A method for detecting short-chain fatty acids in mouse plasma, feces or tissue samples

Abstract

The present invention discloses a method for analyzing short-chain fatty acids in mouse plasma, feces and tissue samples based on gas chromatography-mass spectrometry for rapid detection, and relates to the application of analytical chemistry in clinical medical research. The specific steps are: taking a fresh sample, adding acetonitrile to extract short-chain fatty acids, and analyzing the sample by gas chromatography-mass spectrometry after centrifugation. The detection method of the present invention is simple, rapid, and accurate in determining the content of short-chain fatty acids in biological samples, and eliminates the interference of other factors in the experiment on the quantitative results.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and specifically relates to a method for analyzing short-chain fatty acids in mouse plasma, feces and tissue samples based on gas chromatography-mass spectrometry. Background Art

[0002] Short-chain fatty acids refer to organic fatty acids with less than 6 carbon atoms, including Acetic acid, Propionicacid, Butyric Acid, Isobutyric Acid, Valeric Acid, Isovaleric Acid, Hexanoic Acid, 3-Methylvaleric acid, and 4-Methylvaleric acid. They are produced by intestinal bacteria that degrade carbohydrates and proteins, 95% of which are absorbed by colon cells and provide energy in the form of ATP or enter the systemic circulation through the basolateral membrane, and the other 5% are excreted from the body with feces. It is reported that it can regulate energy balance, protect the immune system, resist inflammatory responses, etc., and plays an important role in the metabolism of intestinal flora. A rapid analysis method for the detection of short-chain fatty acids in mouse plasma, feces and tissue samples was established to provide assistance for the study of intestinal flora metabolism related to short-chain fatty acids.

[0003] Nowadays, there are many reports on the analysis methods of short-chain fatty acids in biological samples. Gas chromatography-mass spectrometry is the most important and most commonly used analytical technique for detecting volatile organic compounds, so short-chain fatty acid analysis mostly uses gas chromatography-mass spectrometry. However, the pretreatment requires extraction through a complex solvent system (such as sodium chloride solution and ether), followed by solid phase extraction column enrichment, or derivatization with MTBSTFA reagent. The pretreatment steps are cumbersome and may introduce contamination or cause short-chain fatty acid loss. Liquid chromatography-mass spectrometry not only has the advantage of high resolution, but also has a wide linear range, which can simultaneously ensure qualitative and quantitative accuracy. It has been reported that the use of 3-nitrophenylhydrazine derivatization combined with liquid chromatography-mass spectrometry can detect short-chain fatty acids in feces. We investigated different brands of ultrapure water and methanol blanks, and all had high short-chain fatty acid detection. If this method is used, it is only suitable for detecting fecal samples with high short-chain fatty acid content, and it will cause great contamination to samples with low content. Therefore, it is necessary to establish a rapid detection method for short-chain fatty acid analysis in mouse plasma, feces and tissue samples. Summary of the invention

[0004] In view of the fact that the existing detection methods have complicated steps and are prone to contamination or loss of short-chain fatty acid detection results, the present invention proposes the use of acetonitrile extraction combined with gas chromatography-mass spectrometry technology to more accurately detect short-chain fatty acids in mouse plasma, feces and tissue samples. This method uses gas chromatography separation combined with mass spectrometry selected ion monitoring mode to simultaneously detect 9 types of short-chain fatty acids. Among them, except for 7 types that can be detected in spleen tissue samples, 9 types can be detected in other types of samples. The pre-treatment method of this method is simple, eliminating the loss caused by the sample processing process and the interference with the measurement, has good repeatability, and can accurately quantify short-chain fatty acids in multiple types of biological samples.

[0005] The technical solution of the present invention is as follows:

[0006] (1) Take 25 μL of blood sample, add 100 μL of acetonitrile containing internal standard extractant (internal standard 2-Ethylbutyric acid 10 μmol / L), vortex for 30 seconds, centrifuge (14000 rpm, 10 min, 4°C), take the supernatant and put it into the injection bottle for gas chromatography-mass spectrometry analysis;

[0007] (2) Weigh about 20 mg of fecal sample, put it into grinding balls, add 200 μL of acetonitrile containing internal standard extractant (internal standard 2-Ethylbutyric acid 10 μmol / L), bead beat (25 Hz, 1 min × 2 times), centrifuge (14000 rpm, 10 min, 4°C), take the supernatant and put it into the injection bottle for gas chromatography-mass spectrometry analysis;

[0008] (3) Weigh about 100 mg of mouse visceral fat sample, put it into grinding balls, add 200 μL of acetonitrile solution containing internal standard (internal standard 2-Ethylbutyric acid 10 μmol / L), bead beat (25 Hz, 1 min × 2 times), centrifuge (14000 rpm, 10 min, 4°C), take the supernatant and put it into the injection bottle for gas chromatography-mass spectrometry analysis;

[0009] (4) About 35 mg of mouse spleen sample was weighed and placed in a grinding ball. 200 μL of acetonitrile solution containing internal standard (internal standard 2-Ethylbutyric acid 10 μmol / L) was added. The sample was bead-beaten (25 Hz, 1 min × 2 times) and centrifuged (14000 rpm, 10 min, 4°C). The supernatant was collected and placed in a sample injection bottle for analysis by gas chromatography-mass spectrometry.

[0010] Gas chromatography conditions: The capillary column used was DB-FFAP (30m×0.25mm×0.25μm), the starting temperature of the program temperature was 50°C, maintained for 1 min, increased to 180°C at a rate of 10°C / min, and then continued to increase to 250°C at a rate of 30°C / min and maintained for 2 min. The linear velocity was 40cm / sec (He, 99.9995%), the injection port temperature was 270°C, the injection volume was 1μL, and no splitting was performed.

[0011] Mass spectrometer conditions: ion source temperature 230°C, quadrupole temperature 150°C, transfer line temperature 270°C, scanning mode SIM (Acetic acid 43, Propionic acid 74, Butyric Acid 60, Isobutyric acid 43, Valeric Acid 60, Isovaleric acid 60, Hexanoic Acid 60, 3-Methylvaleric acid 60, 4-Methylvaleric acid 60, 2-Ethylbutyric acid 73).

[0012] This method uses the extraction technology of analytical chemistry and combines it with the selected ion monitoring technology of gas chromatography-mass spectrometry to detect and quantify short-chain fatty acids in mouse plasma, feces and tissue samples. The detection limit of the nine short-chain fatty acids is 0.2-7nmol / L, the linear range can reach 3-4 orders of magnitude, and the linear correlation coefficient R 2 >0.999. Compared with the existing methods reported in the literature, this method is fast and simple, and can quantitatively detect short-chain fatty acids in mouse plasma, feces and tissue samples, providing assistance for research related to intestinal flora metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Total ion current of short-chain fatty acid standard sample;

[0014] Figure 2 Total ion current of short-chain fatty acids in plasma samples;

[0015] Figure 3 Total ion chromatogram of short-chain fatty acids in stool samples;

[0016] Figure 4 Total ion current of short-chain fatty acids in visceral fat samples;

[0017] Figure 5 Total ion current of short-chain fatty acids in spleen samples;

[0018] Table 1 Detection limit, quantification limit, linear range and correlation coefficient of the invented method. DETAILED DESCRIPTION

[0019] Example 1 Detection of short-chain fatty acids in mouse plasma samples

[0020] 25 μL of plasma samples were taken from two mice respectively, and 100 μL of acetonitrile containing internal standard extractant and 10 μmol / L of internal standard 2-Ethylbutyric acid was added to each sample. The samples were vortexed for 30 s, centrifuged at 14000 rpm for 10 min, and the temperature was controlled at 4°C. The supernatant was taken and placed in a sample injection bottle for instrument analysis.

[0021] The gas chromatograph used a capillary column DB-FFAP, the column parameters were 30m×0.25mm×0.25μm, the program temperature was raised, the starting temperature was 50℃, maintained for 1min, raised to 180℃ at a rate of 10℃ / min, and then continued to rise to 250℃ at a rate of 30℃ / min and maintained for 2min. The carrier gas was helium with a purity of 99.9995% and a linear speed of 40cm / sec. The injection port temperature was 270℃, the injection volume was 1μL, and there was no splitting. The mass spectrometer used the selected ion monitoring mode (the characteristic ions used for various short-chain fatty acids are shown in Table 1), the ion source temperature was 230℃, the quadrupole temperature was 150℃, and the transmission line temperature was 270℃.

[0022] After data processing, the ratio of the peak area of ​​each short-chain fatty acid in serum to the peak area of ​​the internal standard was obtained. The relative content of each short-chain fatty acid is as follows: Acetic acid 6.9 / 7.02, Propionic acid 0.17 / 0.18, Isobutyricacid 0.04 / 0.04, Butyric Acid 0.13 / 0.07, Isovaleric acid 0.07 / 0.06, Valeric Acid0.02 / 0.02, 3-Methylvaleric acid 0.01 / 0.01, 4-Methylvaleric acid 0.0046 / 0.0046, Hexanoic Acid 0.06 / 0.05.

[0023] Substituting the standard curve of Example 5, the above analysis method was used to detect that the content of Acetic acid in mouse plasma was the highest, 1825.82 / 1857.21μmol / L. The second was Propionic acid, 29.15 / 32.5μmol / L. Isobutyric acid 2.97 / 2.54μmol / L, Butyric Acid 6.71 / 3.79μmol / L, Isovaleric acid 2.77 / 2.53μmol / L, Valeric Acid 0.99 / 0.92μmol / L, 3-Methylvaleric acid 0.39 / 0.34μmol / L, 4-Methylvaleric acid 1.09 / 1.09μmol / L, Hexanoic Acid 2.57 / 2.2μmol / L. (See Figure 2 ).

[0024] Example 2 Detection of short-chain fatty acids in mouse feces samples

[0025] Weigh 40 mg of fecal samples from two mice respectively, put them into grinding balls, add 200 μL of acetonitrile containing internal standard extractant and 10 μmol / L of internal standard 2-Ethylbutyric acid, grind by bead beating at a frequency of 25 Hz for 1 min, bead beat twice, centrifuge at 14000 rpm for 10 min, control the temperature at 4°C, take the supernatant and put it into a sampling bottle for gas chromatography-mass spectrometry analysis.

[0026] After data processing, the relative contents of short-chain fatty acids in feces were: Acetic acid 120.55 / 115.98, Propionic acid 50.40 / 48.51, Isobutyric acid 5.59 / 5.28, Butyric Acid84.48 / 82.58, Isovaleric acid 3.92 / 3.77, Valeric Acid 10.40 / 10.42, 3-Methylvaleric acid 0.11 / 0.096, 4-Methylvaleric acid 0.064 / 0.067, Hexanoic Acid0.49 / 0.45.

[0027] Substituting the standard curve of Example 5, the test results show that the content of Acetic acid in mouse feces is the highest, 39885.44 / 38374.08μmol / g. The second is Propionic acid, 11082.72 / 10666.99μmol / g. Isobutyric acid 505.55 / 478.08μmol / g, Butyric Acid 5586.15 / 5460.62μmol / g, Isovaleric acid 195.84 / 188.58μmol / g, Valeric Acid 531.95 / 532.74μmol / g, 3-Methylvaleric acid 5.00 / 4.44μmol / g, 4-Methylvaleric acid 18.89 / 19.59μmol / g, Hexanoic Acid 26.67 / 24.35μmol / g. (See Figure 3 ).

[0028] Example 3 Detection of short-chain fatty acids in mouse visceral fat samples

[0029] 100 mg of visceral fat samples from two mice were weighed respectively, put into grinding balls, and 200 μL of internal standard acetonitrile and internal standard 2-Ethylbutyric acid 10 μmol / L were added to each sample. The samples were ground by bead beating at a frequency of 25 Hz for 1 min, and the beads were beaten twice. The samples were centrifuged at 14000 rpm for 10 min and the temperature was controlled at 4°C. The supernatant was taken and placed in a sample injection bottle for analysis by gas chromatography-mass spectrometry. The relative results are Aceticacid 2.48 / 2.20, Propionic acid 0.045 / 0.046, Isobutyric acid 0.064 / 0.064, ButyricAcid0.39 / 0.22, Isovaleric acid 0.08 / 0.08, Valeric Acid 0.02 / 0.02, 3-Methylvaleric acid 0.002 / 0.001, 4-Methylvaleric acid 0.003 / 0.002, Hexanoic acid 0.17 / 0.12.

[0030] Substituting the standard curve of Example 5, the test results show that the content of Acetic acid is 264.25 / 305.00nmol / g, the content of Propionic acid is 3.18 / 4.27nmol / g, Isobutyric acid is 1.85 / 2.43nmol / g, Butyric Acid is 8.34 / 5.99nmol / g, Isovaleric acid is 1.22 / 1.65nmol / g, Valeric Acid is 0.30 / 0.36nmol / g, 3-Methylvaleric acid is 0.03 / 0.03nmol / g, 4-Methylvaleric acid is 0.31 / 0.20nmol / g, Hexanoic Acid is 2.91 / 2.68nmol / g (see Figure 4 ).

[0031] Example 4 Detection of short-chain fatty acids in mouse spleen samples

[0032] Weigh about 35 mg of spleen samples from two mice respectively, put them into grinding balls, add 200 μL of internal standard acetonitrile and internal standard 2-Ethylbutyric acid 10 μmol / L, grind by bead beating at a frequency of 25 Hz for 1 min, bead beating twice, centrifuge at 14000 rpm for 10 min, control the temperature at 4°C, take the supernatant and put it into a sampling bottle for gas chromatography-mass spectrometry analysis. The relative results are Acetic acid 4.8 / 4.2, Propionic acid 0.11 / 0.08, Isobutyric acid 0.05 / 0.03, Butyric Acid 0.14 / 0.07, Isovaleric acid 0.06 / 0.05, Valeric Acid 0.06 / 0.04, 3-Methylvaleric acid0.03 / 0.02, 4-Methylvaleric acid 0.004 / 0.016, Hexanoic acid 0.14 / 0.11.

[0033] Substituting the standard curve of Example 5, the test results showed that the content of Acetic acid in the mouse spleen tissue sample was the highest, which was 1478.34 / 1554.92nmol / g, Propionic acid 22.36 / 20.00nmol / g, Isobutyricacid 4.30 / 3.24nmol / g, Butyric Acid 8.86 / 5.15nmol / g, Isovaleric acid 2.92 / 2.63nmol / g, Valeric Acid 2.70 / 2.43nmol / g, 3-Methylvaleric acid 1.33 / 0.80nmol / g, 4-Methylvaleric acid 1.22 / 5.25nmol / g, Hexanoic Acid 7.00 / 6.97nmol / g (see Figure 5 ).

[0034] Example 5 Evaluation of analytical methods

[0035] Accurately weigh and prepare 10 mg / mL of short-chain fatty acid mother solution with acetonitrile. Pipette each short-chain fatty acid mother solution to prepare 400 μg / mL mixed standard, and dilute step by step to concentration points of 200 μg / mL, 40 μg / mL, 20 μg / mL, 4 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.2 μg / mL, 0.04 μg / mL, 0.02 μg / mL. Pipette 25 μL of each concentration point, add 200 μL of acetonitrile extractant containing 10 μmol / L 2-Ethylbutyric acid as internal standard, vortex mix, take the supernatant and put it into the injection bottle for gas chromatography-mass spectrometry analysis. The typical standard sample spectrum obtained by detection is attached. Figure 1 The linear range was selected according to the actual sample content and instrument sensitivity. The quantitative limit, detection limit, and linear correlation coefficient of the method are shown in Table 1. The detection limit is 0.2-7nmol / mL, the quantitative limit is 0.5-30nmol / mL, the linear range can reach 3-4 orders of magnitude, and the linear correlation coefficient R 2 >0.999, this method can accurately quantify mouse plasma, feces and tissue samples.

[0036] Table 1

[0037]

Claims

1. A method for detecting short-chain fatty acids in mouse plasma, feces or tissue samples, characterized in that: The process steps include: (1) Take 20-50 μL of blood sample, add 80-200 μL of acetonitrile containing internal standard extractant, vortex, centrifuge, take the supernatant and put it into the injection bottle for gas chromatography-mass spectrometry analysis. After data processing, the ratio of the peak area of ​​each short-chain fatty acid in the serum to the peak area of ​​the internal standard is obtained, that is, the relative content result; (2) Weigh 10-50 mg of fecal sample, put it into grinding balls, add 5-200 μL of acetonitrile containing internal standard extractant per mg of sample, grind, centrifuge, take the supernatant and put it into injection bottle for gas chromatography-mass spectrometry analysis. After data processing, obtain the ratio of the peak area of ​​each short-chain fatty acid in the feces to the peak area of ​​the internal standard, that is, the relative content result; (3) Weigh 30-100 mg of tissue sample, put it into grinding balls, add 2-10 μL of acetonitrile containing internal standard extractant per mg of sample, grind, centrifuge, take the supernatant and put it into injection bottle for gas chromatography-mass spectrometry analysis. After data processing, obtain the ratio of the peak area of ​​each short-chain fatty acid in the tissue to the peak area of ​​the internal standard, that is, the relative content result; The internal standard extractant acetonitrile contains 5-10 μmol / L 2-Ethylbutyric acid; The reagents used for extraction were all acetonitrile, and the gas chromatography needle washing solution was also pure acetonitrile.

2. The detection method according to claim 1, characterized in that: In step (3), 2 μL of acetonitrile containing the internal standard extractant is added to each mg of visceral fat sample, and 6 μL of acetonitrile containing the internal standard extractant is added to each mg of spleen sample.

3. The detection method according to claim 1, characterized in that: The gas chromatography conditions used were as follows: DB-FFAP capillary column, size 30 m × 0.25 mm × 0.25 μm, programmed temperature starting at 50 °C, maintained for 1 min, increased to 180 °C at a rate of 10 °C / min, and then continued to increase to 250 °C at a rate of 30 °C / min and maintained for 2 min; linear velocity 40 cm / sec, He, 99.9995%, injection port temperature 270 °C, injection volume 1 μL, no split; The mass spectrometry detection conditions adopted were: ion source temperature 230 ℃, quadrupole temperature 150 ℃, transfer line temperature 270 ℃, scanning mode SIM, Acetic acid 43, Propionic acid 74, Butyric Acid 60, Isobutyric acid43, Valeric Acid 60, Isovaleric acid 60, Hexanoic Acid 60, 3-Methylvaleric acid60, 4-Methylvaleric acid 60, 2-Ethylbutyric acid 73.

4. The detection method according to claim 1, characterized in that: The grinding frequency is 15-20 times / s, and each grinding time is 1-3 min.

5. The detection method according to claim 1, characterized in that: All samples were extracted with acetonitrile and directly loaded into injection bottles for analysis by gas chromatography-mass spectrometry.

6. The detection method according to claim 1, characterized in that: All samples were calibrated with an internal standard and quantified according to the linear curve; The linear curve configuration method is as follows: prepare a mixed standard of each short-chain fatty acid at a concentration of 200-1000 μg / mL, and dilute it step by step to at least 5 different concentrations of 0.1-0.01 μg / mL; draw samples at each concentration point, add 2-6 times the volume of an extractant containing an internal standard, take the supernatant and put it into a sample injection bottle for gas chromatography-mass spectrometry analysis, and obtain the ratio of the peak area of ​​each short-chain fatty acid to the peak area of ​​the internal standard; The horizontal axis is the ratio of each short-chain fatty acid peak area to the internal standard peak area, and the vertical axis is the configuration concentration. A standard curve is made, and the ratio of each short-chain fatty acid peak area in the sample to the internal standard peak area is substituted into the linear equation to obtain the absolute quantitative results of short-chain fatty acids in each sample.

7. The detection method according to claim 1, characterized in that: The short-chain fatty acids include one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isopropionic acid, hexanoic acid, 3-methylvaleric acid, and 4-methylvaleric acid.

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