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

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-d(3)) 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.