Development and Application of a Selective Analytical Method for Indole Metabolites in Urine: Dietary Exposure Biomarkers for Broccoli Consumption

The identification of dietary exposure biomarkers is crucial for advancing our understanding of the health benefits of specific foods. Broccoli, a vegetable with well-known anticancer properties, contains active ingredients, such as isothiocyanates with indole side chains. Hence, indole metabolites related to broccoli consumption have the potential to serve as biomarkers of dietary exposure. In this work, we developed a new analytical method for indole metabolites in urine using a poly(deep eutectic solvents)-molecularly imprinted polymer/vinyl-functionalized graphene oxide (PDESs-MIP/VGO) in miniaturized centrifugal pipet-tip solid-phase extraction (CPT-SPE) coupled with liquid chromatography. This method integrates the strengths of PDESs-MIP/VGO, including rich adsorption interactions, high adsorption capacity, and excellent selectivity, with the simplicity and cost-effectiveness of CPT-SPE. The proposed method demonstrated low limits of quantification (1.2-2.5 ng mL^-1), high accuracy (91.7-104.8%), and good precision (relative standard deviation ≤4.4%). By applying this method to analyze indole metabolites in urine, our results suggested that indole-3-carbinol and indole-3-acetonitrile have the potential to emerge as reliable dietary exposure biomarkers for broccoli intake. Furthermore, highly selective analytical methods based on molecular imprinting technology are advantageous for precise screening and analysis of dietary exposure biomarkers associated with food consumption.