Method for measuring short-chain fatty acid content in intestinal contents or excrement

Abstract

The present invention relates to the technical field of quantitative detection, and provides a method for measuring short-chain fatty acid content in intestinal contents or excrement. The method comprises a sample pre-treatment step, and in said step, first extracting short-chain fatty acids in an intestinal content or excrement sample using acetone as an extraction solvent, then, by means of a solid-phase extraction column, separating and purifying an extracted short-chain fatty acid acetone extraction solution; a filler of the solid-phase extraction column uses C8 and SAX. The method of the present invention has the characteristics of high recovery rate, low detection limit, and short time consumption.

Description

A method for determining the content of short-chain fatty acids in intestinal contents or feces

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2023117460425 filed on December 19, 2023, and this application cites the full text of the above-mentioned Chinese patent application. Technical Field

[0003] The present invention relates to the field of quantitative detection technology, and in particular to a method for measuring the content of short-chain fatty acids in intestinal contents or feces. Background Art

[0004] Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota in the large intestine through fermentation of unabsorbed components of food. The most abundant SCFAs are acetic acid, propionic acid, and butyric acid, accounting for 90%-95% of the total SCFA in the colon. They are primarily derived from carbohydrates, but some amino acids (particularly valine, leucine, and isoleucine) can be converted into branched-chain SCFAs, such as isobutyric acid and isovaleric acid, which contribute slightly to the total SCFA content. The presence of SCFAs in the intestine has several advantages, such as lowering luminal pH, facilitating nutrient absorption, and reducing the formation of some pathogenic microorganisms.

[0005] Currently, there are a variety of analytical methods for detecting SCFA, such as gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE) or nuclear magnetic resonance (NMR). Among them, GC is the most commonly used technique because of the volatility of SCFA and the high sensitivity of this technique and sample pretreatment, good resolution and relatively low cost. GC can be coupled with a flame ionization detector (FID). In addition, the coupling of GC with a mass spectrometer (MS) can further improve the selectivity and sensitivity of the method. Before SCFA analysis, sample pretreatment usually includes extraction and, in some cases, purification and / or derivatization. For example, CN110045040A, CN116124905A, etc. disclose methods for determining the content of short-chain fatty acids, but the accuracy and efficiency of the above methods need to be improved.

[0006] Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the present invention provides a method for measuring the content of short-chain fatty acids in intestinal contents or feces.

[0008] The present invention provides a method for determining the content of short-chain fatty acids in intestinal contents or feces, comprising a sample pretreatment step, wherein acetone is first used as an extraction solvent to extract short-chain fatty acids from the intestinal contents or feces sample, and then a short-chain fatty acid acetone extract obtained by separation and purification is obtained by using a solid phase extraction column, wherein C8 and SAX are used as fillers for the solid phase extraction column.

[0009] Among them, C8 is a silica gel bonded octyl group, and SAX is a strong anion exchange filler bonded with a quaternary amino functional group.

[0010] Preferably, the mass ratio of C8 to SAX is 1:3 to 3:1, and the packing is staged, with the C8 packing on top and the SAX packing on the bottom. That is, when the extract passes through the solid phase extraction column, it first passes through the C8 packing and then the SAX packing. The mass ratio of C8 to SAX can be any value within the range of 1:3 to 3:1, such as 1:3, 1.5:2.5, 2:2, 2.5:1.5, or 3:1, but is not limited to the values ​​listed above. Other values ​​not listed within the above range are also applicable.

[0011] The present invention uses acetone to extract short-chain fatty acids, and then purifies them through a solid-phase extraction column with specific fillers, so as to obtain a sample solution with high purity, few impurities and good separation, so as to facilitate subsequent gas chromatography detection and ultimately quickly obtain the accurate content of short-chain fatty acids.

[0012] In some embodiments of the present invention, the amount of the intestinal content or feces sample is 50-150 mg, such as 50 mg, 80 mg, or 100 mg, etc. The amount of acetone is 1 mL.

[0013] In some embodiments of the present invention, the extraction includes: adding 50-150 mg of intestinal contents or fecal sample to a centrifuge tube, adding 300-400 μL of acetone and grinding, then adding 600-700 μL of acetone to vortex the sample evenly, and then performing high-speed centrifugation after vortexing, centrifuging at 10000g-12000g at 4°C for 10-15 minutes.

[0014] Preferably, the intestinal contents or fecal samples are stored at -80°C before processing to avoid volatilization and loss of short-chain fatty acids.

[0015] Preferably, each sample is independently ground with an electric grinding rod, and the used grinding rod is soaked in 75% ethanol, washed and sterilized in an autoclave after the test, and stored for future use after drying.

[0016] In some embodiments of the present invention, the separation and purification includes: first activating the solid phase extraction column with acetone and blowing off the residual acetone, then adding the short-chain fatty acid acetone extract to the solid phase extraction column, and the outflow is the short-chain fatty acid sample solution.

[0017] In some embodiments of the present invention, the method further comprises: using gas chromatography to determine the content of short-chain fatty acids in the sample solution obtained by the sample pretreatment.

[0018] In some embodiments of the present invention, the gas chromatography method adopts a temperature program for determination, and the temperature program is as follows: the initial temperature of the chromatographic column is 50°C, maintained for 1 minute, heated to 120°C at 15°C / min, maintained for 0 minutes, then heated to 200°C at 6°C / min, maintained for 0 minutes, and run for a total of 19 minutes; then heated to 235°C and run for 3 minutes.

[0019] In some embodiments of the present invention, the detection conditions of the gas chromatography are as follows: the chromatographic column is Agilent DB-FFAP (30m, 0.25mm×0.25μm), N2 is used as the carrier gas, the flow rate is 1mL / min, the pressure is 11.258psi, the inlet temperature is 280°C, the FID detector temperature is 250°C, the H2 flow rate is 40mL / min, the air flow rate is 300mL / min, and the N2 tail blow flow rate is 25mL / min; the injection volume is 1μL, the cleaning solvent A is water, and the cleaning solvent B is acetone.

[0020] Preferably, the gas chromatograph inlet liner is ultra-inert, split, low-pressure, and with glass wool (Agilent, 5190-3165). After about 50 samples are injected, it is necessary to check whether the liner is dirty, which can easily cause stray peaks. At this time, the dirty glass wool can be gently removed with pointed tweezers, and the liner can continue to be used.

[0021] Preferably, after about 50 samples are injected, the septum of the gas chromatograph inlet needs to be replaced with a new one in time to avoid instrument leakage errors and contamination of the liner.

[0022] Preferably, a high-temperature cleaning method for the chromatographic column is set up after about 50 samples are injected, and 2 injections of acetone blank are injected to avoid residual peaks interfering with the results. The high-temperature cleaning chromatography method was as follows: the inlet heater of the Agilent 7820A gas chromatograph was set at 280°C, a pressure of 11.258 psi, and a split ratio of 10:1. The chromatographic column was an Agilent DB-FFAP (30 m, 0.25 mm × 0.25 μm) column. N2 was used as the carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi. The column was operated at 50°C for 1 min, then the temperature was increased at 5°C / min to 150°C and held for 10 min, and then the temperature was increased at 5°C / min to 240°C and held for 10 min, for a total of 59 min. The FID detector was heated at 250°C, with an H2 flow rate of 40 mL / min, an air flow rate of 300 mL / min, and a tail gas flow rate (N2) of 25 mL / min. The GC autosampler had an injection volume of 1 μL. The wash solvent A was water, and the wash solvent B was acetone. Before injection, A and B were washed twice to the maximum volume, and the sample was washed twice with 4 μL of water. The sample was aspirated three times.

[0023] The short-chain fatty acids of the present invention include acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid and hexanoic acid.

[0024] The present invention also provides application of the method for determining the content of short-chain fatty acids in intestinal contents or feces in preparing a short-chain fatty acid content detection kit.

[0025] The present invention provides a method for determining the content of short-chain fatty acids in intestinal contents or feces. The short-chain fatty acids are extracted with acetone and then purified by a solid-phase extraction column containing specific fillers, thereby increasing the vaporization time of the sample at the inlet of a gas chromatograph. The method is simple, rapid, and efficient and can accurately determine the content of short-chain fatty acids in intestinal contents or feces. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a gas chromatography detection diagram of a mixed standard of short-chain fatty acids at a concentration of L3;

[0027] FIG2 is a gas chromatography detection diagram of short-chain fatty acids in mouse cecal contents;

[0028] FIG3 is a gas chromatography detection diagram of short-chain fatty acids in mouse feces;

[0029] FIG4 is a gas chromatography detection chart of short-chain fatty acids in pig cecal contents;

[0030] FIG5 is a gas chromatography detection diagram of short-chain fatty acids in pig feces;

[0031] FIG6 is a gas chromatography detection chart of short-chain fatty acids in the intestinal contents of honey bees. DETAILED DESCRIPTION

[0032] In one aspect, the present invention provides a method for determining the content of short-chain fatty acids in intestinal contents or feces, comprising a sample pretreatment step, wherein acetone is first used as an extraction solvent to extract short-chain fatty acids from intestinal contents or feces samples, and then the extracted short-chain fatty acid acetone extract is separated and purified by a solid phase extraction column, wherein the filler of the solid phase extraction column is C8 and SAX.

[0033] In some embodiments of the present invention, the mass ratio of C8 to SAX is 1:3 to 3:1, and the packing is performed in stages, with the C8 filler on the top and the SAX filler on the bottom.

[0034] In some embodiments of the present invention, the amount of the intestinal content or stool sample is 50-150 mg, and the amount of acetone is 1 mL.

[0035] In some embodiments of the present invention, the extraction includes: adding 50-150 mg of intestinal contents or fecal sample to a centrifuge tube, adding 300 μL-400 μL of acetone and grinding, then adding 600 μL-700 μL of acetone to vortex the sample evenly, and then performing high-speed centrifugation after vortexing, centrifuging at 10000g-12000g at 4°C for 10-15 minutes.

[0036] In some embodiments of the present invention, the separation and purification includes: first activating the solid phase extraction column with acetone, blowing off the residual acetone, and then adding the short-chain fatty acid acetone extract to the solid phase extraction column, and the outflow is the short-chain fatty acid sample solution.

[0037] In some embodiments of the present invention, the method further comprises: using gas chromatography to determine the content of short-chain fatty acids in the sample solution obtained by the sample pretreatment.

[0038] In some embodiments of the present invention, the gas chromatography method adopts a temperature program for determination, and the temperature program is as follows: the initial temperature of the chromatographic column is 50°C, maintained for 1 minute, heated to 120°C at 15°C / min, maintained for 0 minutes, then heated to 200°C at 6°C / min, maintained for 0 minutes, and run for a total of 19 minutes; then heated to 235°C and run for 3 minutes.

[0039] In some embodiments of the present invention, the detection conditions of the gas chromatography are as follows: the chromatographic column is Agilent DB-FFAP, N2 is used as the carrier gas, the flow rate is 1 mL / min, the pressure is 11.258 psi, the inlet temperature is 280°C, the FID detector temperature is 250°C, the H2 flow rate is 40 mL / min, the air flow rate is 300 mL / min, and the N2 tail blow flow rate is 25 mL / min; the injection volume is 1 μL, the cleaning solvent A is water, and the cleaning solvent B is acetone.

[0040] In some embodiments of the present invention, the short-chain fatty acids include acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid, and hexanoic acid.

[0041] The present invention also provides an application of the method for determining the content of short-chain fatty acids in intestinal contents or feces in the preparation of a short-chain fatty acid content detection kit. On the other hand, the present invention provides a method for determining the content of short-chain fatty acids in intestinal contents or feces, comprising a sample pretreatment step, wherein acetone is first used as an extraction solvent to extract short-chain fatty acids from intestinal contents or feces samples, and then the extracted short-chain fatty acid acetone extract is separated and purified by a solid phase extraction column, wherein the filler of the solid phase extraction column uses C8 and SAX, the mass ratio of C8 to SAX is 1:3 to 3:1, and the filling is segmented, with the C8 filler on the top and the SAX filler on the bottom;

[0042] The extraction operation is as follows: 50 mg to 150 mg of intestinal contents or fecal sample is added to a centrifuge tube, 300 μL to 400 μL of acetone is added and the sample is ground, 600 μL to 700 μL of acetone is added and the sample is vortexed evenly, and then high-speed centrifugation is performed at 10000 g to 12000 g at 4° C. for 10 min to 15 min;

[0043] The separation and purification operation is as follows: first, activate the solid phase extraction column with acetone, blow off the residual acetone, then add the short-chain fatty acid acetone extract into the solid phase extraction column, and the outflow is the short-chain fatty acid sample solution;

[0044] The method further comprises: using gas chromatography to determine the content of short-chain fatty acids in the sample solution obtained by the sample pretreatment, wherein the chromatographic column used in the gas chromatography is Agilent DB-FFAP and the detector is FID;

[0045] The short-chain fatty acids are acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid and hexanoic acid.

[0046] In some embodiments of the present invention, the gas chromatography method adopts a temperature program for determination, and the temperature program is as follows: the initial temperature of the chromatographic column is 50°C, maintained for 1 minute, heated to 120°C at 15°C / min, maintained for 0 minutes, then heated to 200°C at 6°C / min, maintained for 0 minutes, and run for a total of 19 minutes; then heated to 235°C and run for 3 minutes.

[0047] In some embodiments of the present invention, the detection conditions of the gas chromatography are as follows: the chromatographic column is Agilent DB-FFAP, 30m, 0.25mm×0.25μm, N2 is used as the carrier gas, the flow rate is 1mL / min, the pressure is 11.258psi, the inlet temperature is 280°C, the FID detector temperature is 250°C, the H2 flow rate is 40mL / min, the air flow rate is 00mL / min, and the N2 tail blow flow rate is 25mL / min; the injection volume is 1μL, the cleaning solvent A is water, and the cleaning solvent B is acetone.

[0048] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0049] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0050] Method validation

[0051] 1. Determination of standard curve, detection limit and quantification limit

[0052] Preparation of short-chain fatty acid standards: Weigh 60 μL of each of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid into a 10-mL volumetric flask and record the mass of each acid. Then, add acetone to the flask to make the volume 10 mL. Calculate the molar concentration of each acid, as shown in Table 1. This short-chain fatty acid stock solution can be stored at -20°C for one month.

[0053] Table 1 Preparation of short-chain fatty acid mother liquor

[0054] L1: 990 μL acetone + 10 μL SCFA stock solution (Table 1), vortex to mix evenly;

[0055] L2: 500 μL acetone + 500 μL L1, vortex to mix evenly;

[0056] L3: 990 μL acetone + 10 μL L1, vortex to mix evenly;

[0057] L4: 990 μL acetone + 10 μL L2, vortex to mix evenly;

[0058] L5: 990 μL acetone + 10 μL L3, vortex to mix evenly;

[0059] L6: 990 μL acetone + 10 μL L5, vortex to mix evenly.

[0060] Table 2 Preparation of gradient short-chain fatty acid standards

[0061] Each concentration of the standard analyte was measured in triplicate, and the limit of detection (LOD) for each short-chain fatty acid was set at 3 times the baseline noise; the limit of quantification (LOQ) for each short-chain fatty acid was set at 10 times the baseline noise. (See Table 3.)

[0062] Table 3 Response time, detection limit, quantification limit and standard curve of short-chain fatty acids in standard solution

[0063] 2. Repeatability testing

[0064] For the L3 concentration standard, the test was repeated three times in parallel, and the precision (RSD, %) was calculated to observe repeatability. The L3 concentration standard was injected in the morning, afternoon, and evening, and the precision (RSD, %) was calculated to observe intraday stability. The L3 concentration standard was injected on days 1, 2, and 3, and the precision (RSD, %) was calculated to observe interday stability. The results are shown in Table 4.

[0065] Table 4 Repeatability test results of L3 concentration standard

[0066] As can be seen in Table 4, after three injections of the short-chain fatty acid standard at a concentration of L3, the RSD of the peak area was between 0.36% and 0.85%, indicating good repeatability and precision of the instrument method. After measuring the short-chain fatty acid standard at a concentration of L3 in the morning, afternoon, and evening, the RSD of the peak area was between 1.04% and 1.30%, indicating good intraday stability of this chromatographic method. After measuring the short-chain fatty acid standard at a concentration of L3 on the first, second, and third days, the RSD of the peak area was between 0.88% and 1.23%, indicating good interday stability of this chromatographic method.

[0067] 3. Recovery determination

[0068] (1) Obtaining matrix samples:

[0069] The pig feces were dried and ground at high temperature, then crudely extracted with acetone, centrifuged and the supernatant was discarded to remove short-chain fatty acids in the feces, and the precipitate was dried at high temperature to obtain a fecal matrix sample.

[0070] (2) Dissolve the matrix sample and standard:

[0071] H1: 50 mg fecal matrix sample + 990 μL acetone + 10 μL SCFA stock solution (Table 1), vortex mix evenly;

[0072] H2: 50 mg fecal matrix sample + 500 μL acetone + 500 μL L1, vortex to mix evenly;

[0073] H3: 50 mg fecal matrix sample + 990 μL acetone + 10 μL L1, vortex mix thoroughly;

[0074] H4: 50 mg fecal matrix sample + 990 μL acetone + 10 μL L2, vortex mix thoroughly;

[0075] H5: 50 mg fecal matrix sample + 990 μL acetone + 10 μL L3, vortex mix thoroughly;

[0076] The samples were placed symmetrically in a high-speed centrifuge and centrifuged at 12000 g for 10 min at 4°C.

[0077] (3) Matrix spiked sample purification:

[0078] A C8+SAX solid-phase extraction (SPE) cartridge (C8:SAX mass ratio = 1:1, with C8 on top and SAX on the bottom) was activated with 1 mL of acetone, and any residual acetone was blown off. A clean 10 mL centrifuge tube was placed under the SPE cartridge, and the acetone extract of the matrix-spiked sample was added to the SPE cartridge. Impurities were adsorbed onto the SPE cartridge's filler, and the sample that flowed down was the short-chain fatty acids.

[0079] (4) Matrix spiked sample testing:

[0080] Chromatographic analysis was performed using an Agilent 7820A gas chromatograph and an Agilent DB-FFAP gas chromatography column. Gas phase analysis conditions were as follows: inlet heater at 280°C, pressure at 11.258 psi, split ratio 10:1; Agilent DB-FFAP column (30 m, 0.25 mm × 0.25 μm); N2 carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi; column oven at 50°C, hold for 1 min, ramp at 15°C / min to 120°C, hold for 0 min, then ramp at 6°C / min to 200°C, hold for 0 min, for a total of 19 min; post-run at 235°C for 3 min; FID detector heating at 250°C, H2 flow rate of 40 mL / min, air flow rate of 300 mL / min, and makeup flow rate (N2) of 25 mL / min; GC autosampler, injection volume 1 μL; wash solvent A was water, and wash solvent B was acetone. Before injection, A and B were washed twice to the maximum volume, sample washes were performed twice with 4 μL each, and the sample was aspirated three times.

[0081] (5) Recovery rate calculation formula:

[0082] Recovery rate = (concentration of spiked matrix - concentration of matrix) / (concentration of spiked standard) × 100%. The results are shown in Table 5.

[0083] Table 5

[0084] The results in Table 5 show that the recoveries of short-chain fatty acids at different concentrations in treated pig feces ranged from 81.94% to 137.83%, indicating that this method has a good recovery rate and the results are accurate and reliable.

[0085] Example 1

[0086] This embodiment provides a method for determining the content of short-chain fatty acids in mouse feces and pig feces, respectively, comprising the following steps:

[0087] 1) Sample Dissolution: Weigh 50 mg of feces into a 1.5 mL centrifuge tube. Add 300 μL of acetone and thoroughly grind the fecal residue with an electric grinding rod. Add 700 μL of acetone and vortex the sample until evenly distributed. Centrifuge the sample symmetrically at 12,000 g at 4°C for 10 min.

[0088] 2) Sample purification: Activate a C8+SAX solid-phase extraction cartridge (C8:SAX mass ratio = 1:1, with C8 on top and SAX on the bottom) with 1 mL of acetone and blow off any remaining acetone. Place a clean 10 mL centrifuge tube under the solid-phase extraction cartridge and add the acetone extract of the sample to the cartridge. The impurities are adsorbed into the packing of the cartridge, and the sample that flows down is the short-chain fatty acids.

[0089] 3) Sample analysis: Chromatographic analysis was performed using an Agilent 7820A gas chromatograph and an Agilent DB-FFAP gas chromatograph column. The gas phase analysis conditions were as follows: inlet heater at 280°C, pressure at 11.258 psi, split ratio 10:1; chromatographic column: Agilent DB-FFAP (30 m, 0.25 mm × 0.25 μm); N2 as carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi; column oven: 50°C, hold for 1 min, ramp to 120°C at 15°C / min, hold for 0 min, then ramp to 200°C at 6°C / min, hold for 0 min, for a total of 19 min; post-run at 235°C for 3 min; FID detector: heating at 250°C, H2 flow rate 40 mL / min, air flow rate 300 mL / min, and tail gas flow rate (N2) 25 mL / min; GC autosampler: injection volume 1 μL; wash solvent A: water, wash solvent B: acetone; AB washes were performed twice before injection, the sample was washed twice with a maximum volume of 4 μL, and the sample was aspirated three times.

[0090] Example 2

[0091] This embodiment provides a method for determining the content of short-chain fatty acids in mouse cecal contents, pig cecal contents, and bee intestinal contents, respectively, comprising the following steps:

[0092] 1) Sample acquisition: Use clean scissors and forceps to dissect the intestines of mice, pigs, or bees on the operating table. The removed intestinal contents are quickly placed in liquid nitrogen and finally transferred to -80°C for storage.

[0093] 2) Sample Dissolution: Weigh 50 mg of intestinal contents into a 1.5 mL centrifuge tube. Add 300 μL of acetone and thoroughly grind the fecal residue with an electric grinding rod. Add 700 μL of acetone and vortex the sample until evenly distributed. Place the sample symmetrically in a high-speed centrifuge and centrifuge at 12,000 g for 10 min at 4°C.

[0094] 3) Sample purification: Activate a C8+SAX solid-phase extraction cartridge (C8:SAX mass ratio = 1:1, with C8 on top and SAX on the bottom) with 1 mL of acetone and blow off any residual acetone. Place a clean 10 mL centrifuge tube under the solid-phase extraction cartridge and add the acetone extract of the sample to the solid-phase extraction cartridge. The impurities are adsorbed into the packing of the solid-phase extraction cartridge, and the sample that flows down is the short-chain fatty acids.

[0095] 4) Sample analysis: Chromatographic analysis was performed using an Agilent 7820A gas chromatograph and an Agilent DB-FFAP gas chromatograph column. The gas phase analysis conditions were as follows: inlet heater at 280°C, pressure at 11.258 psi, split ratio 10:1; chromatographic column: Agilent DB-FFAP (30 m, 0.25 mm × 0.25 μm); N2 as carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi; column oven: 50°C, hold for 1 min, ramp to 120°C at 15°C / min, hold for 0 min, then ramp to 200°C at 6°C / min, hold for 0 min, for a total of 19 min; post-run at 235°C for 3 min; FID detector: heating at 250°C, H2 flow rate 40 mL / min, air flow rate 300 mL / min, and tail gas flow rate (N2) 25 mL / min; GC autosampler: injection volume 1 μL; wash solvent A: water, wash solvent B: acetone; AB washes were performed twice before injection, the sample was washed twice with a maximum volume of 4 μL, and the sample was aspirated three times.

[0096] The results of detecting the content of short-chain fatty acids in mouse intestinal contents, mouse feces, pig intestinal contents, pig feces and honeybee intestinal contents using the method of the present invention are shown in Table 6.

[0097] Table 6

[0098] As can be seen from Table 6, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid can be detected in intestinal contents and feces, and caproic acid is partially detected (not all intestinal contents and feces contain caproic acid).

[0099] In addition, Figure 1 is a gas chromatography detection chart of a mixed standard of short-chain fatty acids at a concentration of L3; Figures 2-6 are gas chromatography detection spectra of mouse intestinal contents, mouse feces, pig intestinal contents, pig feces, and honeybee intestinal contents, respectively. As can be seen from the detection results of the short-chain fatty acid standards in Figure 1, the method of the present invention can ideally separate acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid. As can be seen from Figures 2-6, the method of the present invention can effectively detect acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid in intestinal contents or feces with minimal impurities.

[0100] Comparative Example 1

[0101] This comparative example uses acetone, methanol, and acetonitrile to extract short-chain fatty acids from a pig feces sample matrix spiked with a mixed standard, including the following steps:

[0102] (1) Dissolve the matrix sample and standard:

[0103] Acetone extraction solution: 50 mg fecal matrix sample + 990 μL acetone + 10 μL L1, vortex to mix evenly;

[0104] Methanol extraction solution: 50 mg fecal matrix sample + 990 μL methanol + 10 μL L1, vortex to mix evenly;

[0105] Acetonitrile extraction solution: 50 mg fecal matrix sample + 990 μL acetonitrile + 10 μL L1, vortex to mix evenly;

[0106] The concentrations of standard additions were: acetic acid 107.4069 μM, propionic acid 80.3186 μM, isobutyric acid 63.1065 μM, butyric acid 64.9225 μM, isovaleric acid 53.5581 μM, valeric acid 54.3414 μM, and hexanoic acid 45.8858 μM.

[0107] Each solvent was treated in parallel for 6 times, and the samples were placed symmetrically in a high-speed centrifuge and centrifuged at 12000g for 10 min at 4°C.

[0108] (2) Matrix spiked sample purification:

[0109] A C8+SAX solid-phase extraction cartridge (C8:SAX mass ratio = 1:1, with C8 on top and SAX on the bottom) was activated with 1 mL of acetone, methanol, and acetonitrile, respectively, and the residual solvent was blown dry. A clean 10 mL centrifuge tube was placed under the solid-phase extraction cartridge, and the supernatant extract of the matrix-spiked sample was added to the cleanup cartridge. The impurities were adsorbed into the packing of the solid-phase extraction cartridge, and the sample flowing down was the short-chain fatty acids.

[0110] (3) Matrix spiked sample testing:

[0111] Chromatographic analysis was performed using an Agilent 7820A gas chromatograph and an Agilent DB-FFAP gas chromatography column. The gas phase analysis conditions were as follows: inlet heater at 280°C, pressure at 11.258 psi, split ratio 10:1; chromatographic column: Agilent DB-FFAP (30 m, 0.25 mm × 0.25 μm); N2 as carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi; column oven: 50°C, hold for 1 min, ramp to 120°C at 15°C / min, hold for 0 min, then ramp to 200°C at 6°C / min, hold for 0 min, for a total of 19 min; post-run at 235°C for 3 min; FID detector: heating at 250°C, H2 flow rate 40 mL / min, air flow rate 300 mL / min, and tail gas flow rate (N2) 25 mL / min; GC autosampler: injection volume 1 μL; wash solvent A: water, wash solvent B: acetone; AB washes were performed twice before injection, the sample was washed twice with a maximum volume of 4 μL, and the sample was aspirated three times.

[0112] (4) Recovery rate calculation formula:

[0113] Recovery rate = (concentration of spiked matrix - concentration of matrix) / (concentration of spiked standard) × 100%. The results are shown in Table 7.

[0114] Table 7

[0115] The results in Table 7 show that the spiked recovery rates of short-chain fatty acids extracted by acetone solvent in treated pig feces are 83.42%-97.79%, the spiked recovery rates of short-chain fatty acids extracted by methanol solvent are 66.11%-84.09%, and the spiked recovery rates of short-chain fatty acids extracted by acetonitrile solvent are 70.83%-84.42%, indicating that the recovery rate of the acetone extraction method is better.

[0116] Comparative Example 2

[0117] In this comparative example, acetone was used to extract short-chain fatty acids from a pig feces sample matrix supplemented with a mixed standard, and the short-chain fatty acids were purified using C8+SAX, SAX, and CN cyanide materials, respectively.

[0118] The steps include:

[0119] (1) Dissolve the matrix sample and standard:

[0120] Acetone extraction solution: 50 mg fecal matrix sample + 990 μL acetone + 10 μL L1, vortex to mix evenly;

[0121] The concentrations of standard additions were: acetic acid 107.4069 μM, propionic acid 80.3186 μM, isobutyric acid 63.1065 μM, butyric acid 64.9225 μM, isovaleric acid 53.5581 μM, valeric acid 54.3414 μM, and hexanoic acid 45.8858 μM.

[0122] Each purified material was processed in parallel 6 times, and the samples were placed symmetrically in a high-speed centrifuge and centrifuged at 12000g for 10 min at 4°C.

[0123] (2) Matrix spiked sample purification:

[0124] Use 1 mL of acetone to activate C8+SAX (where the mass ratio of C8:SAX is 1:1, and C8 is on top and SAX is on the bottom), SAX, and CN cyanide material solid-phase extraction cartridges, and blow dry the residual solvent; place a clean 10 mL centrifuge tube under the solid-phase extraction cartridge, and add the supernatant extract of the matrix-spiked sample to the solid-phase extraction cartridge. The impurities are adsorbed into the packing of the solid-phase extraction cartridge, and the sample that flows down is the short-chain fatty acids.

[0125] (3) Matrix spiked sample testing:

[0126] Chromatographic analysis was performed using an Agilent 7820A gas chromatograph and an Agilent DB-FFAP gas chromatography column. The gas phase analysis conditions were as follows: inlet heater at 280°C, pressure at 11.258 psi, split ratio 10:1; chromatographic column: Agilent DB-FFAP (30 m, 0.25 mm × 0.25 μm); N2 as carrier gas at a flow rate of 1 mL / min and a pressure of 11.258 psi; column oven: 50°C, hold for 1 min, ramp to 120°C at 15°C / min, hold for 0 min, then ramp to 200°C at 6°C / min, hold for 0 min, for a total of 19 min; post-run at 235°C for 3 min; FID detector: heating at 250°C, H2 flow rate 40 mL / min, air flow rate 300 mL / min, and tail gas flow rate (N2) 25 mL / min; GC autosampler: injection volume 1 μL; wash solvent A: water, wash solvent B: acetone; AB washes were performed twice before injection, the sample was washed twice with a maximum volume of 4 μL, and the sample was aspirated three times.

[0127] (4) Recovery rate calculation formula:

[0128] Recovery rate = (concentration of spiked matrix - concentration of matrix) / (concentration of spiked standard) × 100%. The results are shown in Table 8.

[0129] Table 8

[0130] The results in Table 8 show that the spiked recovery rates of short-chain fatty acids extracted by C8+SAX materials in treated pig feces are 84.62%-95.73%, the spiked recovery rates of short-chain fatty acids extracted by SAX materials are 72.48%-87.84%, and the spiked recovery rates of short-chain fatty acids extracted by CN cyanide materials are 72.75%-79.29%, indicating that the C8+SAX material extraction method has a better recovery rate.

[0131] It should be noted that the endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0132] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" and the like mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for determining the content of short-chain fatty acids in intestinal contents or feces, characterized in that: The method comprises a sample pretreatment step, wherein acetone is first used as an extraction solvent to extract short-chain fatty acids in intestinal contents or fecal samples, and then a short-chain fatty acid acetone extract obtained by separation and purification is obtained by a solid phase extraction column, wherein the fillers of the solid phase extraction column are C8 and SAX, the mass ratio of C8 to SAX is 1:3 to 3:1, and the filling is performed in sections, with C8 filler on the top and SAX filler on the bottom; The extraction operation is as follows: 50 mg to 150 mg of intestinal contents or feces sample is added to a centrifuge tube, 300 μL to 400 μL of acetone is added and then ground, 600 μL to 700 μL of acetone is added and the sample is vortexed evenly, and then high-speed centrifugation is performed after vortexing, and centrifugation is performed at 10000 g to 12000 g at 4° C. for 10 min to 15 min; The separation and purification operation is as follows: first, activating the solid phase extraction column with acetone, blowing off the residual acetone, and then adding the short-chain fatty acid acetone extract into the solid phase extraction column, and the outflow is the short-chain fatty acid sample solution; The method further comprises: using gas chromatography to determine the content of short-chain fatty acids in the sample solution obtained by the sample pretreatment, wherein the chromatographic column used in the gas chromatography is Agilent DB-FFAP and the detector is FID; The short-chain fatty acids are acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid and caproic acid.

2. The method for determining the content of short-chain fatty acids in intestinal contents or feces according to claim 1, characterized in that: The gas chromatography method adopts a temperature program for determination, and the temperature program is as follows: the initial temperature of the chromatographic column is 50°C, maintained for 1 minute, heated to 120°C at 15°C / min, maintained for 0 minutes, then heated to 200°C at 6°C / min, maintained for 0 minutes, and operated for a total of 19 minutes; then heated to 235°C and operated for 3 minutes.

3. The method for determining the content of short-chain fatty acids in intestinal contents or feces according to claim 1 or 2, characterized in that: The detection conditions of the gas chromatography method are as follows: the chromatographic column is Agilent DB-FFAP, 30m, 0.25mm×0.25μm, N2 is used as the carrier gas, the flow rate is 1mL / min, the pressure is 11.258psi, the injection port temperature is 280°C, the FID detector temperature is 250°C, the H2 flow rate is 40mL / min, the air flow rate is 00mL / min, and the N2 tail blow flow rate is 25mL / min; the injection volume is 1μL, the cleaning solvent A is water, and the cleaning solvent B is acetone.

Images