CN110045040A - A method of measurement intestinal contents or excrement Short-Chain Fatty Acids content - Google Patents
A method of measurement intestinal contents or excrement Short-Chain Fatty Acids content Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
Abstract
The invention discloses a kind of measurement intestinal contents or the methods of excrement Short-Chain Fatty Acids content, are pre-processed using ether to sample, then with the short-chain fat acid content in gas chromatography measurement sample；The pretreatment temperature is 2~8 DEG C.The present invention does not need other derivatization pre-treatment steps, and the pre-treatment time is shorter than 15min, and length of testing speech is shorter than 20min；Low temperature can effectively reduce the volatilization loss of short chain fatty acids, the rate of recovery a height of 98.10 ± 0.29%~101.30 ± 0.27%；It can be more accurate for the detection of short chain fatty acids and efficient down to 0.0041 μ L/mL down to 0.0036 μ L/mL, quantitative limit using the method for the present invention detection limit.
Description
Technical field
The present invention relates to a kind of measurement intestinal contents or the methods of excrement Short-Chain Fatty Acids content, belong to biological sample
Detection field.
Background technique
Enteron aisle is not only the important place of human consumption's absorption, while being also the largest immune organ, is maintaining normally to exempt from
Extremely important effect is played in epidemic disease defense function.Human body intestinal canal provides good habitat for microorganism, lives in
Micropopulation in human gastrointestinal tract is 10 times of human body cell quantity up to hundreds of thousands of hundred million.Have more than 1000 strains
Through identified confirmation, existing about 160 kinds of microorganism in all individuals.It include about in the genome of enteric microorganism
3000000 genes are 150 times of human genome, the metabolic function not having with human body itself.Intestinal microecology is duodenum 12
Interaction, interactional unified entirety between road normal flora and its host.Currently, more and more researches show that, intestines
Road Tiny ecosystem and host health there is directly or indirectly contacting, intestinal microecology function it is unbalance, it is strong to affect host
Health promotes the generation of many diseases such as inflammatory bowel disease, cardiovascular and cerebrovascular disease, kidney trouble and depressed class disease.As human body
Inside most huge, most complicated microecosystem, enteric microorganism are not only adjustable nutrient metabolism, and metabolite is that is, short
Chain fatty acid plays important Link role also between diet and host health, has received widespread attention in recent years.
Short chain fatty acids are also known as volatile organic acids, are the organic aliphatic acids that carbochain is 1~6, how much can be divided by carbon number
Formic acid, acetic acid, propionic acid, butyric acid, valeric acid etc., and the intracorporal short chain fatty acids of biology are mainly acetic acid, propionic acid and butyric acid, are accounted for about short
The 90%~95% of chain fatty acid total amount is generated by carbohydrate through enteron aisle bacterial degradation, remaining short chain fatty acids only accounts for very
Fraction (about 5%~10%), wherein the branches short chain fatty acids such as isobutyric acid and isovaleric acid derive from the decomposition of protein.It is short
Chain fatty acid is organic anion important in enteric cavity, is absorbed by ion or non-ionic form by mucous membrane of colon, is colon
With the main Energy supply material of intestinal epithelial cell.The study found that short chain fatty acids can promote sodium ion to absorb, enteric epithelium is maintained
The inside and outside osmotic pressure of cell promotes colon cell proliferation and mucous membrane growth, provides the metabolism energy, increase enteron aisle blood flow, stimulates stomach
Intestinal hormones generates, and is the important nutrient of mucous membrane of colon.In addition, intestinal mucosa and lamina propria is immunized in certain short chain fatty acids
Cell also has important regulating and controlling effect, for maintaining enteron aisle normal physiological function and enterocyte form, function etc. to have pole
Its important role.
Short-chain fat acid content can reflect the activity of internal flora and the health status of body entirety, by measuring enteron aisle
Content or excrement Short-Chain Fatty Acids contents level can become evaluation and help sentenced with the variation of anaerobic bacteria in holistic diagnosis body
One of the primary dcreening operation index for a variety of chronic diseases of breaking, the study found that reducing for short chain fatty acids synthesis and having for a variety of diseases
It closes, for example, the diseases such as ulcerative colitis inflammation, Crohn disease, rheumatic arthritis and allergy.Therefore, it establishes a kind of simple, high
The method of effect and Accurate Determining intestinal contents or excrement Short-Chain Fatty Acids, for carrying out chronic disease in great amount of samples
The screening of (such as inflammatory bowel disease), it appears particularly important.The method of measurement short chain fatty acids mainly has gaschromatographic mass spectrometry at present
Combination method, gas chromatography, the chromatography of ions and liquid chromatography, but there is certain disadvantage in these methods before.Gas
Phase combined gas chromatography mass spectrometry instrument cost is higher, and data result processing is comparatively laborious；Gas chromatography and liquid phase before
Chromatography, derivatization step or the pre-treatment temperature for being related to short chain fatty acids are higher, these result in sample
The volatilization loss of product Short-Chain Fatty Acids causes measurement result inaccurate.
(king is color, Gao Peixin, Xu Jingnan, and et al. stool in mice Short-Chain Fatty Acids are extracted and detection method for king's coloured silk et al.
Foundation and application [J] modern biomedical be in progress, 2017 (06): 21-24.) establish using high effective liquid chromatography for measuring
The method of excrement Short-Chain Fatty Acids equally directlys adopt ether extraction short chain fatty acids, using isocratic elution program (phosphoric acid
With acetonitrile as mobile phase) it is measured object, but its treatment process is completed in room temperature, and is related to sodium hydroxide water
Solution neutralizes and short chain fatty acids this process of extraction, thus, the pre-treatment time is longer, and short chain fatty acids is caused to lose, and leads
Cause recovery of standard addition range (86.6%~105.8%) bigger than normal.In addition, liquid phase elution program is more than 25min, and only determine
Three kinds of acetic acid, butyric acid and isovaleric acid target products.
(Geng Meimei, Xu Liwei, Yuan Hongchao, et al. gas chromatography measure short in hog middle content Geng Meimei et al.
Chain fatty acid content [J] modern biomedical progress, 2015,15 (6): 1010-1014.) using gas chromatography measurement pig knot
Short-chain fat acid content in intestinal contents finally determines acetic acid, propionic acid, isobutyric acid, butyric acid, mesh in isovaleric acid and valeric acid 5
Object is marked, although minute is shorter (12min or so), method directlys adopt ultrapure water extraction short chain fatty acids, with inclined
Gas chromatograph is directly entered after phosphoric acid processing.Firstly, ultrapure water extraction short chain fatty acids are inefficient, this can be to the inspection of sample
Measured value is lower than actual value；Secondly, metaphosphoric acid effect is to reduce short chain fatty acids loss, but metaphosphoric acid handles the time in this method
Long (being greater than 2h), greatly reduce conventional efficient；Finally, containing a large amount of moisture in its sample, direct injected is not only resulted in
Chromatography column life seriously reduces, and can also seriously damage detector.Zhang Mengjie et al. is also using short in gas chromatography quantitative fecal
Chain fatty acid content equally uses dilute acid soln (2 Ethylbutanoic acid+hydrochloric acid) to extract short chain fatty acids, crosses the direct air inlet phase of film
Detection, it is low (recovery of standard addition 83.75%~94.95%) that this method equally exists short chain fatty acids recovery rate, when chromatography
Between long (about 40min), easily damage the shortcomings such as chromatographic column and instrument.
Summary of the invention
In view of the above-mentioned problems, being extracted, with solvent (such as ether) compared with low temperature after the present invention only needs to dissolve test sample
The lower centrifugation of degree, after taking supernatant anhydrous sodium sulfate dry, can directly carrying out gas phase, sample introduction is analyzed, before not needing other derivatizations
Processing step, pre-treatment time greatly shorten short, only need 15min or so time, the pre-treatment step simplified saves the time；
In addition, low-temperature treatment and pretreatment process in short-term, can effectively reduce the volatilization loss of short chain fatty acids, make analysis method more
It is accurate and efficiently, that is, the present invention develops short-chain fat in a kind of simple, measurement intestinal contents efficiently and accurately or excrement
The method of acid content.
The first purpose of the invention is to provide a kind of methods for measuring short-chain fat acid content, which is characterized in that uses
Ether directly extracts the short chain fatty acids in sample, is pre-processed at low temperature, then in gas chromatography measurement sample
Short-chain fat acid content, the pretreatment temperature be 2~8 DEG C.
A second object of the present invention is to provide a kind of methods for measuring short-chain fat acid content, comprising the following steps:
(1) sample pretreatment: after sample is mixed with saturated sodium chloride solution, acid solution acidification is added, keeps sample abundant
Dissolution adds the short chain fatty acids in ether extraction sample；Extracting solution extracted is centrifuged at 2~8 DEG C, takes supernatant
Dry anhydrous sodium sulfate is added, centrifugation, crosses film；
(2) gas chromatography measures: being measured using temperature program.
In one embodiment of the present invention, sample dissolution and acidification step condition in step (1) are as follows: saturated sodium-chloride adds
Enter 5-10 times that volume is sample；Acidification is using sulfuric acid or hydrochloric acid solution, and concentration is 5~15% (V/V), additional amount 0.05
~0.1 times of saturated sodium-chloride volume.
In one embodiment of the present invention, short chain fatty acids are extracted with ether in step (1), ether dosage is 10-20 μ
L/mg sample.
In one embodiment of the present invention, centrifugal condition after being extracted in step (1) are as follows: 8000~15 000r/ of revolving speed
Min, 5~10min of centrifugation time, 2~8 DEG C of temperature.
In one embodiment of the present invention, anhydrous sodium sulfate drying, centrifugal condition are added in step (1) are as follows: anhydrous sulphur
Sour sodium additional amount is 3~5 times of test sample quality；4000~6000r/min of centrifugal rotational speed, 3~5min of centrifugation time, temperature
2~8 DEG C.
In one embodiment of the present invention, gas chromatograph temperature program described in step (2) are as follows: chromatographic column is initially warm
Degree is 80~120 DEG C, is warming up to 150~200 DEG C by 5~10 DEG C/min heating rate.
In one embodiment of the present invention, step (2) gas chromatography condition are as follows: 1.8~2.5mL/min of flow velocity, into
1~2 μ L of sample amount, split ratio 1:(10~50), 250~270 DEG C of injector temperature, detector temperature is 260~280 DEG C.
In one embodiment of the present invention, the sample is intestinal contents or excrement.
In one embodiment of the present invention, the short chain fatty acids include acetic acid, propionic acid, isobutyric acid, n-butyric acie, isoamyl
Sour, positive valeric acid.
The application that third object of the present invention is to provide the methods in terms of biology sample detection.
Beneficial effects of the present invention:
After the present invention is by using extracted by ether, low-temperature centrifugation, can carrying out gas phase, sample introduction is analyzed, does not need other derivatives
Change pre-treatment step, pre-treatment time about 15min, machine analysis time about 15min on sample, about 30min (is less than in total
40min), it is more suitable for while the quantitative analysis of short time, a large amount of colonic contents or fecal specimens Short-Chain Fatty Acids content；
Low temperature can effectively reduce the volatilization loss of short chain fatty acids, the rate of recovery a height of 98.10 ± 0.29%~101.30 ± 0.27%；It adopts
Can be down to 0.0036 μ L/mL with the method for the present invention detection limit, quantitative limit can be more accurate and efficient down to 0.0041 μ L/mL.
Detailed description of the invention
Fig. 1 is short chain fatty acids hybrid standard product gas chromatogram.
Fig. 2 is SD rat colon contents samples Short-Chain Fatty Acids gas chromatogram.
Fig. 3 is SD rat fresh excreta sample Short-Chain Fatty Acids gas chromatogram.
Fig. 4 is to measure short chain fatty acids hybrid standard product gas chromatogram after changing temperature program.
Fig. 5 is measurement intestinal contents Short-Chain Fatty Acids sample gas chromatogram after replacement chromatographic column.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that embodiment is in order to preferably explain this hair
It is bright, it is not used in the limitation present invention.
Embodiment 1: measurement short chain fatty acids hybrid standard product standard curve
The sample is the mixed standard solution of short chain fatty acids, and every kind of short-chain fat acidity scale product are dense in standard curve
Spend range setting are as follows: 0.015~0.200 μ L/mL (short chain fatty acids standard items: solvent；V/V)；It is each short in corresponding standard items
The mass-volume concentration range of chain fatty acid are as follows: 15.738~209.840 μ g/mL of acetic acid, 14.850~198.000 μ g/ of propionic acid
ML, 14.235~189.800 μ g/mL of isobutyric acid, butyric acid 14.460~192.800 μ g/mL, 13.962~186.160 μ of isovaleric acid
G/mL, 14.085~187.800 μ g/mL of valeric acid.7 concentration of each standard analysis substance setting (for example, respectively 0.015,
0.025,0.050,0.075,0.100,0.150,0.200 μ L/mL)；Each concentration standard analysis substance is measured in parallel 3 times.It takes
3 times of peak area standard deviation are the minimum detection limit (LOD) of every kind of short chain fatty acids；Taking 10 times of peak area standard deviation is every kind
The minimum quantitative limit (LOQ) of short chain fatty acids.
Standard curve is drawn, for the ease of the difference of more different Short-Chain Fatty Acids in Biological Samples contents, by standard song
The concentration that line computation goes out test sample Short-Chain Fatty Acids is C0(unit: μ L/mL) can become C by following formula scales
(unit: μm ol/g)
Wherein, C is the concentration (μm ol/g) of test sample Short-Chain Fatty Acids after conversion, C0To be calculated from standard curve
The concentration (μ L/mL) of the test sample Short-Chain Fatty Acids arrived, ρ indicate the concentration (g/mL) of short chain fatty acids, and V indicates extraction sample
The volume (μ L) of solvent is added in product Short-Chain Fatty Acids, and m indicates the weight in wet base (g) of test sample, and M indicates mole of short chain fatty acids
Quality (g/mol).
The rate of recovery by measuring sample determines the accuracy of measuring method, and the rate of recovery (RR%) passes through following formula meter
It calculates:
Wherein, c1Indicate the concentration measured, c2Indicate the concentration for the standard substance being added in initial sample.Parallel independent survey
5 times fixed, the rate of recovery is expressed as mean+SD (SD).
It is the retention times of 1 standard solution Short-Chain Fatty Acids of table, standard curve, the range of linearity, minimum detection limit, minimum fixed
Amount limit and the rate of recovery
Note: a. standard deviation is calculated by 5 measurement results；B.LOD is minimum detection limit (μ L/mL), takes peak face
3 times of product standard deviation；The minimum quantitative limit of LOQ (μ L/mL), takes 10 times of peak area standard deviation；1 is acetic acid；2 be propionic acid；3 be different
Butyric acid；4 be n-butyric acie；5 be isovaleric acid；6 are positive valeric acid.
As shown in Figure 1, Shimadzu GC-2030Nexis gas chromatograph cooperates the highly polar column capillary of Agilent DB-FFAP
Pipe chromatographic column shows preferably to divide for six kinds of short chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid)
From effect, the corresponding peak of every kind of short chain fatty acids is sharp and discrete.As shown in Table 1, this method has different short chain fatty acids
Good analysis ability, response time stablize, calibration curve coefficient correlation height (R2> 0.9975), detection be limited to 0.0036~
0.0054 μ L/mL (being scaled mass-volume concentration is 3.351~5.666 μ g/mL)；Quantitatively it is limited to 0.0041~0.0066 μ L/
ML (reduced mass volumetric concentration is 3.816~6.925 μ g/mL), rate of recovery height (98.10 ± 0.29%~101.30 ±
0.27%), illustrate that this method has good accuracy and precision, and detect limit and quantitative limit is low, illustrate that it can be quasi-
The really less sample of analysis short-chain fat acid content meets quick and Accurate Determining short chain fatty acids needs well.
Embodiment 2: the content of measurement SD rat colon content Short-Chain Fatty Acids
(1) sample dissolves: about 50mg sample being mixed with 500 μ L saturated sodium chloride solutions, 0.1 times of saturated sodium-chloride is added
10% sulfuric acid solution of volume is acidified, and concussion dissolves sample sufficiently；
(2) short chain fatty acids are extracted: 800 μ L ether are added, shakes 30s, sufficiently extracts the short chain fatty acids in sample；
(3) sample preparation: extracting solution is centrifuged 10min at 12 000r/min, 4 DEG C, takes supernatant, and it is anhydrous that 0.25g is added
Solid sodium sulfate, after shaking 30s, 4500r/min 4 DEG C, is centrifuged 3min, removes traces of moisture, crosses 0.22 μm of organic micropore filter
Film is analyzed into chromatograph；
(4) gas chromatography measures: chromatographic column: the highly polar column capillary tube chromatographic column of Agilent DB-FFAP.Each every sample
After carrying out 3 independent replications, experimental data is analyzed.
Analysis condition: fid detector, carrier gas are high-purity N2, flow velocity 2.0mL/min, sample volume 2 μ L, split ratio 1:50, into
260 DEG C of sample mouth temperature, 270 DEG C of detector temperature；
Temperature program: after 100 DEG C of holding 1min, being warming up to 150 DEG C by 5 DEG C/min heating rate, is kept for 5 DEG C, when measurement
Between total 16min.
The sample chromatogram figure measured is as shown in Figure 2.
It is compared according to short chain fatty acids standard curve each in mixed mark, measures each short chain rouge in the SD rat colon content
The concentration of fat acid: 45.224 ± 1.111mmol/g of acetic acid；24.199 ± 3.253mmol/g of propionic acid；Isobutyric acid 1.198 ±
0.127mmol/g；14.975 ± 2.331mmol/g of n-butyric acie；1.710 ± 0.009mmol/g of isovaleric acid；Positive valeric acid 1.774 ±
0.104mmol/g.Experiment precision the results are shown in Table 2.
2 rat colon content Short-Chain Fatty Acids of table measure precision experiment result
Embodiment 3: the content of measurement SD rat fresh excreta Short-Chain Fatty Acids
(1) sample dissolves: about 50mg sample being mixed with 500 μ L saturated sodium chloride solutions, 0.05 times of saturation chlorination is added
10% sulfuric acid solution of sodium volume is acidified, and concussion dissolves sample sufficiently；
(2) short chain fatty acids are extracted: 800 μ L ether are added, shakes 30s, sufficiently extracts the short chain fatty acids in sample；
(3) sample preparation: extracting solution is centrifuged 10min at 12 000r/min, 4 DEG C, takes supernatant, and it is anhydrous that 0.25g is added
Solid sodium sulfate, after shaking 30s, 4500r/min 4 DEG C, is centrifuged 3min, removes traces of moisture, crosses 0.22 μm of organic micropore filter
Film is analyzed into chromatograph；
(4) gas chromatography measures: chromatographic column: the highly polar column capillary tube chromatographic column of Agilent DB-FFAP (chromatographic column ginseng
Number: the μ m 0.25mm of 30m × 0.25 i.d.).After every sample carries out 3 independent replications every time, experimental data is divided
Analysis.
Analysis condition: fid detector, carrier gas are high-purity N2, flow velocity 2.0mL/min, sample volume 2 μ L, split ratio 1:20, into
270 DEG C of sample mouth temperature, 280 DEG C of detector temperature；
Temperature program: after 100 DEG C of holding 1min, being warming up to 150 DEG C by 5 DEG C/min heating rate, is kept for 5 DEG C, when measurement
Between total 16min.
The sample chromatogram figure measured is as shown in Figure 3.
It is compared according to short chain fatty acids standard curve each in mixed mark, measures each short chain rouge in the SD rat colon content
The concentration of fat acid: 39.825 ± 2.556mmol/g of acetic acid；24.459 ± 4.684mmol/g of propionic acid；Isobutyric acid 1.211 ±
0.175mmol/g；14.116 ± 0.774mmol/g of n-butyric acie；1.358 ± 0.186mmol/g of isovaleric acid；Positive valeric acid 1.734 ±
0.195mmol/g.Experiment precision the results are shown in Table 3.
3 rat fresh excreta Short-Chain Fatty Acids of table measure precision experiment result
Comparative example 1: change temperature program
The method for using embodiment 2 measures short-chain fat acid concentration as the hybrid standard product of 0.015 μ L/mL, and difference exists
In: (4) gas chromatography measures: will heat up program and is changed to: after initial temperature is 150 DEG C of holding 1min, heating up by 2 DEG C/min fast
Rate is warming up to 180 DEG C, keeps 0min, the total 16min of minute.
Other conditions are as follows:
Chromatographic column: the highly polar column capillary tube chromatographic column of Agilent DB-FFAP；
Analysis condition: fid detector, carrier gas are high-purity N2, flow velocity 2.0mL/min, sample volume 2 μ L, split ratio 1:50, into
260 DEG C of sample mouth temperature, 270 DEG C of detector temperature；
The sample chromatogram figure measured is as shown in Figure 4.After comparison diagram 1 and 4 is it is found that change gas chromatographic detection condition, although
Detection time remains as 16 minutes, but since heating rate reduces, each short chain fatty acids appearance time is seriously lagged；It is 6 kinds short
Chain fatty acid peak sequence is constant, but separating effect is very poor, and each target short chain fatty acids peak type is widened, and goes out between peak and peak
Now more serious is adhered phenomenon, and it is quantitative to show that this method cannot be used for.When chromatographic condition changes, peak
Type and retention time will receive influence, and when heating rate reduces, the retention time of sample can postpone, and peak type can widen,
This also results in Fig. 4 as a result, being adhered between peak and peak, and peak figure is not sharp, therefore, can not be used for sample amounts.
Inventor proves by many experiments, and when initial temperature and too high or too low heating rate, peak type and retention time be will receive
It influences.
Comparative example 2: change chromatographic column
Intestinal contents Short-Chain Fatty Acids content is measured using the method for embodiment 2, difference is: (4) gas phase color
Spectrometry measurement: chromatographic column is changed to TR-FAME capillary chromatographic column (chromatography column parameter: the μ m of 60m × 0.25 0.25mm
i.d.)。
Other conditions are constant, are provided that
Analysis condition: fid detector, carrier gas be high-purity N 2, flow velocity 2.0mL/min, sample volume 2 μ L, split ratio 1:50, into
260 DEG C of sample mouth temperature, 270 DEG C of detector temperature；
Temperature program: after 100 DEG C of holding 1min, being warming up to 150 DEG C by 5 DEG C/min heating rate, is kept for 5 DEG C, when measurement
Between total 16min.
The sample chromatogram figure measured is as shown in Figure 5.After comparison diagram 2 and Fig. 5 are it is found that replace chromatographic column, even if gas phase color
Spectrum analysis condition and temperature program are constant, but the peak figure of the test sample come out, are still not separated by, between object mass peak
It is adhered, and peak type is not sharp, not discrete, unsightly, it is quantitative which cannot be used for sample Short-Chain Fatty Acids to peak figure
Analysis.The wire feeding of chromatographic column is different to the affinity of short chain fatty acids, and short chain fatty acids polarity is strong, it should with highly polar
Chromatogram column analysis, TR-FAME chromatography column polarity is relatively weak, is not so good as highly polar chromatography for the separating capacity of short chain fatty acids
Column, such as DP-FFAP, this is also situation about why occurring in Fig. 5.I.e. after the weaker TR-FAME chromatographic column of more polar exchanging, even if
Chromatographiccondition and temperature program are constant, can not reach separation purpose.
Comparative example 3: change pretreatment Extracting temperature
Using the concentration of each short chain fatty acids in the method measurement SD rat colon content of embodiment 2, difference is:
Sample preparation temperature in embodiment 2 is changed to room temperature (25 DEG C) by embodiment, other conditions measure SD rat knot with embodiment 2
The concentration of each short chain fatty acids in intestinal contents: 10.454 ± 0.380mmol/g of acetic acid；7.456 ± 1.965mmol/g of propionic acid；
0.949 ± 0.212mmol/g of isobutyric acid；5.630 ± 0.285mmol/g of n-butyric acie；1.157 ± 0.284mmol/g of isovaleric acid；Just
1.138 ± 0.461mmol/g of valeric acid.
For comparative example 2 with this as a result, illustrating pre-treatment sample under normal temperature condition, can lead seriously leads to short chain fatty acids
Devolatilization influences final measurement result, so that measurement result is relatively low.
Comparative example 4: it is compared with the prior art
Using existing short chain fatty acids pre-treating method, (" gas chromatography detects in excrement reference literature Zhang Mengjie etc.
The pre-treating method of short chain fatty acids ", contain Compound-acid solution pre-treatment step) processing SD rat colon contents samples, into
And measure the concentration of each short chain fatty acids, the specific steps are as follows:
(1) it takes 0.50g colon contents samples in the disposable centrifuge tube of 20mL, 8mL mixed acid dilute aqueous solution is added
(preparation method: 2-ethyl butyric acid of 15mL, 100mmol/L solution and 50mL, the mixed liquor of 5mmol/L hydrochloric acid solution).Vortex
After mixing 2min, 15000r/min room temperature is centrifuged 20min.
(2) transfer supernatant in 10mL test tube, take 1mL supernatant with 0.45 μm of water phase fibrous filters filter into gas phase into
In sample bottle, using chromatographic.
(3) gas chromatography measures: chromatographic column: the highly polar column capillary tube chromatographic column of Agilent DB-FFAP.Each every sample
After carrying out 3 independent replications, experimental data is analyzed.
Analysis condition: fid detector, carrier gas be high-purity N 2, flow velocity 2.0mL/min, sample volume 2 μ L, split ratio 1:50, into
260 DEG C of sample mouth temperature, 270 DEG C of detector temperature；
Temperature program: after 100 DEG C of holding 1min, being warming up to 150 DEG C by 5 DEG C/min heating rate, is kept for 5 DEG C, when measurement
Between total 16min.
The sample is in the same manner as in Example 2, time for sample pretreatment about 25min, 16 minutes upper machine testing time, total score
Analysing the time is about 41min.Measure the concentration of each short chain fatty acids in SD rat colon content: acetic acid 28.218 ±
1.792mmol/g；17.476 ± 4.171mmol/g of propionic acid；1.603 ± 0.147mmol/g of isobutyric acid；N-butyric acie 16.190 ±
3.805mmol/g；1.707 ± 0.018mmol/g of isovaleric acid；2.238 ± 0.472mmol/g of positive valeric acid.
Compared with the data in embodiment 2, in pre-treatment step, this method is mentioned at room temperature using mixing dilute acid solution
Short chain fatty acids are taken, and stablize short chain fatty acids, uses organic reagent ether at 2~8 DEG C with the present invention, handles short chain rouge
Fat acid is compared, and extraction efficiency reduces, and therefore, the short-chain fat acid content of measurement is relatively low.In comparative example 4 when sample pre-treatments
Between more than 20min, be much higher than the method for the invention.In addition, method described in comparative example 4, will directly extract short chain fatty acids
Sour dilution cross film after, be directly entered gas chromatograph.The aqueous sample of Long-term analysis, will cause chromatograph and chromatographic column
Damage, increases the maintenance cost cost of equipment.
In short, step is more simple using the method for the present invention under the premise of can guarantee test accuracy, the used time is shorter,
Test process is lower to the loss of gas chromatograph.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for measuring short-chain fat acid content, which is characterized in that directly extract the short chain rouge in sample using ether
Fat acid, is pre-processed at low temperature, then with the short-chain fat acid content in gas chromatography measurement sample, the pretreatment
Temperature is 2~8 DEG C.
2. the method according to claim 1, wherein the following steps are included:
(1) sample pretreatment: after sample is mixed with saturated sodium chloride solution, being added acid solution acidification, dissolve sample sufficiently,
Add the short chain fatty acids in ether extraction sample；Extracting solution extracted is centrifuged at 2~8 DEG C, supernatant is taken to be added
Anhydrous sodium sulfate centrifugation, crosses film at drying；
(2) gas chromatography measures: being measured using temperature program.
3. according to the method described in claim 2, it is characterized in that, sample dissolves and acidification step condition in step (1) are as follows: full
5-10 times that volume is sample is added with sodium chloride；Acidification is using sulfuric acid or hydrochloric acid solution, and concentration is 5~15% (V/V), is added
Entering amount is 0.05~0.1 times of saturated sodium-chloride volume.
4. according to the method described in claim 2, it is characterized in that, step (1) in extraction after centrifugal condition are as follows: revolving speed 8000~
15000r/min, 5~10min of centrifugation time, 2~8 DEG C of temperature.
5. according to the method described in claim 2, it is characterized in that, being centrifuged item after anhydrous sodium sulfate drying is added in step (1)
Part are as follows: 4000~6000r/min of revolving speed, 3~5min of centrifugation time, 2~8 DEG C of temperature.
6. according to the method described in claim 2, it is characterized in that, gas chromatograph temperature program described in step (2) are as follows: color
Composing column initial temperature is 80~120 DEG C, is warming up to 150~200 DEG C by 5~10 DEG C/min heating rate.
7. method according to claim 1 or 2, which is characterized in that gas chromatography condition are as follows: chromatographic column Agilent
The highly polar column capillary tube chromatographic column of DB-FFAP, 1.8~2.5mL/min of flow velocity, 1~2 μ L of sample volume, split ratio 1:(10~50),
250~270 DEG C of injector temperature, 260~280 DEG C of detector temperature.
8. method as claimed in claim 1 to 7, which is characterized in that the sample is intestinal contents or excrement.
9. any method of claim 1-8, which is characterized in that the short chain fatty acids include acetic acid, propionic acid, isobutyl
Acid, n-butyric acie, isovaleric acid, positive valeric acid.
10. application of any method of claim 1-9 in terms of biology sample detection.
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