Gas Chromatography Detection Protocol of Short-chain Fatty Acids in Mice Feces

Materials and Reagents

1. 100-1,000 µl pipette tip (Biosharp, catalog number: BS-1000-T)
2. Micro-centrifuge tube, 5 ml (Biosharp, catalog number: BS-50-M)
3. Disposable sterile syringe, 1 ml (Zhengzhou Kangjia Medical Equipment Co., Ltd)
4. Clear short thread wide mouth sample bottle, 2 ml, vial with patch (32*11.6 mm) (Agilent Technologies, catalog number: 5182-0715)
5. Blue screw cap (9 mm) (Agilent Technologies, catalog number: 5182-0717)
6. Disposable sterile needle filter, Nylon 66, Aperture: 0.22 μm (Tianjin cypress Experimental Equipment Co., Ltd, catalog number: SFNF1322)
7. Mice feces samples, stored at -20 °C after collection
8. Hydrochloric acid, 37% (v/v) (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. CAS: 7647-01-0)
9. Acetic acid, standard for GC, ≥ 99.8% (GC) (Aladdin Industrial Corporation, catalog number: A116165)
10. Propionic acid, standard for GC, ≥ 99.5% (GC) (Aladdin Industrial Corporation, catalog number: P110443)
11. Butyric acid, Standard for GC, > 99.5% (GC) (Aladdin Industrial Corporation, catalog number: B110439)
12. Isobutyric acid, Standard for GC, ≥ 99.5% (GC) (Aladdin Industrial Corporation, catalog number: I103521)
13. Valeric acid, Standard for GC, ≥ 99.5% (GC) (Aladdin Industrial Corporation, catalog number: V108269)
14. Isovaleric acid, Standard for GC, ≥ 99.5% (GC) (Aladdin Industrial Corporation, catalog number: I108280)
15. Ethyl acetate, Analytical purity, ≥ 99.5% (Tianjin Fuchen Chemical Reagent Co., Ltd.)
16. 1 M HCl solution (see Recipes)
17. SCFAs single standard solution (see Recipes)
18. SCFAs mixed standard solution (see Recipes)
19. Dilute SCFAs single standard solution with a dilution factor of 2, 4, 8, 10 times (see Recipes)
20. SCFAs mixed standard solution (see Recipes)
21. Dilute SCFAs mixed standard solution with 2, 4, 8, 10 Dilution Times (see Recipes)
    

Equipment

1. 50 ml volumetric flask
2. Beaker
3. 100-1,000 µl pipette gun (Eppendorf, model: research plus)
4. A gas chromatograph (Agilent 7890A, USA) equipped with flame ionization detector and an automatic sampler (Autosampler, model: 7683B)
5. DB-FFAP capillary column with polyethylene glycol modified by terephthalic acid as stationary phase (30 m x 320 µm x 0.5 µm) (Agilent Technologies, USA, catalog number: 123-3233)
6. Stainless steel spoon, 180 mm long, with the widest diameter of 3 mm
7. Analytical balance (METTLER TOLEDO Instruments (Shanghai) Co., Ltd., model: ME204/02)
8. Vortex mixer (Aoran Science and Technology Co., Ltd., model: SI-0246-G560E)
9. Centrifuge (Changsha High-Tech Industrial Development Zone Xiangyi Centrifuge Instrument Co., Ltd., model: H1650R)
10. Ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd., model: KH-500E)

Software

1. Agilent ChemStation was used to analyze data (B.0403)
   

Procedure

1. Sample collection
1. Select mice in similar growth status at 6-8 weeks.
2. Collect fecal samples from 12 mice, approximately 300 mg per sample (stored at -20 °C). Used for the determination of SCFAs and recovery.
3. Mix 12 samples fully, weigh about 100 mg of the mixed sample and place it in a 5 ml centrifuge tube. Repeat the weighing of the 22 samples. Use three of them to test SCFAs in parallel, and use the remaining 19 to determine the rate of recovery. Number and record the sample quality (stored at -20 °C).


2. Sample pretreatment
1. Keep all biological samples frozen at -20 °C until the day of analysis.
2. Add 3 ml HCl 1 M to each sample, vortex for 1 min, mix thoroughly until no obvious clumping.
3. Place the well-mixed centrifuge tube in an ice-water bath in the beaker, and conduct ultrasonic extraction for 10 min (see Video 1). Set the temperature to room temperature and the frequency is 40k Hz.



Video 1. Ice water bath


4. Centrifuge the sample at 4 °C, 9,710 x g for 15 min.
5. Use a pipette to transfer 1 ml of supernatant into a 5 ml centrifuge tube (see Figure 1).
   
   

Figure 1. Absorb the supernatant


6. Add 1 ml of ethyl acetate to the supernatant, invert the centrifuge tube to completely mix the supernatant (see Video 2).



Video 2. Extraction


7. Centrifuge the sample at 4 °C, 9,710 x g for 15 min.
8. Aspirate the supernatant with disposable sterile syringe and filter through a 0.22 μm organic filter membrane. Transfer the filtered supernatant to Clear short thread wide mouth sample bottle, and inject into GC for analysis (If the sample stored in the bottle cannot be analyzed as soon as possible, it can be stored at -20 °C for 1-2 days) (see Figure 2).
   
   
   Figure 2. Aspirate the supernatant and filter
   


3. Gas Chromatography (GC) experimental conditions
 Set the GC program according to conditions mentioned in Table 1.

 Table 1. GC experimental conditions


4. Standard preparation and accuracy analysis
1. Prepare SCFAs single standard solution: using ethyl acetate as solvent, the maximum concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were adjusted to 2, 0.5, 0.1, 0.5, 0.5 and 0.1 g/L, respectively (see Recipe 2 for specific operation).
2. Dilute the mixed solution of the maximum concentration with ethyl acetate 2, 4, 8 and 10 times (see Recipe 3 for specific operation).
3. Prepare SCFAs mixed standard solution: using ethyl acetate as solvent, the maximum concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in the mixed solution were adjusted to 2, 0.5, 0.1, 0.5, 0.5 and 0.1 g/L, respectively (see Recipe 4 for specific operation).
4. Dilute the mixed solution of the maximum concentration with ethyl acetate 2, 4, 8 and 10 times (see Recipe 5 for specific operation).
5. SCFAs were injected into GC in accordance with the concentration gradient (Table 2) to determine the calibration curve of the SCFAs.

Table 2. Standard Solution Concentration of 6 SCFAs


6. Establish correction maps using an external standard method, R2 can be obtained according to the standard curve to confirm the accuracy.
7. The standard SCFAs solutions (6 kinds of acetic acid, propionic acid, etc.) from high to low concentration were sequentially analyzed by GC. The signal-to-noise ratio (S/N) of the standard SCFA was obtained according to the analysis of the GC workstation software.


5. Recovery
1. Calculation of recovery rate by standard addition recovery method (Park et al., 2018).
2. Take 19 samples from the "A. Sample collection" for testing recovery.
3. Use 18 samples as parallel experiment and the other as a blank control without any treatment.
4. Six samples as a group，add 1 ml of SCFAs standard solution of level 1 in Table 2. Select six samples as a group again and add 1 ml of SCFAs standard solution of level 3 in Table 2. Then select 6 samples as a group and add 1 ml of SCFAs standard solution of grade 5 in Table 2. The purpose is to check the accuracy of recovery.
5. Treat the 19 samples in accordance with the sample pretreatment (Procedure B).
6. Analyze the processed samples according to GC experimental conditions.
7. Obtain the content of SCFAs according to the standard curve of SCFAs.
   


6. Sample analysis

      The 3 samples were analyzed according to the GC analysis method, and the SCFAs content in the samples could be obtained according to the standard
      curves of various SCFAs.

Data analysis

1. GC analysis of SCFAs
1. The standard value of SCFA was determined by GC. According to the peak time obtained by GC for a single standard substance, the peak time of each substance of the mixed standard substance can be determined.
2. The result is shown in Figure 1 (Zhang et al., 2019). Each elute should be clearly separated and the shape of the peal sharp and symmetrical. The peak of solvent ethyl acetate is 5 min, and the peak times of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were 7.886, 8.531, 8.724, 9.174, 9.472, and 9.998 min, respectively.
3. Total GC analysis time was 13.5 min.
   
   
2. Linearity and Sensitivity
1. LOD and LOQ were determined from the S/N values of each SCFA.
2. The signal/noise(S/N) values of the standard SCFAs were 3 and 10, respectively, which function as the limit of detection (LOD) and limit of quantification (LOQ) of each SCFA.
3. The results should be almost the same as in Table 1 (Zhang et al., 2019).
   
   
3. Recovery and accuracy
   
   1. The results should be almost the same as in Table 2 (Zhang et al., 2019).
   2. According to the peak area of SCFAs in GC analysis results and the standard curve of SCFAs, the content of SCFAs in blank samples was obtained.
   3. Use the following formula to calculate the recovery rate:
      
      
      

  In the equation, R denotes recovery, %; C₁, is concentration of SCFAs in the standard addition and the samples; C₂, is the concentration of SCFAs in the
  samples; and C₃, is the concentration of  SCFAs in the standard addition.

Notes

1. In this experiment, young mice aged 6-8 weeks were selected. At this time, all organs of the mice were mature and the health index was good. In the following 1-2 months of the experimental cycle, the mice were healthy status.
2. In the sample collection step, after weighing the feces of mice, avoid the repeated freeze-thaw of samples. There are two benefits to storing at -20 °C. One is to freeze the sample into clumps for easy weighing, and the other is to reduce the volatilization of the material in the feces.
3. There are many ways to collect mice feces. This article chooses a method to deal with stimulus. The operation is as follows: fix the mouse and lift its tail, then press the mouse's lower abdomen with your fingers, collect fresh feces in a micro-centrifuge tube, store at -20 °C.
4. The number of samples is not a rigid requirement. It should be judged according to the specific experimental conditions. The purpose of selecting 12 in this article is to increase the randomness of the sample selection as much as possible to make the experimental results more accurate. If there is a clear experimental group sample, the sample size is the experimental group sample number. With regard to the amount of mice feces, it is necessary to judge at least 100 mg for one test. If the recovery rate of six kinds of SCFAs is tested, and one SCFA is added with three different concentrations of SCFA standard solution, it will need to be tested 18 times. When the final detection of SCFAs in the sample, ensure the accuracy, test 3 times, take the average value, and include blank Group, such a system operation will need to be tested 22 times, conservatively requires 2,200 mg. In calculating the loss during weighing and other experiments, at least 3,000 mg is collected.
5. When using 1 M HCI, configure the new reagent according to the recipes. Do not use reagents configured long ago.
6. When using a vortex mixer to mix HCl with the sample, the centrifuge tube should be tightly covered to prevent the mixture from flying out.
7. The reason for apparent lumping is that during pretreatment, the sample is frozen, and there may be caking. Another possibility is that the fecal sample itself has caking, which needs to be shaken to separate the caking and fully reflect with HCl.
8. During ultrasonic treatment in the ice water bath, add ice in time to maintain the temperature.
9. The purpose of using the pipette gun is to operate easily and sensitively, and to control the suction at any time.
10. During the extraction operation, the centrifuge tube needs to be inverted up and down several times to fully react the supernatant with ethyl acetate. The step lasts 3 min.
11. Pay attention to the liquid level dividing line, and do not over aspirate. The disposable syringe is not sensitive enough for operation, so special attention should be paid to it. Alternatively, use a pipette tip to transfer the supernatant to another tube and then filter to avoid disturbing the sediment or the bottom layer.
12. When using 0.22 μm organic filter membrane to filter the supernatant, it is necessary to control the pressure of the needle tube to prevent the filter membrane from being broken.
13. When configuring a single standard solution or a mixed standard solution, try to configure a large-capacity solution or a high-concentration solution as much as possible to reduce the concentration error in the solution preparation.
14. When performing a gradient dilution operation, select the appropriate volumetric flask for constant volume.
15. According to the content of SCFAs in mice feces, the setting range of SCFAs concentration in single standard solution or mixed standard solution was determined. 
16. The purpose of analyzing a single standard solution is to determine the species of each substance in the mixed standard solution.
17. Because the GC injection volume is very small, when performing gradient dilution, you can choose a small volume volumetric flask to make the volume, the purpose is to reduce the waste of solution.
18. When analyzing a single standard solution, you can choose the order from low concentration to high concentration, we only need to obtain the peak time.
19. The retention time and the detection range of each analyte may vary according to capillary column type and length and equipment brands.

Recipes

1. 1 M HCl solution (100 ml)
  8.35 ml HCl 37% (v/v) into a 100 ml volumetric flask
  Add distilled water to constant volume to scale

2. SCFAs single standard solution (100 ml)
  The highest concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid are 2, 0.5, 0.1, 0.5, 0.5, and 0.1 g/L.
  (The label is NO.1)
  190.48 µl analytical standard acetic acid, GC assay ≥ 99.8%
  50.51 µl analytical standard propionic acid, GC assay ≥ 99.8%
  10.53 µl analytical standard isobutyric acid, GC assay ≥ 99.8%
  52.08 µl analytical standard butyric acid, GC assay ≥ 99.8%
  53.72 µl analytical standard isovaleric acid, GC assay ≥ 99.8%
  10.65 µl analytical standard valeric acid, GC assay ≥ 99.8%
  Add ethyl acetate to 100 ml volumetric flask respectively for constant volume. (Six single standard solutions are placed in six volumetric flasks
  respectively); Vortex for 1 min


  In the equation, ρ is the density of substance, g/ml; m is the mass of substance, g; v is the volume of substance, ml

3. Dilute SCFAs single standard solution with a dilution factor of 2, 4, 8, 10 times
 Dilute 2 times: accurately aspirate 25 ml of No. 1 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.2)
 Dilute 4 times: accurately aspirate 25 ml No. 2 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.3)
 Dilute 8 times: accurately aspirate 25 ml No. 3 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.4)
 Dilute 10 times: accurately aspirate 5 ml of No. 1 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.5)
4. SCFAs mixed standard solution
 The maximum concentration of mixed stock solution of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were 2, 0.5,
  0.1, 0.5, 0.5 and 0.1 g/L, respectively (The labeling is No.6)
 190.48 µl analytical standard acetic acid, GC assay ≥ 99.8%
 50.51 µl analytical standard propionic acid, GC assay ≥ 99.8%
 10.53 µl analytical standard isobutyric acid, GC assay ≥ 99.8%
 52.08 µl analytical standard butyric acid, GC assay ≥ 99.8%
 53.72 µl analytical standard isovaleric acid, GC assay ≥ 99.8%
 10.65 µl analytical standard valeric acid, GC assay ≥ 99.8%
 Add ethyl acetate to a 100 ml volumetric flask for constant volume (Six single standard solutions are put together in a volumetric flask), Vortex for 1 min
5. Dilute SCFAs mixed standard solution with 2, 4, 8, 10 Dilution Times
   
 Dilute 2 times: accurately aspirate 25 ml of No. 6 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.7)
 Dilute 4 times: accurately aspirate 25 ml of No. 7 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.8)
 Dilute 8 times: accurately aspirate 25 ml of No. 8 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.9)
 Dilute 10 times: accurately aspirate 5 ml of No. 6 solution, add it to a 50 ml volumetric flask, and add ethyl acetate to the mark. (The label is No.10)

Acknowledgments

This work has been supported by the Foundation (No. ZXKF20180304) of Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control.

Competing interests

All authors declare that there are no conflict of interests.

Ethics

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
  We use animal excrement for our experiment. Animals are not ours. They are samples provided to us by other research groups.

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