This study investigated methods for sampling bile acids in saliva, a potential non-invasive diagnostic biofluid. Bile acids have been implicated in causing damage and permanent changes to the esophageal mucosa and increasing the risk of developing Barrett's esophagus, a condition that can potentially progress to esophageal cancer. Three saliva collection methods were compared: spitting, Salivette® swabs, and Salivette Cortisol® swabs. Spitting emerged as the superior method with the highest recoveries and the least interference, likely due to Salivette swabs retaining bile acids or introducing unknown interferences. All saliva samples were analyzed by UHPLC-MS/MS using the Zorbax RRHD Eclipse Plus C18 column (3 × 50 mm, 1.8 µm) in gradient elution of 0.1 % formic acid in water and methanol. Saliva sample stability was assessed over 14 days, reflecting typical storage times. The levels of detected bile acids were stable for the measured period (RSD ≤ 22 %) and no degradation was observed. Bile acid levels in saliva fluctuated throughout the day, with the greatest changes observed for glycine-conjugated bile acids after meals. To minimize sampling variability, saliva collection by spitting after overnight fasting is recommended for future studies. Our findings are applicable for standardized bile acid sampling and are currently applied in a large clinical study evaluating bile acids as potential susceptibility markers for Barrett's esophagus diagnostics.

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

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

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

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