期刊
TALANTA
卷 222, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.talanta.2020.121677
关键词
Bacterial metabolites; Pyrrolobenzodiazepines; Non-ribosomal peptides; High performance liquid chromatography; High-resolution mass spectrometry; MS/MS; Stable isotope dilution; Intestinal disease
资金
- Austrian Science Fund FWF [W901]
- Doctoral Academy Graz
- NAWI Graz
A fast and reliable method for quantifying bacterial enterotoxins tilimycin and tilivalline in complex biological matrices was developed in this study. By optimizing sample preparation and applying high resolution mass spectrometry, the method overcame the disadvantages and achieved high sensitivity and low matrix interference.
Non-ribosomal peptides are one class of bacterial metabolites formed by gut microbiota. Intestinal resident Klebsiella oxytoca produces two pyrrolobenzodiazepines, tilivalline and tilimycin, via the same nonribosomal biosynthesis platform. These molecules cause human disease by genotoxic and tubulin inhibitory activities resulting in apoptosis of the intestinal epithelium, loss of barrier integrity and ultimately colitis. Here we report a fast, reliable, HPLC-HR-ESMS2 method for quantifying simultaneously the bacterial enterotoxins tilimycin and tilivalline in complex biological matrices. We synthesized and applied stable isotopically labeled internal stan-dards for precise quantification of the metabolites. Sample preparation was optimized using clinical and laboratory specimens including serum, colonic fluid and stool. The developed method overcame the disadvantage of low selectivity by applying high resolution mass spectrometry in MS2 mode. High sensitivity and low interference from matrices were achieved and validated. We show that the approach is suitable for detection and quantification of the enterotoxic metabolites produced in vivo, in infected human or animal hosts, and in bacterial culture in vitro.
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