Epoxy/dihydroxy-oxylipins are important biologically active compounds that are mainly formed from polyunsaturated fatty acids (PUFAs) in the reactions catalyzed by the cytochrome P450 (CYP 450) enzyme. The analysis of epoxy/dihydroxy-oxylipins would be helpful to gain insights into their landscape in living organisms and provide a reference for the biological studies of these compounds. In this work, we employed chemical labeling-assisted liquid chromatography (LC) coupled with high-resolution mass spectrometry (CL-LC-HRMS) to establish a highly sensitive and specific method for screening and annotating epoxy/dihydroxy-oxylipins in biological samples. The isotope reagents 2-dimethylaminoethylamine (DMED) and DMED-d 4 were employed to label epoxy/dihydroxy-oxylipins containing carboxyl groups so as to improve the analysis selectivity and MS detection sensitivity of epoxy/dihydroxy-oxylipins. Based on a pair of diagnostic ions with a mass-to-charge ratio (m/z) difference of 15.995 originating from the fragmentation of derivatives via high-energy collision dissociation (HCD), the potential epoxy/dihydroxy-oxylipins were rapidly screened from the complex matrix. Furthermore, the epoxy/dihydroxy groups could be readily localized by the diagnostic ion pairs, which enabled us to accurately annotate the epoxy/dihydroxy-oxylipins detected in biological samples. The applicability of our method was demonstrated by profiling epoxy/dihydroxy-oxylipins in human serum and heart samples from mice with high-fat diet (HFD). By the proposed method, a total of 32 and 62 potential epoxy/dihydroxy-oxylipins including 42 unreported ones were detected from human serum and the mice heart sample, respectively. Moreover, the relative quantitative results showed that most of the potential epoxy/dihydroxy-oxylipins, especially the oxidation products of linoleic acid (LA) or α-linolenic acid (ALA), were significantly decreased in the heart of mice with HFD. Our developed method is of high specificity and sensitivity and thus is a promising tool for the identification of novel epoxy/dihydroxy-oxylipins in biological samples.

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Gut microbiota–host co-metabolites serve as essential mediators of communication between the host and gut microbiota. They provide nutrient sources for host cells and regulate gut microenvironment, which are associated with a variety of diseases. Analysis of gut microbiota–host co-metabolites is of great significance to explore the host–gut microbiota interaction. In this study, we integrated chemical derivatization, liquid chromatography–mass spectrometry, and molecular networking (MN) to establish a novel CD-MN strategy for the analysis of carboxylated metabolites in gut microbial–host co-metabolism. Using this strategy, 261 carboxylated metabolites from mouse feces were detected, which grouped to various classes including fatty acids, bile acids, N-acyl amino acids, benzoheterocyclic acids, aromatic acids, and other unknown small-scale molecular clusters in MN. Based on the interpretation of the bile acid cluster, a novel type of phenylacetylated conjugates of host bile acids was identified, which were mediated by gut microbiota and exhibited a strong binding ability to Farnesoid X receptor and Takeda G protein-coupled receptor 5. Our proposed strategy offers a promising platform for uncovering carboxylated metabolites in gut microbial–host co-metabolism.

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