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
Urine analysis is an important indicator that is used to assess human health as it
contains many metabolic breakdown products from a wide range of foods, drinks, drugs,
environmental contaminants, endogenous waste metabolites, and bacterial byproducts [1],
which disclose many physiological and health indicators. Metabolites produced by the
intestinal flora after metabolizing food residues, such as organic acids, are absorbed through
the intestinal tract, metabolized, and excreted in urine [2]. Previous studies have found that
the content of organic acids detected in urine is related to diet [3] and chronic conditions,
such as allergies [4], diabetes [5], and obesity [6].
Benzoic acid (BA) is a product of phenylalanine [7] and polyphenol metabolism [8]
from intestinal bacteria. In addition, BA is also used as an additive for preservative
applications in food [9]. After absorption by the intestine, BA is transferred to the liver
and detoxified by combination with glycine to form hippuric acid (HA), which is then
excreted from urine [8]. Elevated levels of benzoic acid in urine are associated with
glycine deficiency or abnormal liver function [10]. Some studies have shown that exposure
to toluene in occupational environments leads to an increase in urinary HA [11]. In
patients with type 2 diabetes, decreased levels of HA are associated with obesity and
hypertension [5,6]. Phenylacetic acid (PAA) and phenylpropionic acid (PPA) are products
of phenylalanine metabolism caused by intestinal
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