TY - JOUR
T1 - Simultaneous quantification of enterotoxins tilimycin and tilivalline in biological matrices using HPLC high resolution ESMS2 based on isotopically 15N-labeled internal standards
AU - Glabonjat, Ronald A.
AU - Kitsera, Maksym
AU - Unterhauser, Katrin
AU - Lembacher-Fadum, Christian
AU - Högenauer, Christoph
AU - Raber, Georg
AU - Breinbauer, Rolf
AU - Zechner, Ellen L.
N1 - Publisher Copyright:
© 2020 The Authors
PY - 2021/1/15
Y1 - 2021/1/15
N2 - 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.
AB - 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.
KW - Bacterial metabolites
KW - High performance liquid chromatography
KW - High-resolution mass spectrometry
KW - Intestinal disease
KW - MS/MS
KW - Non-ribosomal peptides
KW - Pyrrolobenzodiazepines
KW - Stable isotope dilution
UR - http://www.scopus.com/inward/record.url?scp=85091204631&partnerID=8YFLogxK
U2 - 10.1016/j.talanta.2020.121677
DO - 10.1016/j.talanta.2020.121677
M3 - Article
AN - SCOPUS:85091204631
SN - 0039-9140
VL - 222
JO - Talanta
JF - Talanta
M1 - 121677
ER -