Funding informationDeutsche Forschungsgemeinschaft; We acknowledge support by the German Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO 2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and singlequadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C-and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R 2 = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating
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