Colorimetric Detection of Indoxyl Sulfate With Sodium Hypochlorite Pentahydrate Analyzed by Spectrophotometry and ESI‐MS

Indoxyl sulfate (indican) is a tryptophan‐derived metabolite generated by intestinal bacteria and subsequently excreted in urine. Urinary indican is considered an indicator of gut microbial imbalance and small‐intestinal dysfunction because its production closely reflects the extent of microbial activity. In this study, we improved the classical indican color reaction—originally based on the Obermayer and Jaffé methods—by developing a simple colorimetric method using sodium hypochlorite pentahydrate (NaOCl·5H ₂ O) as a novel oxidizing agent that is characterized by its high stability, strong reactivity, and ease of handling. By using this new oxidizing agent, the overall detection procedure becomes simpler to perform than the classical methods. The addition of hydrochloric acid followed by NaOCl·5H ₂ O produced a blue pigment with an absorbance peak at approximately 600 nm, which was identified as indigo by electrospray ionization mass spectrometry. The method exhibited a visual detection limit of 6 mg/dL, comparable to that of the Obermayer and Jaffé methods. In the proposed method, a linear response between 6 and 12 mg/dL ( R ²  = 0.9369) was obtained, and the recovery in spiked human urine samples was 69.3%; however, these results indicate that further improvement is required for reliable quantification of elevated indican levels. Moreover, the chromogenic reaction was susceptible to interference from ascorbic acid although partial suppression was achieved using ascorbate oxidase. Overall, this work provides a useful analytical approach for detecting elevated urinary indican levels in clinical and research settings, serving as an alternative to classical methods that rely on ferric chloride and calcium hypochlorite.