Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.

Applied Analytical Chemistry University of Duisburg Essen Universitaetsstrasse 5 45141 Essen Germany

Central European Institute of Technology and Faculty of Science Masaryk University Kamenice 5 62500 Brno Czechia

Department of Bioanalytical Instrumentation Institute of Analytical Chemistry of the Czech Academy of Sciences Veveří 967 97 60200 Brno Czechia

Department of Environmental Analytical Chemistry Institute of Analytical Chemistry of the Czech Academy of Sciences Veveří 967 97 60200 Brno Czechia

Internal Gastroenterology Department University Hospital Brno and Faculty of Medicine Masaryk University Jihlavská 20 625 00 Brno Czechia

Teaching and Research Center for Separation University of Duisburg Essen Universitaetsstrasse 5 45141 Essen Germany

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