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A Simplified Method for the Quantitation of Short-Chain Fatty Acids in Human Stool

B. Loye Eberhart, II, Annette S. Wilson, Stephen J.D. O’Keefe, Matsepo C. Ramaboli,
Lucky T. Nesengani

PII: S0003-2697(20)30548-0
DOI: https://doi.org/10.1016/j.ab.2020.114016
Reference: YABIO 114016

To appear in: Analytical Biochemistry

Received Date: 25 September 2020

Revised Date: 1 November 2020
Accepted Date: 3 November 2020

Please cite this article as: B.L. Eberhart II., A.S. Wilson, S.J.D. O’Keefe, M.C. Ramaboli, L.T. Nesengani,
A Simplified Method for the Quantitation of Short-Chain Fatty Acids in Human Stool, Analytical
Biochemistry, https://doi.org/10.1016/j.ab.2020.114016.

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© 2020 Published by Elsevier Inc.

Author Contributions

B. Loye Eberhart II, B.S.

• Designed and performed the experiments.

• Analyzed all the samples.
• Compiled all the data.
• Wrote the manuscript.

Annette S. Wilson, Ph.D.

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• Provided expertise with designing the experiments.
• Ordered all the supplies necessary to do the experiments.

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• Edited the manuscript.

Stephen J.D. O’Keefe, MBBS, M.D., M.Sc.

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• Edited the manuscript.
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• Provided expertise.

Matsepo C. Ramaboli, Ph.D.

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• Provided pilot-study samples.

• Edited the manuscript.
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• Provided expertise.
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Lucky T. Nesengani, Ph.D.

• Provided pilot-study samples.

• Edited the manuscript.
• Provided expertise.
 1

A Simplified Method for the Quantitation of Short-Chain Fatty Acids in

Human Stool
B. Loye Eberhart II*†, Annette S. Wilson†, and Stephen J. D. O’Keefe†, Matsepo C. Ramaboli‡,
Lucky T. Nesengani‡
†
University of Pittsburgh, United States of America
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, 200 Lothrop
Street, Room W1111/2, Biomedical Science Tower, Pittsburgh, PA 15213
eberhart.bl@pitt.edu, aswilson@pitt.edu, and sjokeefe@pitt.edu
‡
Stellenbosch University, South Africa

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Department of Internal Medicine, Room 3055, Clinical Building, Faculty of Medicine & Health
Sciences, Francie Van Zjil Drive, Tygerberg, 7505
matsepor@sun.ac.za, ltnesengani@sun.ac.za

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* -p
Author to whom correspondence should be addressed. Complete contact information:
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Blaine Loye Eberhart II, B.S.
Researcher III
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Department of Medicine
Division of Gastroenterology, Hepatology, and Nutrition
University of Pittsburgh
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Lab: W1111 Biomedical Sciences Tower

200 Lothrop St
Pittsburgh, PA 15213
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Mobile: 412-610-6867
Email: eberhart.bl@pitt.edu
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 2

ABSTRACT: A one-vial extraction method for the quantitation of short-chain fatty acids

(SCFAs) in human stool was developed. Samples were extracted with an acidified aqueous

internal standard solution, sodium sulfate, and diethyl ether, followed by analysis with GC-FID.

Accuracy, in terms of relative recovery, was typically between 90 and 110% for most analytes;

without internal standard, the accuracy was about 5-34%; the linear dynamic range (LDR) was

0.05 to 50 micromoles per gram; the limit of detection (LOD) was less than or equal to 0.05

micromoles per gram; and the (lower) limit of quantitation (LOQ) was 1 micromole per gram.

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The method is suitable for quantitating acetic acid, propanoic acid, isobutyric acid, butyric acid,

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isovaleric acid, valeric acid, isohexanoic acid, hexanoic acid, and heptanoic acid. It is not
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suitable for the quantitation of formic acid. Application to human biological research was tested
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by the measurement of SCFA in heathy humans. This confirmed that the method performed
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adequately, and even better than expected, with values up to 150 micromoles per gram.
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Keywords: Gas Chromatography, Human Stool, Short-Chain Fatty Acids, Gut Health
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Graphical Abstract:
 3

Highlights

• This method uses one-vial extraction approach for quantitation of short-chain fatty acids

in human stool.

• GC run time is 21.5 minutes.

• Extracts do not foul the injection port or the syringe.

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1.1 Introduction

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There is intense current interest in the potential benefits of fiber-rich foods on reducing the risk
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of non-communicable diseases – the major threat to health in the USA today. The chief
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mechanism of action is thought to be via the fermentation of fiber by the colonic microbiota to
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short chain fatty acids, particularly as acetate, propionate and butyrate which have all been

shown to maintain colonic mucosa health and defense, and to prevent inflammation and
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neoplastic change in the colon (1,2). If generation is high, they are absorbed and can express
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epigenetic histone deacetylase inhibitory functions throughout the body, and metabolic
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regulatory functions by suppressing appetite, reducing weight and diabetes risk, and decreasing

lipoprotein synthesis (1-14). Short-chain fatty acids are defined as carboxylic acids containing

two to eight carbon atoms. This paper describes a method for quantifying acetic (AA), propanoic

(PA), isobutyric (IBA), butyric (BA), isovaleric (IVA), valeric (VA), hexanoic (HA),

isohexanoic (IHA), and heptanoic (HPA) acids in human stool. Among these acids, acetic,

propanoic, and butyric acids are the most significant and among these three, butyric acid is the

most biologically active SCFA produced during fermentation of fiber by the colonic microflora

(1-14). Branched SCFAs (BSCFAs), such as isobutyric and isovaleric acids, are produced by

fermentation of branched amino acids, valine, leucine, and isoleucine generated from undigested
 4

proteins reaching the colon (4). The main butyrate producing bacteria are from the Firmicutes

phylum, the genera in Clostridium cluster XIVa in particular – Faecalibacterium, Eubacterium,

and Roseburia (5). Butyric acid is a histone deacetylase (HDAC) inhibitor and binds to several G

protein-coupled receptors (GPCRs). Levels of butyric acid (as well as acetic and propanoic

acids) are directly proportional to fiber intake; additionally, butyric acid has anti-inflammatory

properties and acts as a food source for colonic mucosal cells (1-14). The BSCFAs isobutyric

acid and isovaleric acid were shown to inhibit lipolysis in rat adipocytes (6).

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Previously described quantitative analytical methods for SCFAs involve various extraction
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techniques, including derivatizations, and require use of instruments such as GC-MS, LC-MS,
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LC-MS-MS, NMR, and capillary electrophoresis (15-30). Tao’s method of quantitating colonic
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contents in mice uses a diethyl ether extraction with GC-FID (27); however, the calibration range
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was too low for levels typically seen in human stool. As a result, we developed a simplified

method of extracting human stool samples into an organic solvent, followed by analysis by gas
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chromatography with flame ionization detection (GC-FID), with an expanded calibration range.
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1.2 Material and Methods

Safety: Sample and standard preparations were carried out under a fume hood, including

weighing the samples or standards. Safety glasses, with side shields, a lab jacket, and nitrile

gloves, were worn at all times. After the procedure, surfaces were sanitized with a 10% bleach

solution in water.
 5

1.2.1 Equipment

Apparatus: Agilent GC-FID (Palo Alto, CA, USA)—GC Conditions: Agilent 6890 with 7683

autosampler; GC Column, Agilent DB-FFAP, 30 m x 0.53 mm ID x 0.50 µm film (125-3237)

operated in splitless mode; Oven temperature program: 40°C for 1 minute ramped at

20°C/minute to 250°C, holding for 10 minutes; carrier gas was helium with a flow of 3.0

mL/min; inlet temperature of 240°C.

Detector Conditions: Temperature was 250°C; air flow was 400 mL/minute; hydrogen flow was

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40 mL/minute; makeup gas flow (helium) was 40 mL/minute.

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Autosampler Conditions: Solvent A and B Washes were methylene chloride, three times, pre-
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and post-injection; Sample Wash, one time; sample pumps, three times.
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Software: Agilent ChemStation (Version D.01.02.16)
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(b) Vortex-Genie Mixer (Model #21515), (Bohemia, NY, USA)

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(c) 40-mL vials with Teflon/Silicone lined Caps, Thermo Scientific, B7800-6, (Rockwood, TN,

USA)
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(d) Micropipettes capable of delivering 1-5000 µL, Eppendorf, (Enfield, CT, USA)
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(e) Transfer Pipettes, 3-mL, Samco, #225, (San Diego, CA, USA)

(f) 100-mL volumetric flasks, 250-mL beakers

(g) Autosampler vials with Teflon/Silicone Septa (Thermo Scientific, #CERT5000-78),

(Langerwehe, Germany)

(h) Ohaus Explorer Analytical Balance (Model #E10640), (Parsippany, NJ, USA)

(i) Microliter Syringe, 50-µL, fixed needle (Hamilton), (Reno, NV, USA)

(j) Tilt Dispensing Flask, 5-mL (Kimble™Kontes™ #7593000005), (Vineland, NJ, USA)
 6

1.2.2 Reagents

(a) Volatile Free Acid Mix (VFAM), Certified Reference Material (CRM), Sigma-Aldrich,

CRM46975, (St. Louis, MO, USA)

(b) Sodium Sulfate, Sigma-Aldrich, 239313, (St. Louis, MO, USA)

(c) Hydrochloric Acid, Certified ACS Plus, Fisher, A144 SI-212, (Fairlawn, NJ, USA)

(d) Diethyl Ether, Merck, SupraSolv®, 1.00931.2500, (Billerica, MA, USA)

(f) Deionized Water, 18.2 MΩ∙cm @ 25℃ (Milli-Q System), (St. Louis, MO, USA)

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(g) Methylene Chloride, Fisher, D143-4, (Fairlawn, NJ, USA)

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(i) 2,2-Dimethylbutyric Acid, Sigma-Aldrich, D152609, (St. Louis, MO, USA)
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1.2.3 Standards
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Internal Standard (ISTD) Stock Solution. An amount (0.116 g) of 2,2-dimethylbutyric acid

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(116.16 g/mol) was weighed into a glass vessel (e.g., vial or tube) and quantitatively transferred

into a 100-mL volumetric flask with deionized water. The solution was diluted to volume with
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deionized water and homogenized using a Vortex Genie mixer. The resulting solution was 10.0
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mM. The solution was transferred to 40-mL vials and refrigerated (4°C) when not in use.

Before use, the solution was allowed to equilibrate to room temperature and then

rehomogenized.

Internal Standard Extraction Solution. Deionized water (12.00 ± 0.01 g) was weighed into a 40-

mL vial followed by 20.00 ± 0.01 g of the internal standard stock solution. Then, 8000 µL of

concentrated hydrochloric acid was added. The solution was capped and homogenized.
 7

Calibrations. Aliquots of the Sigma-Aldrich VFAM Certified Reference Material (1, 250, 500,

750, and 1000 µL) were added to five 40-mL vials for five calibration standards which

corresponded to 0.01, 2.5, 5.0, 7.5, and 10 µmol, respectively, followed by 1000 µL of the

Internal Standard Extraction Solution (5.0 µmol 2,2-dimethylbutyric acid). Then, 4-5 mL of

diethyl ether was added. The vials were capped tightly, shaken vigorously by hand, and vortexed

for 10 seconds. Then, about 5-10 g of sodium sulfate was added, shaken by hand vigorously,

and vortexed again for 10 seconds. (Care must be taken in adding the sodium sulfate such that

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enough diethyl ether remains for analysis, but also such that no bottom water layer remains.)

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With a transfer pipette, an aliquot was transferred to an autosampler vial and capped, being
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careful to avoid any granules of sodium sulfate. Five-point calibration curves were prepared by
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plotting peak area ratios vs. molar ratios and performing a linear regression. Amounts of SCFAs
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in the samples were calculated using the following equation, followed by normalization to the
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sample weight (0.200 g) to obtain analyte amounts:

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= −
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Where Amountanalyte is the amount of analyte in micromoles; Peak Areaanalyte is the peak area of

the analyte; Peak AreaISTD is the peak area of the internal standard; b is the y-intercept;

AmountISTD is the amount of internal standard spiked into the sample in micromoles (5.0 µmol);

and m is the slope of the curve. This amount was then divided by the sample weight (0.200 g).

The equation does not include the sample weight, as it is possible, with actual clinical samples,

that 0.200 g of sample may not be available. In that case, the amount would be divided by some

other sample weight less than 0.200 g.

 8

1.2.4 Sample Preparation and Extraction.

University of Pittsburgh Institution Review Board (PRO 08100243); Stellenbosch University

Health Research Ethics Committee (REF: N19/02/024); and the University of KwaZulu-Natal

Medical Ethics and Research Committee (REF: BE006/01) provided ethical review and approval

for the analysis of human samples in this study. Volunteer stool samples from healthy controls

were used to establish a method that can be used for participants who add or remove fiber from

their diets. Samples were stored at -80°C and allowed to thaw on water ice before preparation. A

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sample of human stool (0.200 ± 0.001 g) was weighed into a 40-mL vial. Some stool samples

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contained what appeared to be undigested material such as seeds, plant material, and shell
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fragments from shellfish. Whenever possible, these types of materials were avoided when
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weighing out the sample. Then, 1000 µL of the internal standard extraction solution was added,
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followed by 4-5 mL diethyl ether. The tube was capped, shaken vigorously by hand, and
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vortexed for 10 seconds. Afterwards, about 5-10 g of sodium sulfate was added to the vial.

(Care must be taken in adding the sodium sulfate such that enough diethyl ether remains for
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analysis, but also such that no bottom water layer remains.) The vial was recapped tightly and
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then vigorously shaken by hand, followed by vortexing again for 10 seconds. With a transfer

pipette, an aliquot was transferred to an autosampler vial and capped, being careful to avoid any

granules of sodium sulfate.

In some cases, stool was very dry and/or hard. Shaking and vortexing times were increased to

ensure dispersion, as gauged by observation.

 9

After addition of sodium sulfate, followed by vortexing, the upper organic layer (diethyl ether)

would typically turn pale to dark yellow. Sometimes, the organic layer would turn green or pink,

or other colors, presumably based on the dietary habits of the subject.

Chromatogram of a Mid-Range Standard

Response

8000000

7500000 HPA
7000000 HA
ISTD IHA
6500000

6000000
IVA

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VA
5500000

5000000

IBA

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4500000 BA
4000000

3500000

3000000

2500000

2000000
PA
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1500000 AA
1000000

500000
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6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Time
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Figure 1a. Example Chromatogram (Mid-Range Calibration Standard).

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Chromatogram of a Stool Sample

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Response

7500000 ISTD
7000000

6500000

6000000

5500000

5000000

4500000

4000000
BA
3500000

3000000
AA PA
2500000

2000000
IVA
1500000 VA
IBA IHA
1000000
HA HPA
500000

6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Time
Figure 1b. Example Chromatogram (Stool Extract).
 10

1.2.5 Accuracy (Relative Recovery, %) and Precision (CV, %) (Table 1)

A human stool evacuate was spiked at several different levels with the 10 mM VFAM: 10 (0.1

µmol), 250 (2.5 µmol), 500 (5.0 µmol), 750 (7.5 µmol) and 1000 (10.0 µmol) µL, corresponding

to 0.5, 12.5, 25.0, 37.5, and 50.0 µmol/g. Five replicates were done at each level, including an

unspiked sample, and extracted according to the sample preparation procedure described above.

Percent recoveries were determined by subtracting the concentrations of the unspiked sample

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from each spiked level and dividing the result by the spiked concentrations, for each analyte, and

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then multiplying by 100.
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 11

Accuracy (Relative Recovery, %) and Precision (CV, %)

Spike,
µmol/g Parameter AA PA IBA BA IVA VA IHA HA HPA
0.5 Interday Relative Recovery 133 85 91 90 94 89 91 89 84
Day 1, Relative Recovery 180 89 93 94 98 94 96 95 91
Day 2, Relative Recovery 85 80 89 85 91 84 86 83 78
Interday CV 21 4 8 4 17 13 10 6 2
Day 1, CV 6 1 1 2 1 1 1 1 1
Day 2, CV 6 2 2 2 2 2 2 2 2
12.5 Interday Relative Recovery 104 101 101 102 103 103 103 103 103
Day 1, Relative Recovery 104 101 100 102 101 101 101 100 100
Day 2, Relative Recovery 103 102 103 103 105 105 105 105 105

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Interday CV 2 2 2 2 2 3 3 3 4
Day 1, CV 2 2 2 2 2 2 2 2 2

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Day 2, CV 2 2 1 1 1 2 2 2 3
25.0 Interday Relative Recovery 99 100 102 101 102 102 102 102 102
Day 1, Relative Recovery 94 99 102 101 102 102 102 102 102
Day 2, Relative Recovery
Interday CV
103
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102
1
102
1
102
1
102
1
102
1
102
1
102
1
102
1
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Day 1, CV 1 1 1 1 1 1 1 1 1
Day 2, CV 0 1 0 1 0 1 1 1 1
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37.5 Interday Relative Recovery 103 101 101 102 102 102 103 103 103
Day 1, Relative Recovery 105 102 102 102 102 102 102 102 102
Day 2, Relative Recovery 100 100 100 101 102 103 104 104 105
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Interday CV 3 2 2 1 0 1 1 1 2
Day 1, CV 3 2 1 1 0 0 1 1 1
Day 2, CV 2 1 0 0 0 0 0 0 0
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50.0 Interday Relative Recovery 96 99 100 100 100 101 101 101 102
Day 1, Relative Recovery 91 98 100 100 101 101 101 101 102
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Day 2, Relative Recovery 100 100 100 100 100 101 100 101 101
Interday CV 5 1 0 0 0 0 0 0 0
Day 1, CV 2 1 1 1 0 0 0 0 0
Day 2, CV 1 1 0 0 0 0 0 0 0

Table 1. Accuracy (Relative Recovery, %) and Precision (CV, %), N=10 for each level (N=5 on
two different days).

1.2.6 Long-Term Precision (Tables 2a and 2b).

An evacuate stool sample was chosen for long-term precision. It was thawed and small aliquots

were deposited into 1.5 mL plastic centrifuge tubes. After capping, the samples were refrozen at

-80°C. When a sample was run, one of the vials was removed from the freezer and thawed. This
 12

was done so that the entire sample was not continuously thawed and refrozen, thus preventing

sample degradation. Samples were prepared on different days according to the method described

above. Additionally, a solid stool sample was chosen for a precision study, but since it was only

going to be used for about one month, it was sampled out of one container.

Precision Data for Evacuate

Parameter AA PA IBA BA IVA VA IHA HA HPA

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Mean 36.6 5.3 2.3 1.1 2.3 1.2 0.2 0.1 0.2
σ 1.1 0.3 0.2 0.2 0.2 0.2 0.3 0.3 0.3

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2σ 2.2 0.5 0.4 0.4 0.4 0.4 0.5 0.5 0.5
CV, % 3 5 9 19 10 19 160 205 139
Mean-2σ 34.4 4.8 1.9 0.7 1.8 0.7 -0.3 -0.4 -0.3
Mean+2σ 38.7 5.8 2.7 1.6 -p 2.7 1.6 0.7 0.6 0.7
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Table 2a. Evacuate Precision data, N=32 over five months. Concentrations are µmol/g for each
analyte. (NOTE: “σ” is the population standard deviation. The mean ±2σ is the 95% confidence
interval.)
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Precision Data for Solid

Parameter AA PA IBA BA IVA VA IHA HA HPA
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Mean 57.7 20.4 3.5 18.7 5.6 3.6 -0.1 0.8 0.2
σ 3.6 0.8 0.2 0.7 0.2 0.2 0.1 0.2 0.2
2σ 7.2 1.6 0.3 1.4 0.5 0.4 0.3 0.3 0.4
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CV, % 6 4 4 4 4 5 -145 21 94
Mean-2σ 50.5 18.8 3.2 17.4 5.1 3.2 -0.4 0.5 -0.2
Mean+2σ 64.9 22.0 3.8 20.1 6.1 4.0 0.2 1.2 0.7

Table 2b. Solid Precision data, N=20 over four different weeks. Concentrations are µmol/g for
each analyte. (NOTE: “σ” is the population standard deviation. The mean ±2σ is the 95%
confidence interval.)
 13

1.2.7 Efficacy of the Internal Standard

Because of the way the calibration standards were prepared—i.e., as samples—the efficacy of

the internal standard was evaluated by preparing 2-mL solutions of Standards 1-5, with

corresponding levels of the VFAM (1, 250, 500, 750, and 1000 µL) and internal standard stock

(500 µL), were prepared with deionized water (1499, 1250,1000,750, and 500 µL). These

standards were used to prepare a calibration curve to quantitate previously run diethyl ether

calibration standards. (On the autosampler, Solvent A and B washes were deionized water and

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methanol, respectively. Other conditions were the same as described above.)

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Quantitation with Internal Standard

Standard AA PA IBA BA
-p IVA VA IHA HA HPA
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STD-3 Mean 23.3 25.7 26.4 25.9 25.8 24.9 24.5 24.0 23.1
s 1.2 0.8 0.7 0.6 0.5 0.8 1.1 1.3 1.7
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CV, % 5 3 3 2 2 3 5 5 7
Actual 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0
Relative Recovery, % 93 103 106 104 103 100 98 96 93
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Table 3a. Efficacy of the internal standard using aqueous standards and with internal standard
correction, N=18. Concentrations are µmol/g for each analyte. (NOTE: “s” is the sample
standard deviation.
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Quantitation without Internal Standard

Standard AA PA IBA BA IVA VA IHA HA HPA

STD-3 Mean 1.2 8.3 8.4 8.4 8.3 8.1 8.0 7.8 7.6
s 0.5 2.7 2.8 2.8 2.8 2.9 2.9 2.9 2.9
CV, % 47 33 33 34 34 35 36 37 38
Actual 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0
Relative Recovery, % 5 33 34 34 33 32 32 31 30
Table 3b. Efficacy of the internal standard using aqueous standards and without internal
standard correction, N=18. Concentrations are µmol/g for each analyte. (NOTE: “s” is the
sample standard deviation.)
 14

1.2.8 The African Pilot Study

A set of stool samples were sent to the University of Pittsburgh, USA, from Stellenbosch

University, South Africa. These samples were from Southern Africa and were analyzed using

the method described here. It was found that many samples exceeded the highest level of the

calibration curve. The calibration curve was based on our experience with lower-fiber diets. As

a result, several higher standards were prepared, and quantitated as samples, to check for

linearity up to 150 µmol/g.

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African Pilot Study

Sample
001
AA
84
PA
19
IBA
1
-p
BA
10
IVA
2
VA
3
IHA
<1
HA
1
HPA
<1
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002 66 17 3 3 4 2 <1 <1 <1
003 101 42 5 25 8 9 <1 3 1
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004 67 20 2 15 2 3 <1 1 <1

005 75 24 1 12 2 3 <1 1 <1
006 92 53 2 36 2 4 <1 1 <1
007 89 38 3 25 4 7 <1 3 1
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008 49 11 4 17 8 3 <1 1 <1

009 68 27 1 26 1 2 <1 <1 <1
010 115 78 2 23 3 5 <1 <1 <1
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011 96 37 5 29 9 6 <1 1 <1

012 60 14 3 7 4 2 <1 <1 <1
013 70 33 2 14 3 3 <1 <1 <1
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014 96 66 1 21 1 2 <1 <1 <1

015 102 66 4 37 5 6 <1 1 <1
016 73 19 2 32 3 5 <1 3 <1
017 72 24 1 8 2 4 <1 1 <1
018 62 29 1 13 2 8 <1 5 5
019 88 50 2 21 2 11 <1 5 3
020 52 29 1 19 1 2 <1 1 <1
021 46 11 2 7 3 2 <1 <1 <1
022 74 20 5 15 8 5 <1 <1 <1
023 43 9 1 2 2 1 <1 <1 <1
024 48 16 2 7 3 2 <1 <1 <1
STD, 75.0 µmol/g 71 75 76 76 76 77 77 77 77
(Relative Recovery, %) (95) (100) (102) (101) (102) (102) (102) (103) (103)
STD, 112 µmol/g 114 114 115 115 115 116 116 117 117
(Relative Recovery, %) (101) (101) (102) (102) (102) (103) (103) (104) (104)
STD, 150 µmol/g 136 147 150 151 152 154 154 155 156
(Relative Recovery, %) (90) (98) (100) (101) (102) (102) (103) (103) (104)
Table 4. Pilot study using this method for stool samples from South Africa. Results are in
µmol/g, unless otherwise indicated.
 15

1.3 Results and Discussion

1.3.1 Calibration Curves and Chromatography

Good linearity was obtained over the calibration range (0.01 to 10 µmol) with R2 values typically

better than 0.9990 for all compounds. (Additional experiments showed good linearity up to 30

µmol.) Peak shape and separation were both excellent (Figures 1a and 1b).

Preliminary experiments (data not shown) with organic solvent extractions showed that both

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sodium sulfate and acid were necessary to efficiently extract acetic acid and propanoic acid. It is

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likely that the acid ensures protonation of the SCFA, as a dissociated acid would tend to be
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hydrophilic. The sodium sulfate removes the water, but likely provides a salting-out effect
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forcing the SCFAs into the organic solvent. Additionally, during the second vortexing step, the
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abrasiveness of the sodium sulfate helped disperse the stool samples, increasing the surface area
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in contact with the organic solvent.

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The diethyl ether contains an interference for acetic acid which is ≤ 1 µmol/g. Method blanks
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typically have much less than this concentration, typically negative, showing that the method is

calibrated for such an interference.

Small concentrations (typically, <0.3 µmol/g) of SCFAs were found to carry over from the

highest standard into the next sample. As a result, diethyl ether blanks were run immediately

afterwards and during various points in the sequence—roughly every ten samples—to monitor

for carryover.
 16

There is some accumulation of additional material in the GC-FID system. As a result, a bake-out

method was added at the end of each sequence. The bake-out step was simply an injection of

diethyl ether, with the injection port temperature increased to 250°C, and the run time extended

to ten hours. The bake out could be stopped once a flat baseline was obtained, typically two to

three hours.

1.3.2 Accuracy and Precision

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Typically, interday and intraday accuracy results, in terms of relative recovery, were similar for

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most compounds at each spike level, and ranged from 91 to 105%, except at the at the 0.5
-p
µmol/g level, where the range was 78 to 180%. Interday and intraday precision, in terms of CVs,
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followed a similar trend (Table 1). Two samples were chosen for repeated analysis (evacuate
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and solid stool) to evaluate long-term precision. The CVs (coefficients of variation) showed
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good precision over time, but also showed a substantial degradation in precision below 1 µmol/g

(Tables 2a and 2b). Typically, IBA, IVA, VA, IHA, HA, and HPA (especially the latter three)
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are not in high abundance, and the peaks in the chromatograms were very close to the baseline
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noise.

1.3.3 Efficacy of the Internal Standard

The efficacy of the internal standard was evaluated with standards prepared in water and a mid-

range standard presented in Tables 3a and 3b. When quantitating with the internal standard, the

data show good precision (CVs 2-7%) and recovery (93-106%).

Quantitation without the internal standards resulted in poor precision (CVs 33-47%) and relative

recoveries around 33%, with the recovery of acetic acid only 5%. Additionally, the recovery of
 17

AA is disproportionate compared to all the other analytes. The good recoveries and precision

that were obtained for AA were mostly likely the result of the combination of the internal

standard and the fact that the standards were prepared in the same manner as the samples.

1.3.4 Formic Acid

The Sigma-Aldrich VFAM contains 10 mM formic acid, which according to chromatogram in

the Certificate of Analysis (figure not shown), elutes immediately before propanoic acid. In this

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work, no peak for formic acid was observed in either the aqueous or diethyl ether calibration

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standards. So, an aqueous VFAM standard was re-analyzed by GC-FID, but at only 5°C/minute,

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instead of 20°C/minute, to check for coelution. Still, no peak for formic acid was observed.

Additionally, several different concentrations of aqueous formic acid were injected, but no
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discernable peak for formic acid was found.
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1.3.5 The African Pilot Study

As can be seen from the Table 4, a wide range of analyte concentrations were found, with many
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analytes exceeding the highest standard. Additional standards, to check for linearity, were
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prepared and analyzed as samples, by extrapolation. These results were in good agreement with

their target values. However, preparing these higher standards required adding substantially

more sodium sulfate, and was, therefore, cumbersome.

The most prominent SCFAs found were AA, PA, and BA. The results for IBA, IVA, VA, were

typically lower than 5 µmol/g. Additionally, IHA, HA, and HPA were mostly <1 µmol/g, with

some exceptions.
 18

1.4 Conclusions

Although the lowest calibration level in this method is 0.01 µmol, data show a substantial

degradation in precision below 0.2 µmol (1 µmol/g) as well as increased interferences. As a

result, the limit of quantitation (LOQ) is 0.2 µmol (1 µmol/g), but the limit of detection (LOD) is

≤0.01 µmol ( ≤0.05 µmol/g)—at this level, the signal-to-noise ratio is about 10:1 (by visual

estimation); the LDR is 0.01 µmol to 10 µmol (0.05 to 50 µmol/g).

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This organic extraction method for measuring SCFAs provides a simple method of extraction

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with very good chromatography and an expanded calibration range.
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1.5 Acknowledgements
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The analyses were done at the University of Pittsburgh, and funded by the National Institutes of
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Health (NIH), United States of America; The African Pilot Study, officially “The AMI Pilot

Study” at Stellenbosch University, South Africa, was funded by The Rector’s Strategic Fund,
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2018-2020.
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1.6 Conflicts of Interest

The authors assert that they have no conflicts of interest relevant to this work.
 19

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