的准确定量。该方法已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中的 AQ 浓度。此外，我们证实了细胞培养物中存在几种 AQ 同系物的不饱和形式。图形摘要
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