Quantification of Gut Microbiota Dysbiosis-Related Organic

• 09 September 2024

Article
Quantification of Gut Microbiota Dysbiosis-Related Organic
Acids in Human Urine Using LC-MS/MS
Yu-Tsung Lee 1 , Sui-Qing Huang 1 , Ching-Hao Lin 1, Li-Heng Pao 1,2,* and Chun-Hui Chiu 1,3,*
1 Graduate Institute of Health Industry and Technology, Research Center for Food and Cosmetic Safety,
College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan
2 Department of Gastroenterology and Hepatology, Linkuo Chang Gung Memorial Hospital,
Taoyuan 33305, Taiwan
3 Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung 20401, Taiwan
* Correspondence: paolhaa@gmail.com (L.-H.P.); chchiu@mail.cgust.edu.tw (C.-H.C.);
Tel.: +886-3-211-8999 (L.-H.P. & C.-H.C.); Fax: +886-3-211-8866 (L.-H.P. & C.-H.C.)
Abstract: 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.
Keywords: gut microbiota; LC-MS/MS; organic acid; human urine
1. Introduction

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