A method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens

Abstract

The present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, comprising the following steps: a) dividing the biological specimen into a tissue specimen and a body fluid sample; alkalizing the tissue specimen, freeze-drying, acidifying, and liquid-liquid extraction to obtain a first extract; using acetonitrile to precipitate protein from the body fluid sample and taking the supernatant to obtain a second extract; b) concentrating and re-dissolving the first extract and / or the second extract, and then adding a derivatization solution for chemical derivatization to obtain a sample solution; c) adding a derivatized internal standard to the sample solution as a marker, and using gas chromatography-mass spectrometry to measure and determine the content of short-chain fatty acids and their hydroxylated derivatives in the sample solution. The present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, based on N-methylbenzylamine-d 0 / d 3 The chemical isotope labeling strategy has the advantages of small sample usage, high detection sensitivity and wide application range.

Description

Technical Field

[0001] The present invention belongs to the technical field of component detection in biological samples, and relates to a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological samples, and specifically to a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological samples based on stable isotope labeling GC-MS technology. Background Art

[0002] Short-chain fatty acids (SCFA) usually refer to organic fatty acids with less than 6 carbon atoms, which are produced by fermentation of indigestible dietary fiber under the action of intestinal flora, mainly including acetic acid, propionic acid, butyric acid, valeric acid and their derivatives. In recent years, a large number of studies have shown that SCFAs play an important role in various physiological and pathological processes, including maintaining the innate intestinal barrier function, affecting the blood-brain barrier and neuro-immune endocrine function, and regulating immune function. In addition, some branched-chain fatty acids and hydroxylated short-chain fatty acids have also been shown to be potential diagnostic markers for a variety of complex diseases (including colitis, obesity, metabolic syndrome and infection).

[0003] At present, there are many methods for quantitative analysis of short-chain fatty acids in biological samples, including gas chromatography, nuclear magnetic resonance and high-performance liquid chromatography. In the prior art, China's patent application CN111239291A provides a GC-MS-based method for detecting short-chain fatty acids in mouse gut-brain axis-related tissue samples. Brain tissue, colon tissue, colon contents and feces are used as samples, pre-cooled ultrapure water is added to the tissue samples, and they are ground evenly at low temperature, and then vortexed, ultrasonically extracted, and centrifuged to obtain the supernatant to obtain the extract; the internal standard 2-ethylbutyric acid is added to each extract, and a derivatization reaction is performed; after the derivatization solution is extracted respectively, GC-MS is used to detect acetic acid, propionic acid and butyric acid in each extract. This method has the characteristics of simple pretreatment and is suitable for brain-gut axis-related tissues. However, the water content in biological sample tissues varies greatly, and only 2-ethylbutyric acid is used as a single internal standard reagent, which is very easy to produce analytical errors and is difficult to meet the high accuracy and high precision requirements of clinical tests. Summary of the invention

[0004] In view of the shortcomings of the prior art mentioned above, the object of the present invention is to provide a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, a method for determining the contents of 19 short-chain fatty acids and their hydroxylated derivatives in biological specimens based on isotope-labeled GC-MS, which improves the accuracy, compactness and sensitivity of quantitative analysis of short-chain fatty acids and their hydroxylated derivatives.

[0005] To achieve the above-mentioned object and other related objects, the present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in a biological specimen, comprising the following steps:

[0006] a) dividing the biological specimen into a tissue specimen and a body fluid sample; alkalizing the tissue specimen, freeze-drying, acidifying, and liquid-liquid extraction to obtain a first extract; using acetonitrile to precipitate protein from the body fluid sample, and taking the supernatant to obtain a second extract;

[0007] b) concentrating and re-dissolving the first extract and / or the second extract, and then adding a derivatization solution containing N-methylbenzylamine-d0 as a label to perform chemical derivatization to obtain a sample solution;

[0008] c) adding a derivatized internal standard containing N-methylbenzylamine-d3 as a marker to the sample solution, and determining the content of short-chain fatty acids and their hydroxylated derivatives in the sample solution by gas chromatography-mass spectrometry (GC-MS).

[0009] Preferably, the short-chain fatty acids and their hydroxylated derivatives are selected from any one or more of formic acid, acetic acid, propionic acid, n-butyric acid, isobutyric acid, 2-methylbutyric acid, lactic acid, hydroxyacetic acid, isovaleric acid, 2-methylvaleric acid, 2-hydroxybutyric acid, 3-methylvaleric acid, 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, hexanoic acid, 2-hydroxy-2-methylbutyric acid, 2-hydroxy-4-methylvaleric acid, and n-heptanoic acid.

[0010] Preferably, there are 19 short-chain fatty acids and their hydroxylated derivatives, including formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, 2-methylbutyric acid, lactic acid, hydroxyacetic acid, isovaleric acid, 2-methylvaleric acid, 2-hydroxybutyric acid, 3-methylvaleric acid, 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, hexanoic acid, 2-hydroxy-2-methylbutyric acid, 2-hydroxy-4-methylvaleric acid, and heptanoic acid.

[0011] Preferably, in step a), the biological specimen is a conventionally used biological specimen.

[0012] Preferably, in step a), the tissue specimens include but are not limited to human or animal feces, brain tissue, intestinal tissue, that is, the tissue specimens are selected from one or more combinations of human or animal feces, brain tissue, intestinal tissue.

[0013] Preferably, in step a), the body fluid sample includes but is not limited to human or animal plasma and urine, that is, the body fluid sample is selected from one or more combinations of human or animal plasma and urine.

[0014] Preferably, in step a), the alkalization is performed by adding alkali solution to the tissue specimen, and the alkali solution is a sodium carbonate solution.

[0015] Preferably, the sodium carbonate solution is an aqueous solution of sodium carbonate, and the concentration of the sodium carbonate solution is 40-60 μg / mL, preferably 50 μg / mL.

[0016] Preferably, the ratio of the added mass of the tissue specimen to the added volume of the alkali solution is 300-500:400-600, preferably 400:500, mg / μL.

[0017] Preferably, in step a), the freeze-drying time is 6-8 hours. The freeze-drying is freeze drying.

[0018] Preferably, in step a), the freeze-drying temperature is -80 to -60°C.

[0019] Preferably, in step a), the tissue specimen is accurately weighed after freeze-drying to facilitate obtaining the accurate mass of the tissue specimen for subsequent quantitative processing.

[0020] Preferably, in step a), the acidification is performed by adding an acid solution to the freeze-dried tissue specimen, and the acid solution is a hydrochloric acid solution.

[0021] Preferably, the hydrochloric acid solution is a hydrochloric acid aqueous solution with a concentration of 4-6M (mol / L).

[0022] Preferably, the ratio of the mass of the tissue specimen added to the volume of the acid solution added is 40-60:205-415, preferably 50:310, mg / μL.

[0023] Preferably, in step a), the extraction reagent used in the liquid-liquid extraction is diethyl ether, and the diethyl ether is anhydrous diethyl ether.

[0024] Preferably, in step a), in the liquid-liquid extraction, the ratio of the mass of the tissue specimen added to the volume of the extraction reagent added is 40-60:50-150, preferably 50:100, mg / μL.

[0025] The liquid-liquid extraction is a conventional liquid-liquid extraction, that is, an extraction reagent is added to the sample and mixed to form layers, and the solvent layer is taken as the first extraction liquid. During the liquid-liquid extraction process, the solid layer can be washed multiple times with the extraction reagent, and the washing liquid is combined with the solvent layer.

[0026] Preferably, in step a), the ratio of the added mass of the body fluid sample to the added volume of acetonitrile is 1:2-4, preferably 1:3, g / mL.

[0027] The body fluid sample is subjected to a conventional acetonitrile protein precipitation method, and the supernatant is taken out by layering to obtain a second extract.

[0028] Preferably, in step b), the concentration is carried out by evaporating to dryness under nitrogen blowing.

[0029] Preferably, in step b), the reconstitution is to dissolve the concentrate in anhydrous acetonitrile, and the volume of the anhydrous acetonitrile is 90-110 μL, preferably 100 μL.

[0030] Preferably, in step b), the volume ratio of the reconstitution solution to the derivatization solution is 40-60:100-200, preferably 50:150.

[0031] Preferably, in step b), the derivatization solution comprises N-methylbenzylamine-d0, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and triethylamine.

[0032] Preferably, the volume ratio of N-methylbenzylamine-d0, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and triethylamine in the derivatization solution is 0.5-1.5:0.5-1.5:0.5-1.5, preferably 1:1:1.

[0033] The CAS number of the above-mentioned N-methylbenzylamine-d0 (N-methyl-1-phenylmethanamine-d0) is 103-67-3.

[0034] The abbreviation of the above-mentioned 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethaneminium hexafluorophosphate N-oxide) is HATU, and the CAS number of the HATU is 148893-10-1.

[0035] The CAS number of the above triethylamine is 121-44-8.

[0036] Preferably, the N-methylbenzylamine-d0 is an acetonitrile solution of N-methylbenzylamine-d0 with a concentration of 5-7 μM (μmol / L), preferably 6 μM.

[0037] Preferably, the HATU is an acetonitrile solution with a concentration of 2-4 μM (μmol / L), preferably 3 μM HATU.

[0038] Preferably, the triethylamine is an acetonitrile solution of triethylamine with a concentration of 0.05-0.15 μM (μmol / L), preferably 0.1 μM.

[0039] Preferably, in step b), the chemical derivatization is performed by oscillating the reconstituted solution and the derivatization solution.

[0040] Preferably, the shaking temperature is 35-40°C, preferably 37°C.

[0041] Preferably, the shaking time is 10-30 minutes, preferably 20 minutes.

[0042] Preferably, in step c), the volume of the derivatized internal standard added to the sample solution is 5-15 μL, preferably 10 μL.

[0043] Preferably, in step c), the derivatized internal standard is obtained by mixing a standard solution of short-chain fatty acids and their hydroxylated derivatives with N-methylbenzylamine-d3, HATU and triethylamine and then shaking.

[0044] The above-mentioned standard solution of short-chain fatty acids and hydroxylated derivatives thereof is a mixed standard solution of 19 short-chain fatty acids and hydroxylated derivatives thereof in biological specimens.

[0045] The structural formula of the above-mentioned N-methylbenzylamine-d3 (N-methyl-1-phenylmethanamine-d3) is:

[0046]

[0047] Preferably, the volume ratio of the standard solution of short-chain fatty acids and hydroxylated derivatives thereof, N-methylbenzylamine-d3, HATU and triethylamine is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5, preferably 1:1:1:1.

[0048] Preferably, the concentration of each short-chain fatty acid and its hydroxylated derivative in the standard solution of short-chain fatty acids and their hydroxylated derivatives is 100-300 ng / mL, preferably 200 ng / mL of acetonitrile solution.

[0049] Preferably, the N-methylbenzylamine-d3 is an acetonitrile solution of N-methylbenzylamine-d3 with a concentration of 5-7 μM (μmol / L), preferably 6 μM.

[0050] Preferably, the HATU is an acetonitrile solution with a concentration of 2-4 μM (μmol / L), preferably 3 μM HATU.

[0051] Preferably, the triethylamine is an acetonitrile solution of triethylamine with a concentration of 0.05-0.15 μM (μmol / L), preferably 0.1 μM.

[0052] Preferably, the shaking temperature is 25-40°C, preferably 37°C.

[0053] Preferably, the shaking time is 10-30 minutes, preferably 20 minutes.

[0054] Preferably, in step c), the determination is performed using gas chromatography-mass spectrometry (GC-MS), comprising the following steps:

[0055] 1) Prepare standard solution: take standard products of short-chain fatty acids and their hydroxylated derivatives, add solvent to dissolve them and make up to volume to prepare standard solution;

[0056] 2) Sample detection: The sample solution with the added derivatized internal standard as a marker and the standard solution in step 1) are detected by gas chromatography-mass spectrometry, and the retention time is compared for qualitative analysis. The internal standard standard curve method is used for quantitative analysis to determine the content of short-chain fatty acids and their hydroxylated derivatives in the sample solution.

[0057] Preferably, in step 1), the solvent is acetonitrile.

[0058] Preferably, in step 1), the concentration range of short-chain fatty acids and hydroxylated derivatives thereof in the standard solution is 50-100 μg / mL.

[0059] Preferably, in step 2), in the gas chromatography-mass spectrometry (GC-MS), the gas chromatography measurement conditions are:

[0060] Chromatographic column: CB capillary column; injection port temperature: 270-290℃; injection volume: 0.5-2.0μL; carrier gas: high-purity helium, carrier gas purity ≥99.999%; carrier gas flow rate: constant flow rate, 1.0-2.0ml / min; split injection, split ratio: 1:10-20; heating program: initial temperature is 90-110℃, maintained for 1-3 minutes, then increased to 130-150℃ at a rate of 30-50℃ / min, maintained for 15-25min, and finally increased to 270-290℃ at a rate of 5-15℃ / min, maintained for 4-6min.

[0061] More preferably, the gas chromatography measurement conditions are:

[0062] Chromatographic column: Agilent J&W CP-Sil 8CB capillary column (30m×0.25mm id×0.25μm df); injection port temperature: 280℃; injection volume: 1.0μL; carrier gas: high-purity helium, carrier gas purity ≥99.999%; carrier gas flow rate: constant flow rate, 1.2ml / min; split injection, split ratio: 1:15; heating program: initial temperature is 100℃, maintained for 2 minutes, then increased to 140℃ at a rate of 40℃ / min, maintained for 20min, and finally increased to 280℃ at a rate of 10℃ / min, maintained for 5min, and the total running time is 42min.

[0063] Preferably, in step 2), in the gas chromatography-mass spectrometry (GC-MS), the mass spectrometry measurement conditions are:

[0064] Ionization mode: electron impact (EI) ion source; ionization energy: 70 eV; ion source temperature: 275-285°C, preferably 280°C; scanning mode: positive ion mode or SIM mode; acquisition rate: 0.1-0.3s / scan, preferably 0.2s / scan; mass range: 50-400m / z.

[0065] Preferably, in step 3), the internal standard curve method comprises the following steps:

[0066] A) preparing a series of standard solutions with different concentrations according to step 1), performing GC-MS detection respectively, obtaining a linear relationship between the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative / internal standard and the content of each corresponding short-chain fatty acid and its hydroxylated derivative, drawing a corresponding standard working curve, and calculating the regression equation of the standard working curve of each short-chain fatty acid and its hydroxylated derivative;

[0067] B) The sample solution is then subjected to GC-MS detection, and the chromatographic peak area ratios of each short-chain fatty acid and its hydroxylated derivative / internal standard in the sample solution are respectively substituted into the regression equations of the standard working curves of each short-chain fatty acid and its hydroxylated derivative in step A), and the content of each short-chain fatty acid and its hydroxylated derivative in the sample solution is calculated.

[0068] More preferably, in the standard working curve, the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative to the internal standard is used as the ordinate (Y-axis), and the corresponding content of each short-chain fatty acid and its hydroxylated derivative is used as the abscissa (X-axis).

[0069] As described above, the present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, which has the following beneficial effects:

[0070] (1) The present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, and establishes a chemical isotope labeling strategy based on N-methylbenzylamine-d0 / d3 for the analysis of SCFAs and their hydroxylated derivatives. It can not only correct the matrix effect, improve the accuracy and precision of the analysis, and improve the problems of low ionization efficiency and insufficient sensitivity when mass spectrometry is used to detect carboxylic acid metabolites, but also overcome the problems of high price, limited variety and difficulty in obtaining commercial isotope internal standards.

[0071] (2) The present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in a biological specimen, which adds sodium carbonate solution for alkalization, converts free SCFAs and their hydroxylated derivatives into SCFA sodium salts, and eliminates the loss of volatile SCFAs during the freeze-drying process of the biological sample.

[0072] (3) The present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens. Combined with GC-MS technology, it can quickly and accurately detect SCFAs and their hydroxylated derivatives in biological specimens with high contents of SCFAs and their hydroxylated derivatives, such as human or animal feces, plasma, urine, brain tissue, intestinal tissue, etc., and has the advantages of small sample dosage, high detection sensitivity, and a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Shown are representative single ion SIM monitoring chromatograms of 19 short-chain fatty acids and their hydroxylated derivatives in the standard sample of the present invention, wherein 1 is formic acid; 2 is acetic acid; 3 is propionic acid; 4 is n-butyric acid; 5 is isobutyric acid; 6 is 2-methylbutyric acid; 7 is lactic acid; 8 is hydroxyacetic acid; 9 is isovaleric acid; 10 is 2-methylvaleric acid; 11 is 2-hydroxyvaleric acid; 12 is 3-methylvaleric acid; 13 is 2-hydroxy-3-methylbutyric acid; 14 is 3-hydroxybutyric acid; 15 is β-hydroxyisovaleric acid; 16 is hexanoic acid; 17 is 2-hydroxy-2-methylbutyric acid; 18 is 2-hydroxy-4-methylvaleric acid; and 19 is n-heptanoic acid. DETAILED DESCRIPTION

[0074] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0075] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0076] The reagents and instruments used in the following examples are as follows:

[0077] 1. Reagents

[0078] Fecal samples, plasma samples, and urine samples were obtained from the Laboratory Department of Zhujiang Hospital of Southern Medical University; intestinal tissue and brain tissue samples were obtained from C57BL / 6 mice.

[0079] Formic acid, acetic acid, propionic acid, n-butyric acid, isobutyric acid, 2-methylbutyric acid, lactic acid, glycolic acid, isovaleric acid, 2-methylvaleric acid, 2-hydroxybutyric acid, 3-methylvaleric acid, 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, hexanoic acid, 2-hydroxy-2-methylbutyric acid, 2-hydroxy-4-methylvaleric acid, n-heptanoic acid (purity>98%, Sigma-Aldrich, USA); N-methylbenzylamine-d0 (purity>98%, Sigma-Aldrich, USA); N-methylbenzylamine-d3 (purity>98%, TRC, Canada); sodium carbonate, anhydrous ether, hydrochloric acid, anhydrous acetonitrile, HATU, triethylamine (purity>98%, Aladdin); pure water (ultrapure water instrument homemade).

[0080] 2. Instruments

[0081] Agilent 5977B gas chromatograph-mass spectrometer (Agilent, USA); Agilent J&W CP-Sil 8CB capillary chromatographic column (Agilent, USA).

[0082] Example 1

[0083] 1. Sample pretreatment:

[0084] A stool sample was selected as a biological specimen, and 400 mg of the stool sample was mixed with 500 μL of 50 μg / mL sodium carbonate solution for alkalization, and then freeze-dried at -70 ° C for 7 hours. After the freeze-dried sample was accurately weighed, 50 mg of the sample was mixed with 310 μL of 5M hydrochloric acid aqueous solution for acidification, and then transferred to a new microtube. After that, 100 μL of anhydrous ether was added to the acidified stool sample in the microtube for liquid-liquid extraction to form layers, the ether layer was collected, and the ether layer was further washed twice with ether, and the washing liquid was combined with the ether layer to obtain the first extract. The first extract was then evaporated to dryness under nitrogen blowing. Subsequently, the residue was dissolved in 100 μL of anhydrous acetonitrile to obtain a complex solution for standby use.

[0085] 50 μL of the complex solution was added with 150 μL of a derivatization solution containing 6 μM N-methylbenzylamine-d0, 3 μM HATU and 0.1 μM triethylamine in a volume ratio of 1:1:1 (v / v). The mixture was shaken at 37°C for 20 minutes to perform a chemical derivatization reaction to obtain a sample solution.

[0086] Add 10 μL of derivatized internal standard to the sample solution and mix for testing. The derivatized internal standard is obtained by mixing the standard solution of short-chain fatty acids and their hydroxylated derivatives with N-methylbenzylamine-d3, HATU, and triethylamine and shaking. That is, a mixed standard solution containing 200 ng / mL of each short-chain fatty acid and its hydroxylated derivative is mixed with 6 μM N-methylbenzylamine-d3, 3 μM HATU, and 0.1 μM triethylamine in a ratio of 1:1:1:1 (v / v), and shaken at 37°C for 20 minutes to prepare.

[0087] 2. Preparation of standard solution

[0088] Take 19 kinds of standard samples of short-chain fatty acids and their hydroxylated derivatives, add solvent to dissolve them and then make up to volume to prepare standard solutions.

[0089] 3. Determination

[0090] Gas chromatography-mass spectrometry (GC-MS) was used to detect the sample solution and standard solution marked with the derivatized internal standard, and the retention time was compared for qualitative analysis. The internal standard standard curve method was used for quantitative analysis. That is, a series of standard solutions with different concentrations were first subjected to GC-MS detection to obtain the linear relationship between the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative / internal standard and the content of each short-chain fatty acid and its hydroxylated derivative. The corresponding standard working curve was drawn, and the regression equation of the standard working curve of each short-chain fatty acid and its hydroxylated derivative was calculated. The sample solution was then subjected to GC-MS detection, and the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative / internal standard in the sample solution was substituted into the regression equation of the standard working curve of each short-chain fatty acid and its hydroxylated derivative, and the content of each short-chain fatty acid and its hydroxylated derivative in the sample solution was calculated. The chromatograms of 19 short-chain fatty acids and their hydroxylated derivatives are shown in Figure 1 .

[0091] The gas chromatography measurement conditions are as follows: chromatographic column: Agilent J&W CP-Sil 8CB capillary chromatographic column (30m×0.25mm id×0.25μm df); injection port temperature: 280°C; injection volume: 1.0μL; carrier gas: high-purity helium, carrier gas purity ≥99.999%; carrier gas flow rate: constant flow rate, 1.2ml / min; split injection, split ratio: 1:15; heating program: initial temperature is 100°C, maintained for 2 minutes, then increased to 140°C at a rate of 40°C / min, maintained for 20min, and finally increased to 280°C at a rate of 10°C / min, maintained for 5min, and the total running time is 42min.

[0092] The mass spectrometry conditions are as follows: ionization mode: electron impact (EI) ion source; ionization energy: 70 eV; ion source temperature: 280°C; scanning mode: positive ion mode or SIM mode; acquisition rate: 0.2 s / scan; mass range: 50-400 m / z.

[0093] Example 2

[0094] 1. Sample pretreatment:

[0095] An intestinal tissue sample was selected as a biological specimen, and 400 mg of the intestinal tissue sample was prepared according to step 1 of Example 1 to add a derivatized internal standard as a labeled sample solution.

[0096] 2. Preparation of standard solution

[0097] Prepare the standard solution according to step 2 of Example 1.

[0098] 3. Determination

[0099] The measurement was carried out according to step 3 of Example 1.

[0100] Example 3

[0101] 1. Sample pretreatment:

[0102] A brain tissue sample was selected as a biological specimen, and 400 mg of the brain tissue sample was prepared according to step 1 of Example 1 to add a derivatized internal standard as a labeled sample solution.

[0103] 2. Preparation of standard solution

[0104] Prepare the standard solution according to step 2 of Example 1.

[0105] 3. Determination

[0106] The measurement was carried out according to step 3 of Example 1.

[0107] Example 4

[0108] 1. Sample pretreatment:

[0109] A urine or plasma sample is selected as a biological specimen, and 50 mg of the urine or plasma sample is precipitated with 3 times the amount of acetonitrile to precipitate the protein, and the supernatant is taken to obtain a second extract. The second extract is then evaporated to dryness under nitrogen. Subsequently, the residue is dissolved in 100 μL of anhydrous acetonitrile to obtain a reconstituted solution for standby use. The reconstituted solution is further chemically derivatized according to step 1 of Example 1 to prepare a sample solution with a derivatized internal standard added as a marker.

[0110] 2. Preparation of standard solution

[0111] Prepare the standard solution according to step 2 of Example 1.

[0112] 3. Determination

[0113] The measurement was carried out according to step 3 of Example 1.

[0114] Example 5

[0115] 1. Sample pretreatment:

[0116] A stool sample was selected as a biological specimen, and 450 mg of the stool sample was mixed with 600 μL of 55 μg / mL sodium carbonate solution for alkalization, and then freeze-dried at -80 ° C for 67 h. After the freeze-dried sample was accurately weighed, 50 mg of the sample was mixed with 400 μL of 5M hydrochloric acid aqueous solution for acidification, and then transferred to a new microtube. After that, 120 μL of anhydrous ether was added to the acidified stool sample in the microtube for liquid-liquid extraction to form layers, the ether layer was collected, and the ether layer was further washed twice with ether, and the washing liquid was combined with the ether layer to obtain the first extract. The first extract was then evaporated to dryness under nitrogen blowing. Subsequently, the residue was dissolved in 110 μL of anhydrous acetonitrile to obtain a complex solution for standby use.

[0117] 50 μL of the complex solution was added with 180 μL of a derivatization solution containing 5 μM N-methylbenzylamine-d0, 2 μM HATU and 0.05 μM triethylamine in a volume ratio of 1:1:1 (v / v). The mixture was shaken at 38°C for 18 minutes to perform a chemical derivatization reaction to obtain a sample solution.

[0118] Add 12 μL of derivatized internal standard to the sample solution and mix for testing. The derivatized internal standard is obtained by mixing the standard solution of short-chain fatty acids and their hydroxylated derivatives with N-methylbenzylamine-d3, HATU, and triethylamine and shaking. That is, a mixed standard solution containing 200 ng / mL of each short-chain fatty acid and its hydroxylated derivative is mixed with 5 μM N-methylbenzylamine-d3, 2 μM HATU, and 0.05 μM triethylamine in a ratio of 1:1:1:1 (v / v), and shaken at 38°C for 18 minutes to prepare.

[0119] 4. Preparation of standard solution

[0120] Take 19 kinds of standard samples of short-chain fatty acids and their hydroxylated derivatives, add solvent to dissolve them and then make up to volume to prepare standard solutions.

[0121] 5. Determination

[0122] Gas chromatography-mass spectrometry (GC-MS) was used to detect the sample solution and standard solution marked with the derivatized internal standard, and the retention time was compared for qualitative analysis. The internal standard standard curve method was used for quantitative analysis. That is, a series of standard solutions with different concentrations were first subjected to GC-MS detection to obtain the linear relationship between the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative / internal standard and the content of each short-chain fatty acid and its hydroxylated derivative. The corresponding standard working curve was drawn, and the regression equation of the standard working curve of each short-chain fatty acid and its hydroxylated derivative was calculated. The sample solution was then subjected to GC-MS detection, and the chromatographic peak area ratio of each short-chain fatty acid and its hydroxylated derivative / internal standard in the sample solution was substituted into the regression equation of the standard working curve of each short-chain fatty acid and its hydroxylated derivative, and the content of each short-chain fatty acid and its hydroxylated derivative in the sample solution was calculated. The chromatograms of 19 short-chain fatty acids and their hydroxylated derivatives are shown in Figure 1 .

[0123] The gas chromatography measurement conditions are as follows: chromatographic column: Agilent J&W CP-Sil 8CB capillary chromatographic column (30m×0.25mm id×0.25μm df); injection port temperature: 290°C; injection volume: 2.0μL; carrier gas: high-purity helium, carrier gas purity ≥99.999%; carrier gas flow rate: constant flow rate, 1.5ml / min; split injection, split ratio: 1:18; heating program: initial temperature is 100°C, maintained for 2 minutes, then increased to 140°C at a rate of 40°C / min, maintained for 20min, and finally increased to 280°C at a rate of 10°C / min, maintained for 5min, and the total running time is 42min.

[0124] The mass spectrometry conditions are as follows: ionization mode: electron impact (EI) ion source; ionization energy: 70 eV; ion source temperature: 282°C; scanning mode: positive ion mode or SIM mode; acquisition rate: 0.3 s / scan; mass range: 50-400 m / z.

[0125] Example 6

[0126] According to step 3 of Example 1, the internal standard standard curve was constructed by comparing the peak area ratio of the sample solution labeled with N-methylbenzylamine-d0 and the corresponding internal standard labeled with N-methylbenzylamine-d3. The specific data are shown in Table 1-2. As can be seen from Table 1-2, within the wide dynamic range of 1.21 to 625 ng / ml, the internal standard standard curve has excellent accuracy and the correlation coefficient (R 2 ) is satisfactory.

[0127] Table 1 Mass spectrometry parameters

[0128]

[0129] Table 2

[0130] Compound Regression equation Formic acid y=368.43x+5.72 acetic acid y=252.79x+2.70 Propionic acid y=511.56x+1,94 Butyric acid y=601.18x+2.96 Isobutyric acid y=414.65x+2.79 2-Methylbutyric acid y=148.15x+0.51 lactic acid y=185.13x+0.60 Glycolic acid y=401.38x+1.24 Isovalerate y=1019.05x-10.83 2-Methylvaleric acid y=449.956x-1.21 2-Hydroxybutyric Acid y=248.53x-1.46 3-Methylvaleric acid y=543.17x-2.51 2-Hydroxy-3-methylbutyric acid y=439.35x-2.16 3-Hydroxybutyric Acid y=1248.13x-7.48 β-Hydroxyisovaleric acid y=630.52x-0.71 Hexanoic acid y=391.74x1.85 2-Hydroxy-2-methylbutyric acid y=293.61x-1.65 2-Hydroxy-4-methylvaleric acid y=369.19x+1.89 n-heptanoic acid y=615.41x+3.40

[0131] Note: y: chromatographic peak area; x: content.

[0132] Example 7

[0133] Accurately weigh one portion of the feces sample in Example 1, prepare the sample solution in step 1 of Example 1, and continuously inject the sample 10 times for analysis according to step 3 of Example 1. The relative standard deviation (RSD) of the content of 19 short-chain fatty acids and their hydroxylated derivatives is less than 2%, indicating that the instrument has good precision. Specific data are shown in Table 3.

[0134] At the same time, 10 portions of fecal samples in Example 1 were accurately weighed, and sample solutions were prepared according to step 1 of Example 1. The samples were injected and analyzed in sequence, and the relative standard deviation (RSD) of the contents of 19 short-chain fatty acids and their hydroxylated derivatives was less than 3%, indicating that the method had good reproducibility. Specific data are shown in Table 3.

[0135] Table 3

[0136] Compound Relative standard deviation (RSD) of precision Relative standard deviation (RSD) of reproducibility Formic acid 1.55 2.93 acetic acid 0.34 2.83 Propionic acid 1.53 2.6 Butyric acid 0.46 0.47 Isobutyric acid 0.67 1.79 2-Methylbutyric acid 2.73 2.69 lactic acid 2.87 1.45 Glycolic acid 2.19 2.22 Isovalerate 0.87 0.99 2-Methylvaleric acid 1.14 3 2-Hydroxybutyric Acid 0.83 0.15 3-Methylvaleric acid 2.03 1.77 2-Hydroxy-3-methylbutyric acid 0.96 2.29 3-Hydroxybutyric Acid 1.92 2.75 β-Hydroxyisovaleric acid 2.19 0.04 Hexanoic acid 1.6 0.34 2-Hydroxy-2-methylbutyric acid 2.38 2.77 2-Hydroxy-4-methylvaleric acid 2.87 1.86 n-heptanoic acid 2.52 0.5

[0137] Example 8

[0138] Accurately weigh one portion of the stool sample in Example 1 and prepare the sample solution in step 1 of Example 1. According to the chromatographic conditions of step 3 of Example 1, the sample was injected and analyzed at room temperature at 0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, and the peak area was determined. The specific results of stability are shown in Table 4. The results show that the relative standard deviation (RSD) of 19 short-chain fatty acids and their hydroxylated derivatives is less than 3%, and the sample solution is basically stable within 24 hours, indicating that the method has good stability.

[0139] Table 4

[0140] Compound Relative standard deviation (RSD) of stability Formic acid 1.26 acetic acid 0.63 Propionic acid 2.67 Butyric acid 1.45 Isobutyric acid 2.58 2-Methylbutyric acid 2.59 lactic acid 0.32 Glycolic acid 2.6 Isovalerate 0.32 2-Methylvaleric acid 2.21 2-Hydroxybutyric Acid 2.21 3-Methylvaleric acid 1.96 2-Hydroxy-3-methylbutyric acid 2.61 3-Hydroxybutyric Acid 2.08 β-Hydroxyisovaleric acid 0.46 Hexanoic acid 0.51 2-Hydroxy-2-methylbutyric acid 1.48 2-Hydroxy-4-methylvaleric acid 0.08 n-heptanoic acid 1.52

[0141] Example 9

[0142] One portion of the fecal sample in Example 1 was accurately weighed, and 9 portions were prepared in parallel. A mixed standard solution of 19 short-chain fatty acids and hydroxylated derivatives thereof was precisely added to each sample at a ratio of about 100%, and the sample solution was prepared according to step 1 in Example 1, and the chromatographic conditions of step 3 in Example 1 were respectively injected and analyzed, and the results are shown in Table 5. As can be seen from Table 5, the relative standard deviation (RSD) of the 19 short-chain fatty acids and hydroxylated derivatives thereof is less than 3%, and the recovery rate of the determination results of the 19 short-chain fatty acids and hydroxylated derivatives thereof is good, indicating that the accuracy of this method is good.

[0143] Table 5

[0144]

[0145]

[0146] Example 10

[0147] Meconium and neonatal feces samples were measured according to the method in Example 1, and the contents of 19 short-chain fatty acids and their hydroxylated derivatives in the feces samples are shown in Table 6. The results show that the present invention can accurately measure 19 short-chain fatty acids and their hydroxylated derivatives in biological specimens.

[0148] Table 6

[0149] No. fatty acid Meconium (n=15) Neonatal stool (n=15) 1 Formic acid 1.76±0.66 1.68±0.58 2 acetic acid 2.02±0.51 4.90±1.44* 3 Propionic acid 2.02±0.63 1.97±0.69 4 Butyric acid 1.91±0.75 6.82±1.08* 5 Isobutyric acid 1.69±0.59 7.07±1.29* 6 2-Methylbutyric acid 1.79±0.62 2.18±0.64 7 lactic acid 1.83±0.63 1.84±0.70 8 Glycolic acid 1.94±0.65 2.07±0.65 9 Isovalerate 1.97±0.69 1.94±0.68 10 2-Methylvaleric acid 1.85±0.67 1.79±0.63 11 2-Hydroxybutyric Acid ND 0.13±0.02* 12 3-Methylvaleric acid 2.06±0.72 1.67±0.55 13 2-Hydroxy-3-methylbutyric acid ND 0.17±0.09* 14 3-Hydroxybutyric Acid 0.60±0.08 0.82±0.06* 15 β-Hydroxyisovaleric acid 1.99±0.59 7.01±1.20 16 Hexanoic acid 2.02±0.59 2.01±0.65 17 2-Hydroxy-2-methylbutyric acid ND 0.03±0.007* 18 2-Hydroxy-4-methylvaleric acid 1.65±0.51 1.82±0.72 19 n-heptanoic acid 2.08±0.57 1.98±0.55

[0150] Note: ND: not detected; *: significant difference.

[0151] In summary, the present invention provides a method for detecting short-chain fatty acids and their hydroxylated derivatives in biological specimens, establishes a chemical isotope labeling strategy based on N-methylbenzylamine-d0 / d3, and has the advantages of small sample dosage, high detection sensitivity, and wide application range. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0152] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for detecting short-chain fatty acids and their hydroxylated derivatives in a biological specimen, comprising the following steps: a) dividing the biological specimen into a tissue specimen and a body fluid sample; alkalizing the tissue specimen, freeze-drying, acidifying, and liquid-liquid extraction to obtain a first extract; using acetonitrile to precipitate protein from the body fluid sample, and taking the supernatant to obtain a second extract; b) concentrating and re-dissolving the first extract and / or the second extract, and then adding a derivatization solution containing N-methylbenzylamine-d0 as a label to perform chemical derivatization to obtain a sample solution; c) adding a derivatized internal standard containing N-methylbenzylamine-d3 as a marker to the sample solution, and determining the content of short-chain fatty acids and their hydroxylated derivatives in the sample solution by gas chromatography-mass spectrometry; The short-chain fatty acids and hydroxylated derivatives thereof are selected from any one or more of formic acid, acetic acid, propionic acid, n-butyric acid, isobutyric acid, 2-methylbutyric acid, lactic acid, glycolic acid, isovaleric acid, 2-methylvaleric acid, 2-hydroxybutyric acid, 3-methylvaleric acid, 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, hexanoic acid, 2-hydroxy-2-methylbutyric acid, 2-hydroxy-4-methylvaleric acid, and n-heptanoic acid; In step a), the alkalization is to add alkali solution to the tissue specimen, the alkali solution is sodium carbonate solution; the sodium carbonate solution is an aqueous solution of sodium carbonate, and the concentration of the sodium carbonate solution is 40-60 μg / mL; the ratio of the mass of the tissue specimen added to the volume of the alkali solution added is 300-500:400-600, mg / μL; In step a), the freeze-drying time is 6-8h; In step a), the freeze-drying temperature is -80--60°C; In step a), the acidification is to add acid to the freeze-dried tissue specimen, and the acid is a hydrochloric acid solution; The hydrochloric acid solution is a 4-6M hydrochloric acid aqueous solution; The ratio of the mass of the tissue specimen added to the volume of the acid solution added is 40-60:205-415, mg / μL; In step a), the extraction reagent used in the liquid-liquid extraction is ether; In step a), in the liquid-liquid extraction, the ratio of the mass of the tissue specimen added to the volume of the extraction reagent added is 40-60:50-150, mg / μL; In step a), the ratio of the added mass of the body fluid sample to the added volume of acetonitrile is 1:2-4, g / mL; In step b), the re-dissolving is to dissolve the concentrate in anhydrous acetonitrile; In step b), the volume ratio of the reconstitution solution to the derivatization solution is 40-60:100-200; In step b), the derivatization solution includes N-methylbenzylamine-d0, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and triethylamine; In the gas chromatography-mass spectrometry method, the gas chromatography measurement conditions are: Chromatographic column: CB capillary column; injection port temperature: 270-290℃; injection volume: 0.5-2.0μL; Carrier gas: high-purity helium, carrier gas purity ≥99.999%; Carrier gas flow rate: constant flow rate, 1.0-2.0ml / min; Split injection, split ratio: 1:10-20; Heating program: initial temperature is 90-110℃, keep for 1-3 minutes, then increase to 130-150℃ at a rate of 30-50℃ / min, keep for 15-25min, and finally increase to 270-290℃ at a rate of 5-15℃ / min, keep for 4-6min; In the gas chromatography-mass spectrometry method, the mass spectrometry measurement conditions are: Ionization mode: electron bombardment EI ion source; ionization energy: 70eV; ion source temperature: 275-285℃; scanning mode: positive ion mode or SIM mode; acquisition rate: 0.1-0.3s / scan; mass range: 50-400m / z.

2. The method for detecting short-chain fatty acids and hydroxylated derivatives thereof in a biological specimen according to claim 1, characterized in that: Step a) includes any one or more of the following conditions: A1) the tissue specimen is selected from one or more combinations of feces, brain tissue, and intestinal tissue of humans or animals; A2) The body fluid sample is selected from one or more combinations of plasma and urine of humans or animals.

3. The method for detecting short-chain fatty acids and hydroxylated derivatives thereof in a biological specimen according to claim 1, characterized in that: Step b) includes any one or more of the following conditions: B1) the concentration is evaporation to dryness by blowing nitrogen; B2) the volume of anhydrous acetonitrile is 90-110 μL; B3) The chemical derivatization is to subject the reconstituted solution to an oscillating reaction with the derivatization solution.

4. The method for detecting short-chain fatty acids and hydroxylated derivatives thereof in a biological specimen according to claim 1, characterized in that: Step c) includes any one or more of the following conditions: C1) the volume of the derivatized internal standard added to the sample solution is 5-15 μL; C2) The derivatized internal standard is obtained by mixing a standard solution of short-chain fatty acids and their hydroxylated derivatives with N-methylbenzylamine-d3, HATU and triethylamine and then shaking.

5. The method for detecting short-chain fatty acids and hydroxylated derivatives thereof in a biological specimen according to claim 1, characterized in that: In step c), the determination is performed by gas chromatography-mass spectrometry, comprising the following steps: 1) Prepare standard solution: take standard products of short-chain fatty acids and their hydroxylated derivatives, add solvent to dissolve them and make up to volume to prepare standard solution; 2) Sample detection: The sample solution with the added derivatized internal standard as a marker and the standard solution in step 1) are detected by gas chromatography-mass spectrometry, and the retention time is compared for qualitative analysis. The internal standard standard curve method is used for quantitative analysis to determine the content of short-chain fatty acids and their hydroxylated derivatives in the sample solution.

6. The method for detecting short-chain fatty acids and hydroxylated derivatives thereof in a biological specimen according to claim 5, characterized in that: Step 1) includes any one or more of the following conditions: D1) the solvent is acetonitrile; D2) The concentration range of short-chain fatty acids and their hydroxylated derivatives in the standard solution is 50-100 μg / mL.

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