Simultaneous quantification of enterotoxins tilimycin and tilivalline in biological matrices using HPLC high resolution ESMS2 based on isotopically 15N-labeled internal standards

• Published in: Talanta (Oxford) 2021-01, Vol.222, p.121677-121677, Article 121677
• Main Authors: Glabonjat, Ronald A., Kitsera, Maksym, Unterhauser, Katrin, Lembacher-Fadum, Christian, Högenauer, Christoph, Raber, Georg, Breinbauer, Rolf, Zechner, Ellen L.
• Format: Article
• Language: English
• Subjects: Bacterial metabolites; High performance liquid chromatography; High-resolution mass spectrometry; Intestinal disease; MS/MS; Non-ribosomal peptides; Pyrrolobenzodiazepines; Stable isotope dilution
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2020.121677

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 standards 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. [Display omitted] •Selective and sensitive HPLC-HR-ESMS2 method developed for bacterial metabolites.•Stable 15N-labeled tilimycin (TM) and tilivalline (TV) standards synthesized.•Effective extraction from complex fecal samples optimized.•Precise quantification of microbiota-derived enterotoxins achieved.•Method validated by spiking and standard addition experiments.