Integrating the lactulose-mannitol test for intestinal permeability with untargeted metabolomics for drug monitoring through dual liquid chromatography-mass spectrometry

In this study, we developed a customized high-resolution mass spectrometry metabolomics workflow integrating the dual sugar test employing lactulose and mannitol as test probes for intestinal permeability assessment with untargeted screening of small molecules. Urine samples were collected from patients with major depression and healthy controls as part of a clinical study at the psychiatric department. Using a dual injection/dual chromatography setup, the test probes were quantified by hydrophilic interaction liquid chromatography (HILIC) in a targeted assay, while drugs and their metabolites were profiled in an untargeted manner by reversed-phase separation. Rigorous method development and validation allowed for selective separation of sugar isomers and consequently accurate quantification of lactulose and mannitol in urine. Internal standardization with compound specific stable isotope-labeled standards enabled excellent analytical figures of merit such as high recoveries, precision (< 5%), and working range (5 orders of magnitude). Within one analytical run, intestinal permeability was assessed together with drugs and their metabolites, allowing to screen for confounding drugs and patient compliance to the therapeutic scheme.