A fast and accurate colorimetric assay for quantifying hippuric acid in human urine

• Published in: Analytical biochemistry 2023-11, Vol.680, p.115303-115303, Article 115303
• Main Authors: Bhattacharyya, Dipanjan, LeVatte, Marcia A., Wishart, David S.
• Format: Article
• Language: English
• Subjects: Colorectal cancer; Colorimetric assay; Hippurate; p-toluenesulfonyl chloride; Urine
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2023.115303

Hippuric acid is an abundant metabolite in human urine. Urinary hippuric acid levels change with toxic exposure to aromatic compounds, consumption of fruits and vegetables, cancers, chronic kidney disease, schizophrenia and Crohn's disease. While urinary hippuric acid can be detected and quantified via mass spectrometry or nuclear magnetic resonance spectroscopy, a colorimetric assay would be preferable for a low-cost, point-of care clinical assay. Two colorimetric methods, that use p-dimethylaminobenzaldehyde (DMAB) or benzenesulfonyl chloride (PhSO2Cl), respectively, have been previously developed to detect hippuric acid but these assays have many limitations. We replaced PhSO2Cl with p-toluenesulfonyl chloride (p-TsCl), to create a simpler, faster and more accurate method that works with human urine. This modified colorimetric assay detects from 60 μM to 1000 μM hippuric acid in urine in 2 min. We also corrected for the effects of interfering compounds present in urine such that the assay works across many urine backgrounds. We validated this improved assay on multiple hippurate-spiked urine samples, observing an excellent correlation (R2 > 0.94) between observed and known hippurate concentrations. These data suggest that this colorimetric assay is accurate and should greatly facilitate the measurement of hippuric acid in urine to detect a variety of human conditions. [Display omitted] •Modified assay for hippurate, an exposure, dietary and disease urinary biomarker.•Our assay replaced benzenesulfonyl chloride with solid p-toluenesulfonyl chloride.•Fast, simple, more stable assay works in all urine backgrounds using dilution.•Detects 60–1000 μM urinary hippurate in 2 min.•Correlation coefficient >0.94 between measured and expected hippurate levels.