Metabolic Profile of Indigo Naturalis in Rat Plasma, Bile, Urine, and Feces by UPLC–Q‐TOF MS Method

Indigo naturalis (IN, Qing‐Dai) is a well‐established traditional Chinese medicine (TCM) with extensive biological activities, particularly in the clinical management of ulcerative colitis (UC) and psoriasis. However, the comprehensive in vivo metabolic behavior of IN extract remains inadequately characterized. This research employed ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC–Q‐TOF MS) methodology to comprehensively examine the metabolic characteristics of IN in rat models. Biological samples, including plasma, bile, urine, and feces, were obtained from rats following single oral administration of IN extract. Sample preparation techniques involved protein precipitation, solid‐phase extraction, and ultrasonic extraction for processing plasma, bile, urine, and feces specimens, respectively. Through precise mass measurement and fragment ion analysis, we identified 24 prototype compounds and 37 metabolites and clarified the transformation relationships between prototypes and their metabolites. The biotransformation pathways of IN components in rats primarily involved N‐ or O‐glucuronidation, N‐ or O‐sulfation, hydroxylation, dehydroxylation, demethylation, demethoxylation, decarbonylation, and reduction reactions. Our findings demonstrate that N‐glucuronidation and N‐sulfation represent the predominant metabolic reactions. This investigation marks the first systematic report on metabolite identification and metabolic pathway speculation of major constituents following oral administration of IN extract, providing a scientific foundation for understanding its in vivo processes.