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

TALANTA

卷:222

Article

Glabonjat, Ronald A.¹  Kitsera, Maksym²  Unterhauser, Katrin²  Lembacher-Fadum, Christian³  Hoegenauer, Christoph^4,5  Raber, Georg¹  Breinbauer, Rolf^3,4  Zechner, Ellen L.^2,4,6 

[1] Karl Franzens Univ Graz, NAWI Graz, Inst Chem, A-8010 Graz, Austria

[2] Karl Franzens Univ Graz, Inst Mol Biosci, Humboldtstr 50-1, A-8010 Graz, Austria

[3] Graz Univ Technol, Inst Organ Chem, A-8010 Graz, Austria

[4] BioTechMed, Graz, Austria

[5] Med Univ Graz, Dept Internal Med, Div Gastroenterol & Hepatol, A-8036 Graz, Austria

[6] Karl Franzens Univ Graz, Field Excellence BioHlth, Graz, Austria

关键词: Bacterial metabolites;    Pyrrolobenzodiazepines;    Non-ribosomal peptides;    High performance liquid chromatography;    High-resolution mass spectrometry;    MS/MS;    Stable isotope dilution;    Intestinal disease;   

DOI  :  10.1016/j.talanta.2020.121677

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

Free