MS method for quantitation of gut microbial short-chain fatty acids

Journal Pre-proof Chemical derivatization-based LC-MS/MS method for quantitation of gut microbial short-chain fatty acids Won-Suk Song, Han-Gyu Park, Seong-Min Kim, Sung-Hyun Jo, Byung-Gee Kim, Ashleigh B. Theberge, Yun-Gon Kim

PII:

S1226-086X(19)30639-2

DOI:

https://doi.org/10.1016/j.jiec.2019.12.001

Reference:

JIEC 4885

To appear in:

Journal of Industrial and Engineering Chemistry

Received Date:

13 July 2019

Revised Date:

29 November 2019

Accepted Date:

1 December 2019

Please cite this article as: Song W-Suk, Park H-Gyu, Kim S-Min, Jo S-Hyun, Kim B-Gee, Theberge AB, Kim Y-Gon, Chemical derivatization-based LC-MS/MS method for quantitation of gut microbial short-chain fatty acids, Journal of Industrial and Engineering Chemistry (2019), doi: https://doi.org/10.1016/j.jiec.2019.12.001

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Chemical derivatization-based LC-MS/MS method for quantitation of gut microbial short-chain fatty acids

Won-Suk Song†, Han-Gyu Park‡, Seong-Min Kim‡, Sung-Hyun Jo‡, Byung-Gee Kim†, Ashleigh B. Theberge§,∥ and Yun-Gon Kim‡,*

of Chemical and Biological Engineering, Seoul National University, Seoul 08826,

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†School

Korea

§Department

of Chemical Engineering, Soongsil University, Seoul 06978, Korea

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‡Department

of Chemistry, University of Washington, Box 351700, Seattle, WA 98195,

∥Department

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United States

of Urology, University of Washington School of Medicine, Seattle, WA 98105,

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United States

*

Corresponding author contact information:

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Yun-Gon Kim, Ph.D., Professor

Department of Chemical Engineering

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Soongsil University

369 Sangdo-Ro, Seoul 06978, Korea E-mail: [email protected] Phone: +82-2-828-7099

Graphical abstract

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Abstract Short chain fatty acids (SCFAs) are end products of fermentation by anaerobic gut microbiota. They can be used as beneficial metabolites to regulate the host’s physiological

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processes. Despite their importance, SCFAs are difficult to analyze with mass spectrometric technologies due to their poor ionization efficiency and susceptibility to

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water loss during ionization of low molecular weight organic acids. Here, we developed a

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sensitive and reliable method to quantify SCFAs by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. SCFAs were chemically derivatized by Girard’s reagent T (GT), providing a permanent cationic charge.

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This technique demonstrated an excellent quantitative capacity, showing good linearity (R2 >0.99) and limit of quantification (femtomole levels) for five SCFAs (i.e., acetate,

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propionate, butyrate, valerate, and caproate). Next, we applied this derivatization method to quantitate SCFAs from a small volume of total extracellular metabolites produced by

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Eubacterium rectale (E. rectale), one of main butyrate-producing gut bacteria. GT-labeled SCFAs were quantitated well in small volumes of culture medium (5, 10, 15, and 20 μL), with the amount of SCFA measured being proportional to the volume of culture medium, as expected. We also investigated plant-derived polysaccharides as prebiotics that could enhance the production of butyrate by E. rectale. Finally, the production of butyrate was 2

successfully monitored in a co-culture system for E. rectale and Bifidobacterium longum (B. longum) by analyzing GT-labeled butyrate. Taken together, our results suggest that this highly sensitive method would be useful for quantifying SCFAs extracted from stool in an aqueous solution to monitor gut health.

Key words: Short-chain fatty acid, Chemical derivatization, LC-MS/MS, Multiple reaction

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mornitoring, Gut microbiota

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1. Introduction

Short-chain fatty acids (SCFAs) as end products are produced by fermentation of

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dietary fibers, which cannot be directly digested by humans, via anaerobic gut microbiota

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[1]. Intestinal concentrations of SCFAs derived from gut microbiota range from 50 to 150 mM depending on intestinal conditions [2]. More than 95% of these SCFAs are acetate, propionate, and butyrate [3]. They are not only used as energy sources for colonocytes in

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humans, but also have anti-inflammatory effects through histone deacetylase inhibition with positive effects on host health such as weight loss and glycemic control [4]. Therefore,

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research on the mechanism of SCFA biosynthesis by gut microbiota has been actively conducted, with a particular focus on increasing SCFA production in the gut microbiome

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[5].

Despite the importance of SCFAs in gut microbiome, it is difficult to quantitatively

analyze them using mass spectrometry in experiments to confirm the production of SCFAs because they have their poor ionization efficiency and susceptibility to water loss during ionization of low molecular weight organic acids [6]. SCFAs have been analyzed using gas 3

chromatography-mass spectrometry (GC-MS), which is excellent for analyzing highly volatile molecules. However, the detection limit is approximately 3 to 20 pmol, indicating limited detection sensitivity [7]. To increase detection sensitivity, SCFA have been derivatized with pentafluorobenzyl bromide (PFBBr) prior to analysis by GC-MS. However, the quantification limit is 0.1~1 pmol, which is still insufficient for many biological studies [8]. High-throughput SCFA quantitation systems using several

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microliters (< 10 μL) of cell culture media or human samples require a higher sensitive method that can detect them below a few femtomoles, making these conventional methods

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unsuitable. Therefore, in this study, we developed a novel quantitative method with superior sensitivity to analyze even a small amount (below femtomole) of SCFAs in a small

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volume (down to 5 µL).

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Recently, to increase the sensitivity of SCFA detection, several chemical derivatization methods using LC-MS/MS-based MRM analysis have been reported. Han et al. [9] and Chen et al. [10] have analyzed SCFAs derivatized by each 3 and 2-nitrophenylhydrazine

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(3-NPH and 2-NPH) through EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling reaction in negative ion mode of LC-MS/MS based MRM analysis. Chan et al.

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[11] have analyzed SCFA derivatized with aniline which is relatively inexpensive using LC-MS/MS based MRM analysis and proposed a derivatization method that can more

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easily quantify SCFA by LC-MS/MS. However, these methods showed quantitative limits of detection of several to several tens of femtomoles. Therefore, quantitative limits of detection below a few femtomoles are required to quantify SCFA in small amounts of cell culture media or human samples.

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In this study, we developed a SCFA derivatization method for LC-MS/MS based MRM analysis. It can be applied to high-throughput screening (HTS) system by analyzing them more sensitively. In ESI (electrospray ionization)-MS analysis, ionization in positive ion mode is generally more efficient than negative ion mode, resulting in higher sensitivity [12]. Therefore, we devised a method for quantitatively analyzing SCFAs by chemical derivatization with Girard's reagent T (GT), a molecule with inherently permanent cations,

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in LC-MS/MS. Derivatization with GT is expected to lead to better sensitive ionization in mass spectrometry compared to other derivatives since there is no need to add proton or

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sodium ion for ionization [13]. First, we quantitatively analyzed five representative gut microbial-derived SCFAs (acetate, propionate, butyrate, valerate, and caproate) derivatized

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with GT and optimized MRM analysis and LC separation conditions in LC-MS/MS. To

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demonstrate the quantification of SCFAs using GT derivatization method, we analyzed concentrations of SCFA quantified according to the extraction volume (5-20 μL) of Eubacterium rectale cell culture medium, one of main butyrate producing bacteria in the

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intestine. Next, a variety of plant-derived polysaccharides (fructooligosaccharide, inulin, pullulan, and xylan) sold commercially as food additive and prebiotics were used as sole

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carbohydrate sources in E. rectale culture. Quantitative analysis of butyrate productivity identified the best prebiotic polysaccharides that could increase E. rectale growth and

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butyrate production. Finally, we monitored the increase of butyrate-producing activity in bacterial mixed culture (i.e., cross-feeding effect) by co-culturing E. rectale (that could not directly digest inulin and produce SCFA effectively) with Bifidobacterium longum (B. longum) [14]. Results of this study revealed that GT derivatization based on LC-MS/MS was a sensitive and reliable SCFA analysis method. 5

2. Experimental 2.1. Chemicals and materials Acetate, propionate, butyrate, valerate, caproate, Girard’s reagent T, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), and fructooligosaccharides (FOS) from chicory were purchased from Sigma Aldrich (MO, USA). Butyrate-d7 as internal standard was

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obtained from CDN isotopes (QC, Canada). 3-Nitrophenylhydrazine was purchased from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). HPLC-grade water and acetonitrile

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were products of Fisher Scientific (NH, USA). Xylan from corncob and inulin from dhliatubers were purchased from Avention (Incheon, South Korea). Pullulan was obtained

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from Megazyme (IL, USA).

2.2. Girard’s reagent T (GT) derivatization

To analyze MS/MS fragmentation conditions of six GT-labeled SCFAs (acetate,

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propionate, butyrate, valerate, caproate and butyrate-d7) in LC-MS/MS and separate LC, GT derivatization method was used as follows. Each SCFA standard compound was

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dissolved in 50% ACN to 100 μM. Then 40 μL of standard compounds was mixed with 20 μL of 100 mM GT and 20 μL of 100 mM EDC followed by incubation at 40 °C for 1 hour.

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After incubation, it was diluted 20-fold with 50% ACN and analyzed by LC-MS/MS.

2.3. LC-MS/MS analysis LC-MS/MS analysis was performed using an integrated system composed of

Agilent 1260 Infinity Binary LC (CA, USA) and Agilent 6420 Triple Quadrupole LC/MS 6

(CA, USA). Then 5 μL of reaction mixture of GT-labeled SCFAs was injected into Agilent Zorbax HILIC plus column (4.6 x 100 mm, 3.5 μm). Solvent A comprised water containing 20 mM ammonium acetate and 20 mM acetic acid while solvent B was 100% acetonitrile. GT-labeled SCFAs were separated on the analytical column at a flow rate of 500 μL/min. The LC gradient method was set as follows: t = 0 min, 70% B; 2 min, 70% B; 12 min, 60% B; 12.5 min, 70% B; and 19 min, 70% B. The mass spectrometer was operated in positive

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ion mode. An electrospray ionization spray voltage was used at 4 kV and the capillary

2.4. Calibration curve and sensitivity test

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temperature was 300 °C.

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To analyze the quantitative curve of GT-labeled SCFAs, the SCFA standard

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mixture was dissolved in 50% ACN (100 nmol per 1 mL), serially diluted, derivatized with GT, and injected (5 μL) into LC-MS/MS (n = 3). The number of molecules of GT-labeled SCFA injected into LC-MS/MS was as follows: 450 pmol, 269 pmol, 90 pmol, 27 pmol, 9

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pmol, 2.7 pmol, …, 0.001 fmol. GT derivatization reaction conditions were prepared as follows. First, 180 μL of the prepared SCFA standard mixture was mixed with 20 μL of 2

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mM butyrate-d7. Then 40 μL of standard mixture, 20 μL of 100 mM GT, and 20 μL of 100 mM EDC were mixed and incubated at 40 ° C for 1 hour. After incubation, it was diluted

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20-fold with 50% ACN and analyzed by LC-MS/MS. Butyrate-d7 was used as an internal standard.

2.5. Culture of Eubacterium rectale

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Eubacterium rectale designated as KCTC-5835 used in this research was obtained from Korean Collection for Type Cultures (KCTC). E. rectale was cultured in mYCFA medium supplemented with 0.5% w/v glucose [15]. The composition of mYCFA medium was as follows: casitone 10 g, yeast extract 2.5 g, NaCl 0.9 g, MgSO4.7H2O 0.09 g, CaCl2 0.09 g, K2HPO4 0.45 g, KH2PO4 0.45 g, resazurin 1 mg, biotin 10 μg, cobalamin 10 μg, paminobenzoic acid 30 μg, folic acid 50 μg, and pyridoxamine 150 μg in 1L of water. After

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mixing well, this mixture was autoclaved. Then a mixture of NaHCO3 4 g, L-cysteine.HCl 1 g, hemin 10 mg, thiamine 10 μg, riboflavin 40 μg, acetic acid 1.8 g, sodium propionate

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0.87 g, valeric acid 0.1 g, and caproic acid 0.1 g in 1L of water was prepared and filter sterilized before adding to the basal medium. Before inoculation, oxygen in medium was

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replaced with nitrogen in a 37 °C anaerobic chamber (MI, USA, Coy Laboratory Products)

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containing a mixture of 90% N2, 5% CO2, and 5% H2 overnight. E. rectale was also cultured in an anaerobic chamber containing a mixture of 90% N2, 5% CO2, and 5% H2 at

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37 °C.

2.6. Quantitation test by volume ratio of cell culture medium

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To confirm the quantification of GT labeled SCFAs according to cell culture medium extraction volume, E. rectale was inoculated into 5 mL of mYCFA medium

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containing 0.5% w/v glucose and cultured until the optical density was approximately 1. After 5, 10, 15, or 20 μL of cell culture medium was extracted and diluted with distilled water to a total volume of 100 μL (n = 3), it was centrifuged in a table-top centrifuge at room temperature for 1 minute with maxspeed. Then 50 μL of the supernatant was mixed with 130 μL of 20% ACN and 20 μL of 2 mM butyrate-d7. The extracellular metabolite 8

mixture was centrifuged at 13,200 rpm for 10 minutes at 4 °C in a 3k-Da molecular weight cut-off (MWCO) membrane filter. After 40 μL of flow-through was mixed with 20 μL of 100 mM GT and 20 μL of 100 mM EDC, the mixture was incubated at 40 °C for 1 hour. After incubation, it was diluted 20-fold with 50% ACN and analyzed by LC-MS/MS.

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2.7. Growth and butyrate production analysis of E. rectale according to supplement of various plant-derived oligosaccharides

Four types of mYCFA medium containing 0.5 g/L of FOS, inulin, pullulan, and

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xylan were prepared to analyze the growth rate and butyrate production of E. rectale for various polysaccharide supplementation. After E. rectale was cultured in mYCFA medium

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containing 0.5% w/v glucose as a carbohydrate source to stationary phase, 30 mL of each

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polysaccharide-containing mYCFA medium was inoculated with E. rectale to have OD of 0.05 (n = 3). ODs at 0, 4, 8, and 12h after inoculation were then measured and 5 μL of each

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cell culture medium was derivatized with GT. This was analyzed by LC-MS/MS.

2.8. Analysis of butyrate production of E. rectale co-culture with B. longum

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Bifidobacterium longum designated as KCTC-3128 used in this research was obtained from Korean Collection for Type Cultures (KCTC). Each E. rectale and B.

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longum was incubated overnight in 0.5% w/v glucose supplemented mYCFA medium for preculture (37 °C in anaerobic chamber containing a mixture of 90% N2, 5% CO2 and 5% H2). To analyze butyrate production of E. rectale by co-culture with B. longum, preculture of E. rectale was inoculated into 27 mL of mYCFA medium supplemented with 0.5% w/v inulin such that the OD of E. rectale was 0.05. In addition, preculture of B. longum was 9

inoculated at the same cell number as E. rectale to analyze difference in butyrate production of E. rectale according to co-culture with B. longum (n = 3). As a control, mYCFA medium supplemented with 0.5% w/v inulin was added in the same volume as B. longum culture (n = 3). After incubation for 24 h in an anaerobic chamber, cultures (5 μL each) at 0 h and 24 h were extracted and analyzed by LC-MS/MS after GT chemical

3. Results and Discussion

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3.1. LC-MS/MS analysis of GT-labeled SCFAs

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derivatization.

First, GT derivatization and MS/MS fragmentation condition for LC-MS/MS

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analysis were optimized for five representative SCFAs (acetate, propionate, butyrate,

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valerate, and caproate) produced by gut microbiota (Figs. 1 and 2). The retention time and MS/MS fragmentation conditions in LC-MS/MS were analyzed by GT derivatization of all six SCFAs including butyrate-d7 as an internal standard for absolute quantification (Figs.

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2A and 2B). As a result, m/z 100 was detected as product ion mass in MS/MS fragmentation in all GT-labeled SCFAs. In GT derivatization of SCFAs, the -OH residue

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possessed by SCFAs was replaced with a trimethylammonium group having permanent cations of GT. The amide bond of these GT-labeled SCFAs is the most vulnerable to

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fragmentation when collision induced dissociation (CID) occurs [16]. Therefore, m/z 100 was used as the product ion mass of MRM analysis for all GT-labeled SCFAs. m/z 174 was detected as precursor ion mass when analyzing GT-labeled acetate. However, precursor ion mass of isourea by-product, a by-product produced by EDC coupling reaction, was also detected as m/z 174 in positive ion mode. We isolated GT-labeled acetate from isourea by10

products using LC separation and analyzed MS/MS spectra of GT-labeled acetate more accurately (Fig. S1). Using these parameters for MRM analysis of GT-labeled SCFAs, we established the LC method that these five GT-labeled SCFAs were all separated in 15 minutes by HPLC. It was confirmed that the longer the carbon chain of GT-labeled SCFAs, the faster elution was obtained on hydrophilic interaction liquid chromatography (HILIC) (Fig. 2C). Therefore, we could optimize the LC-MS/MS-based MRM analytical method to

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accurately and quickly analyze GT-labeled SCFAs in subsequent experiments.

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3.2. Sensitivity and quantitation test of GT-labeled SCFAs

We analyzed quantitative linearity, limit of quantitation (LOQ), and limit of

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detection (LOD) of the GT-labeled SCFAs through LC-MS/MS based MRM analysis (Fig.

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3, Table 1). All GT-labeled SCFAs had quantitatively superior linearity (R2 > 0.99) and sensitivity (LODs of all GT-labeled SCFAs except acetate are below femtomole). We compared LODs of GT-labeled SCFAs with those of 3NPH-labeled SCFAs and un-labeled

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SCFAs using the LC-MS/MS in our laboratory (Supporting results). A sensitivity of GT derivatization was 1.2x108 times higher than that of un-derivatization and 100-fold higher

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than 3-NPH derivatization on a basis of propionate. GT derivatization of SCFAs developed in this study showed not only excellent linearity, but also better sensitivity than un-

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derivatization and a previously reported chemical derivatization method [9]. The GT derivatization for maximizing the analytical sensitivity of SCFAs in LC-MS/MS to several femtomoles levels would be highly useful for the development of a high-throughput screening platform to analyze SCFA production by gut microbiome in the future.

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To validate the quantitative reproducibility, we directly extracted SCFAs from gut bacteria culture medium and then performed GT derivatization of the SCFAs. E. rectale, one of the main butyrate-producing bacteria in human gut [17], was used as a model system. SCFAs were extracted from 5, 10, 15, and 20 μL of culture supernatant of E. rectale and derivatized with GT. As a result, we confirmed that these SCFAs in a small volume of culture sample could be uniformly quantified according to the extraction volume of E.

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rectale culture (Fig. 4A). Comparing the total ion chromatogram (TIC) for extraction volume, peak areas of GT-labeled SCFAs increased in proportion to the extraction volume

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(Fig. 4B). Therefore, GT derivatization of SCFA could maintain accurate quantification according to various amounts of SCFA in small volume of gut bacteria culture medium,

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indicating that this method could be used for SCFA quantification with HTS platform.

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Based on these results, 5 μL cell culture was used for SCFA analysis in subsequent experiments.

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3.3. Growth and butyrate production analysis of E. rectale according to supplement of various plant-derived oligosaccharides

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To apply our GT derivatization method into the gut bacterial SCFAs, we investigated the growth of gut microbiota influenced by dietary fiber such as prebiotics and

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quantitatively analyzed the difference in SCFA productivity. First, four plant-derived polysaccharides (i.e., fructooligosaccharide (FOS), inulin, pullulan and xylan) that humans can ingest as food or prebiotics were provided as sole carbohydrate sources in E. rectale culture media. We observed the growth of E. rectale in these four kinds of dietary fibers and monitored the increase of butyrate production. 12

Growth and butyrate concentrations were measured at 0, 4, 8, and 12 hours after inoculation with E. rectale in mYCFA medium supplemented with each polysaccharide. As previous reported, the specific growth rate of E. rectale in mYCFA medium supplemented with xylan was increased markedly at 0.047 h-1 compared to that supplemented with other polysaccharides (Fig. 5) [18]. Moreover, butyrate productivity of E. rectale in media supplemented with xylan was also about 20 times higher than that of media supplemented

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with FOS at 1.1 mmol/L/h. Xylan increased butyrate production of E. rectale as well as the growth rate in comparison with other plant-derived polysaccharides. However,

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concentrations of propionate, valerate, and caproate were almost the same as those of E. rectale culture supernatant cultured on other polysaccharide source media (Fig. S3).

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Propionate production requires propionyl-CoA: succinate CoA transferase contained in the

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succinate biosynthetic pathway [17]. In addition, to produce valerate or caproate, it is necessary to produce specific CoA transferase to link the metabolism to fatty acid biosynthesis [19]. However, E. rectale does not have any gene related to these metabolism

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[17]. Concentrations of propionate, valerate, and caproate added to the initial medium in this study were found to be almost unchanged. Therefore, these results showed that our GT

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derivatization method for gut bacterial SCFAs may have the potential for screening

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prebiotic candidates whose function has not yet been confirmed.

3.4. Analysis of butyrate production in co-cultures of E. rectale and B. longum Gut microbiota can also produce beneficial metabolites for the host via metabolic

interaction between two or more different organisms [20]. Here, we confirmed that SCFA production could be quantitatively and reliably analyzed using GT derivatization when two 13

gut bacterial species were co-cultured. We used Bifidobacterium longum and E. rectale as a model of gut symbiosis. B. longum is one of representative probiotics. It has genes encoding numerous glycoside hydrolases that can digest various kinds of carbohydrates [21]. It has been reported that B. longum can metabolize inulin which cannot be digested solely by E. rectale [14]. Subsequently, the produced short fructooligosacchrides enable enhanced growth and activity of E. rectale [14].

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We measured butyrate productivity by culturing E. rectale with B. longum in inulin carbohydrate source to determine whether SCFA analysis using GT derivatization was

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reliable even with a small culture medium (5 μL) in a mixed culture. We compared butyrate productivity of mono-culture of E. rectale and co-cultures of E. rectale and B. longum at

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0 hr and 24 hr after inoculation. As a result, the concentration of butyrate in the co-culture

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supernatant at 24 hr after inoculation was about 2.6 times higher than that of mono-culture (Fig. 6). However, the concentration of butyrate in the culture supernatant of both 0 hr was similar. Consistent with prior studies, we hypothesis that B. longum supplied nutrients for E.

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rectale by producing short fructooligosacchrides via metabolism of inulin through βfructofuranosidase or acetate using the internal metabolism pathway such as fructose 6-

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phosphate phosphoketolase pathway [22]. However, the production of butyrate using inulin was lower by 4.2-fold at 12 hours after incubation (data not shown) compared to feeding

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xylan as a carbohydrate source to mono-culture of E. rectale. Since inulin is commercially available as prebiotics, unlike xylan, the increase of butyrate production by inulin is important to be studied for health. Taken together, we demonstrated that SCFA analysis using GT derivatization in a small volume of culture medium could be used to analyze reliable results in mixed culture of gut bacteria as well as mono-cultures. In addition, our 14

GT derivatization method may be applicable to reliable quantification of SCFAs in complex environments such as fecal sample or mixed culture.

4. Conclusions Gut microbial SCFAs have beneficial effects on human health in various ways. They can act as energy source and immune response modulator. In this study, we

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developed a GT derivatization method for more sensitive and reliable quantification of gut microbial SCFAs based on LC-MS/MS. The introduction of a permanent cation of SCFA

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by GT derivatization resulted in a more sensitive and quantitative analysis compared to previous methods with LC-MS/MS. In order to demonstrate that this GT derivatization

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method could quantitatively analyze SCFAs from cell culture medium in gut microbial

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experiment, the following three evaluation steps were carried out: i) quantitative evaluation by extraction of different volumes of E. rectale culture supernatant; ii) analysis of butyrate production of E. rectale grown on various plant-derived polysaccharide sources; and iii)

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monitoring of butyrate productivity changes in mixed cultures through co-culturing of E. rectale and B. longum. This multidimensional evaluation showed that GT derivatization of

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SCFA was suitable for quantitative analysis of gut microbial SCFA. Our results suggest that it is a sensitive and quantitatively reliable new SCFA assay method. This SCFA

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quantitative analysis method developed is expected to be useful for gut microbiome highthroughput screening requiring superior sensitivity of SCFA analysis.

Acknowledgements

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This work was supported by the Basic Science Research Program through the National Research

Foundation

of

Korea

(NRF-2018R1D1A1B07048185,

NRF-

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2017M3A9E4077235, NRF-2019M2C8A2058418).

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[22] M. Rossi, C. Corradini, A. Amaretti, M. Nicolini, A. Pompei, S. Zanoni, D. Matteuzzi,

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Figure legends Fig. 1. Derivatization reaction for SCFAs by Girard’s reagent T in this study.

Fig. 2. (A) (+)-ESI-MS/MS fragmentation of 6 GT-labeled SCFAs. (B) MS/MS detection

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parameters of 5 GT-labeled SCFAs. Bold font is the condition used for quantification. (C)

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MRM chromatograms of GT-labeled SCFAs. MRM, multiple reaction monitoring.

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Fig. 3. Calibration curve of GT-labeled SCFAs (n=3).

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Fig. 4. (A) Quantitation test of GT-labeled SCFAs according to extraction volume of E. rectale culture. (B) Comparison of LC-MS total ion chromatogram by extraction volume of

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E. rectale culture.

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Fig. 5. (A) Growth curve of E. rectale on various carbohydrates in modified YCFA media.

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(B) Butyrate production in E. rectale on various carbohydrates.

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Fig. 6. Comparison of butyrate production in E. rectale with inulin contained media according to co-culture with or without B. longum (P values were derived from the two-

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tailed Student’s t test; Error bars show standard deviation).

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Table 1. Quantitative information of each GT-labeled SCFA. R²

LOQ (fmol)

LOD (fmol)

Acetate

0.9982

30

30

Propionate

0.9999

1

0.01

Butyrate

0.9999

0.03

0.03

Valerate

0.9958

0.03

0.01

Caproate

0.9954

0.01

0.001

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na l

Pr

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pr

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SCFA

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