Isoprenoid quinone profiling in complex biological samples using a novel semi-quantitative HPLC-MS/MS method

ABSTRACT Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity are challenging for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 16-quinone standard mixture, we optimised separation within a 14-minute HPLC gradient and achieved femtomole-level sensitivity in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 distinct quinones, revealing stage-specific quinone profiles that reflect shifts in bacterial communities during wastewater treatment. This rapid and sensitive workflow provides a robust tool for accurate quinone profiling in complex samples, opening avenues for the discovery of novel quinones through untargeted approaches. By pushing the boundaries of quinone profiling, our method holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications. Highlights uHPLC-Orbitrap method for the semi-quantitative profiling of isoprenoid quinones Analysis of the widest range of isoprenoid quinones to date Femtomole-level sensitivity in just 14 minutes of chromatographic separation Detection of 57 quinones in complex wastewater sludge matrices Most comprehensive set of quinone standards including purified microbial quinones