Quantification of Gut Microbiota Dysbiosis-Related Organic Acids in Human Urine Using LC-MS/MS

• Published in: Molecules (Basel, Switzerland) Switzerland), 2022-08, Vol.27 (17), p.5363
• Main Authors: Lee, Yu-Tsung, Huang, Sui-Qing, Lin, Ching-Hao, Pao, Li-Heng, Chiu, Chun-Hui
• Format: Article
• Language: English
• Subjects: Acetic acid; Acetonitrile; Amino acids; Bacteria; Benzoic acid; Capillary electrophoresis; Chromatography; Chromatography, Liquid - methods; Creatinine; Deionization; Diabetes; Dysbacteriosis; Dysbiosis; Gastrointestinal Microbiome; gut microbiota; Hippuric acid; human urine; Human wastes; Humans; Indoleacetic acid; Intestinal microflora; Ions; LC-MS/MS; Lipid metabolism; Lipids; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Metabolic rate; Metabolism; Metabolites; Microbiota; Microbiota (Symbiotic organisms); NMR; Nuclear magnetic resonance; organic acid; Organic acids; Organic Chemicals; p-Hydroxybenzoic acid; Phenylacetic acid; Phenylketonuria; Physiological aspects; Polyphenols; Propionic acid; Scientific imaging; Selectivity; Sugar; Tandem Mass Spectrometry - methods; Type 2 diabetes; Urine; Vitamins; Water
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules27175363

Urine organic acid contains water-soluble metabolites and/or metabolites—derived from sugars, amino acids, lipids, vitamins, and drugs—which can reveal a human’s physiological condition. These urine organic acids—hippuric acid, benzoic acid, phenylacetic acid, phenylpropionic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid, 3-hydroxyphenylpropionic acid, 3,4-dihydroxyphenyl propionic acid, and 3-indoleacetic acid—were the eligible candidates for the dysbiosis of gut microbiota. The aim of this proposal was to develop and to validate a liquid chromatography−tandem mass spectrometry (LC-MS/MS) bioanalysis method for the nine organic acids in human urine. Stable-labeled isotope standard (creatinine-d3) and acetonitrile were added to the urine sample. The supernatant was diluted with deionized water and injected into LC-MS/MS. This method was validated with high selectivity for the urine sample, a low limit of quantification (10−40 ng/mL), good linearity (r > 0.995), high accuracy (85.8−109.7%), and high precision (1.4−13.3%). This method simultaneously analyzed creatinine in urine, which calibrates metabolic rate between different individuals. Validation has been completed for this method; as such, it could possibly be applied to the study of gut microbiota clinically.