WO2020232970A1 - 一种测定肠道内容物或粪便中短链脂肪酸含量的方法 - Google Patents

一种测定肠道内容物或粪便中短链脂肪酸含量的方法 Download PDF

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WO2020232970A1
WO2020232970A1 PCT/CN2019/113213 CN2019113213W WO2020232970A1 WO 2020232970 A1 WO2020232970 A1 WO 2020232970A1 CN 2019113213 W CN2019113213 W CN 2019113213W WO 2020232970 A1 WO2020232970 A1 WO 2020232970A1
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chain fatty
acid
short
fatty acids
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刘元法
叶展
曹晨
李进伟
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江南大学
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    • 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
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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

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  • the invention relates to a method for determining the content of short-chain fatty acids in intestinal contents or feces, and belongs to the field of biological sample detection.
  • the intestine is not only an important place for human digestion and absorption, but also the largest immune organ, which plays an extremely important role in maintaining normal immune defense functions.
  • the human intestine provides a good habitat for microorganisms.
  • the microbial population living in the human gastrointestinal tract can reach tens of trillions, which is 10 times the number of human cells. More than 1,000 bacterial species have been identified and confirmed, and there are approximately 160 species of microorganisms present in all individuals.
  • the genome of gut microbes includes about 3 million genes, which is 150 times the human genome and has metabolic functions that the human body does not possess.
  • Intestinal microecology refers to the unified whole of the interaction and mutual influence between the normal intestinal flora and its host.
  • intestinal microecology affects the health of the host and promotes inflammatory bowel disease, cardiovascular and cerebrovascular diseases, and kidney diseases.
  • the occurrence of many diseases such as illness and depression.
  • intestinal microbes can not only regulate the metabolism of nutrients, but their metabolites, namely short-chain fatty acids, also play an important role as a link between diet and host health. Received widespread attention.
  • Short-chain fatty acids also known as volatile organic acids, are organic fatty acids with carbon chains of 1 to 6, which can be divided into formic acid, acetic acid, propionic acid, butyric acid, valeric acid, etc. according to the number of carbons.
  • the short-chain fatty acids in organisms are mainly Acetic acid, propionic acid and butyric acid, which account for about 90%-95% of the total short-chain fatty acids, are generated by the degradation of carbohydrates by intestinal bacteria, and the remaining short-chain fatty acids account for only a small part (about 5%-10%) ), where branched short-chain fatty acids such as isobutyric acid and isovaleric acid come from the decomposition of protein.
  • Short-chain fatty acids are important organic acid anions in the intestinal lumen. They are absorbed by the colonic mucosa in ionic or non-ionic form, and are the main energy supply material for colon and small intestinal epithelial cells. Studies have found that short-chain fatty acids can promote the absorption of sodium ions, maintain the internal and external osmotic pressure of intestinal epithelial cells, promote colon cell proliferation and mucosal growth, provide metabolic energy, increase intestinal blood flow, and stimulate gastrointestinal hormone production. They are important for colonic mucosa. Nutrients.
  • certain short-chain fatty acids also have important regulatory effects on the immune cells of the intestinal mucosa and lamina basement, which play an extremely important role in maintaining the normal physiological functions of the intestine and the morphology and function of intestinal epithelial cells.
  • the content of short-chain fatty acids can reflect the activity of the flora in the body and the overall health of the body.
  • the overall diagnosis of anaerobic bacteria changes in the body can be used to evaluate and help judge a variety of chronic diseases.
  • One of the primary screening indicators for diseases studies have found that the reduction of short-chain fatty acid synthesis is related to the production of many diseases, such as ulcerative colon inflammation, Crohn’s disease, rheumatoid arthritis and allergies.
  • the establishment of a simple, efficient and accurate method for the determination of intestinal contents or short-chain fatty acids in feces is particularly important for the screening of chronic diseases (such as inflammatory bowel disease) in a large number of samples.
  • the current methods for the determination of short-chain fatty acids mainly include gas chromatography-mass spectrometry, gas chromatography, ion chromatography and liquid chromatography, but these previous methods have certain disadvantages.
  • the pretreatment time is longer, resulting in the loss of short-chain fatty acids, resulting in a large range of spike recovery (86.6% ⁇ 105.8) %).
  • the liquid phase elution procedure exceeded 25 minutes, and only three target products of acetic acid, butyric acid and isovaleric acid were detected.
  • Geng Meimei et al. Geng Meimei, Xu Liwei, Yuan Hongchao, et al. Determination of short-chain fatty acid content in pig colon by gas chromatography[J]. Advances in Modern Biomedicine, 2015, 15(6): 1010-1014.
  • gas chromatography The method was used to determine the content of short-chain fatty acids in the contents of the pig’s colon, and finally five target compounds of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were determined.
  • the method directly uses ultrapure water to extract short-chain fatty acids, and then directly enters the gas chromatograph after treatment with metaphosphoric acid.
  • the efficiency of ultrapure water extraction of short-chain fatty acids is not high, which will cause the detection value of the sample to be lower than the actual value;
  • the effect of metaphosphoric acid in this method is to reduce the loss of short-chain fatty acids, but the treatment time of metaphosphoric acid is long (more than 2h) , which greatly reduces the efficiency of the experiment; finally, the sample contains a lot of water, and direct injection will not only cause a serious decrease in the life of the chromatographic column, but also seriously damage the detector.
  • Zhang Mengjie and others also used gas chromatography to quantify the content of short-chain fatty acids in feces. They also used dilute acid solution (2-ethylbutyric acid + hydrochloric acid) to extract short-chain fatty acids, and passed the membrane directly into the gas phase. This method also has short-chain fatty acids.
  • the extraction rate of chain fatty acid is low (the recovery rate of standard addition is 83.75%-94.95%), the chromatographic analysis time is long (about 40min), and it is easy to damage the chromatographic column and instrument.
  • the present invention only needs to dissolve the test sample, extract it with a solvent (such as ether), centrifuge at a lower temperature, take the supernatant and dry it with anhydrous sodium sulfate, and then directly perform gas phase injection analysis.
  • a solvent such as ether
  • Other derivatization pre-treatment steps are required.
  • the pre-treatment time is greatly shortened, only about 15 minutes, which simplifies the pre-treatment steps and saves time; in addition, low-temperature treatment and short-term pre-treatment process can effectively reduce the short-chain fatty acid
  • the volatilization loss makes the analysis method more accurate and efficient, that is, the present invention develops a simple, efficient and accurate method for determining the content of short-chain fatty acids in the intestinal contents or feces.
  • the first object of the present invention is to provide a pretreatment method for determining the content of short-chain fatty acids.
  • the short-chain fatty acids in the sample are directly extracted by ether, and the pretreatment is performed at a low temperature, and the pretreatment temperature is 2-8°C.
  • the second object of the present invention is to provide an application of the above pretreatment method in detecting the content of short-chain fatty acids.
  • the third object of the present invention is to provide a method for determining the content of short-chain fatty acids, which is to process the sample to be tested by the above-mentioned pretreatment method, and then determine the content of short-chain fatty acids in the sample by gas chromatography.
  • the method includes the following steps:
  • Sample pretreatment After mixing the sample with a saturated sodium chloride solution, add acid solution to acidify the sample to fully dissolve the sample, and then add ether to extract the short-chain fatty acids in the sample; Centrifuge at °C, take the supernatant and add anhydrous sodium sulfate to dry, centrifuge, and pass the membrane;
  • the conditions of the sample dissolution and acidification step in step (1) are: saturated sodium chloride is added in a volume of 5-10 times that of the sample; acidification uses sulfuric acid or hydrochloric acid solution with a concentration of 5-15% (V/V), the addition amount is 0.05 to 0.1 times the volume of saturated sodium chloride.
  • step (1) ether is used to extract short-chain fatty acids, and the amount of ether is 10-20 ⁇ L/mg sample.
  • the centrifugation conditions after extraction in step (1) are: rotation speed 8000-150000 r/min, centrifugation time 5-10 min, temperature 2-8°C.
  • anhydrous sodium sulfate is added for drying and centrifugation conditions are: the addition of anhydrous sodium sulfate is 3 to 5 times the mass of the test sample; the centrifugal speed is 4000 to 6000 r/min, Time 3 ⁇ 5min, temperature 2 ⁇ 8°C.
  • the gas chromatograph temperature rising program in step (2) is as follows: the initial temperature of the chromatographic column is 80-120°C, and the temperature is raised to 150-200°C at a heating rate of 5-10°C/min.
  • the gas chromatography conditions of step (2) are as follows: flow rate 1.8 ⁇ 2.5mL/min, injection volume 1 ⁇ 2 ⁇ L, split ratio 1:(10 ⁇ 50), injection port temperature 250 ⁇ 270°C, the detector temperature is 260 ⁇ 280°C.
  • the method includes the following steps:
  • Sample pretreatment After mixing the sample with a saturated sodium chloride solution, add acid solution to acidify the sample to fully dissolve the sample, and then add ether to extract the short-chain fatty acids in the sample; Centrifuge at °C, take the supernatant and add anhydrous sodium sulfate to dry, centrifuge, and pass the membrane;
  • Gas chromatographic determination use a temperature rising program for determination; the gas chromatographic conditions are: chromatographic column: Agilent DB-FFAP strong polar column capillary column; gas chromatograph temperature rising program: the initial temperature of the column is 80 ⁇ At 120°C, the temperature is increased to 150-200°C at a heating rate of 5-10°C/min.
  • the method includes the following steps:
  • Sample dissolution Mix about 50 mg of sample with 500 ⁇ L saturated sodium chloride solution, add 0.1 times the volume of saturated sodium chloride 10% sulfuric acid solution to acidify, shake to dissolve the sample;
  • Heating program After holding at 100°C for 1 min, increase the temperature to 150°C at a heating rate of 5°C/min, and keep it at 5°C for a total of 16 minutes.
  • the sample is intestinal contents or stool.
  • the short-chain fatty acid includes acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid.
  • an external standard method is used to detect the content of short-chain fatty acids.
  • the fourth objective of the present invention is to provide the application of the method in the detection of biological samples.
  • gas-phase sample injection analysis can be carried out without other pre-treatment steps for derivatization.
  • the pre-treatment time is about 15 minutes
  • the sample analysis time is about 15 minutes
  • the detection limit of the inventive method can be as low as 0.0036 ⁇ L/mL, and the limit of quantification can be as low as 0.0041 ⁇ L/mL, which is more accurate and efficient.
  • Figure 1 is a gas chromatogram of a standard mixture of short-chain fatty acids.
  • Figure 2 is a gas chromatogram of short-chain fatty acids in the colon contents of SD rats.
  • Figure 3 is a gas chromatogram of short-chain fatty acids in a fresh stool sample of SD rats.
  • Figure 4 shows the gas chromatogram of the mixed standard of short-chain fatty acids after changing the temperature program.
  • Figure 5 is a gas chromatogram of a sample of short-chain fatty acids in the intestinal contents after replacing the chromatographic column.
  • Example 1 Determination of the standard curve of short-chain fatty acid mixed standards
  • the sample is a mixed standard solution of short-chain fatty acids, and the concentration range of each short-chain fatty acid standard product in the standard curve is set to: 0.015 ⁇ 0.200 ⁇ L/mL (short-chain fatty acid standard product: solvent; V/V);
  • concentration range of each short-chain fatty acid in the corresponding standard is: acetic acid 15.738 ⁇ 209.840 ⁇ g/mL, propionic acid 14.850 ⁇ 198.000 ⁇ g/mL, isobutyric acid 14.235 ⁇ 189.800 ⁇ g/mL, butyric acid 14.460 ⁇ 192.800 ⁇ g/mL mL, isovaleric acid 13.962 ⁇ 186.160 ⁇ g/mL, valeric acid 14.085 ⁇ 187.800 ⁇ g/mL.
  • each concentration of standard analyte is measured 3 times in parallel. Take 3 times the peak area standard deviation as the lowest limit of detection (LOD) for each short-chain fatty acid; take 10 times the peak area standard deviation as the lowest limit of quantification (LOQ) for each short-chain fatty acid.
  • LOD lowest limit of detection
  • LOQ lowest limit of quantification
  • C is the concentration of short-chain fatty acids in the test sample after conversion ( ⁇ mol/g)
  • C 0 is the concentration of short-chain fatty acids in the test sample calculated from the standard curve ( ⁇ L/mL)
  • ⁇ represents the concentration of short-chain fatty acids (g/mL)
  • V represents the volume of the short-chain fatty acid added to the solvent ( ⁇ L) in the extracted sample
  • m represents the wet weight (g) of the test sample
  • M represents the molar mass of the short-chain fatty acid (g/mol).
  • the accuracy of the determination method is determined by measuring the recovery rate of the sample, and the recovery rate (RR%) is calculated by the following formula:
  • c 1 represents the measured concentration
  • c 2 represents the concentration of the standard substance added to the initial sample.
  • a. The standard deviation is calculated from 5 measurements; b. LOD is the lowest detection limit ( ⁇ L/mL), which is 3 times the peak area standard deviation; LOQ is the lowest limit of quantification ( ⁇ L/mL), which is the peak area 10 times the standard deviation; 1 is acetic acid; 2 is propionic acid; 3 is isobutyric acid; 4 is n-butyric acid; 5 is isovaleric acid; 6 is n-valeric acid.
  • this method has a good analytical ability for different short-chain fatty acids, stable response time, high correlation coefficient of the standard curve (R 2 >0.9975), and detection limit of 0.0036 ⁇ 0.0054 ⁇ L/mL (converted to mass volume
  • concentration is 3.351 ⁇ 5.666 ⁇ g/mL
  • limit of quantification is 0.0041 ⁇ 0.0066 ⁇ L/mL
  • mass volume concentration is 3.816 ⁇ 6.925 ⁇ g/mL
  • recovery rate is high (98.10 ⁇ 0.29% ⁇ 101.30 ⁇ 0.27%), indicating this
  • the method has good accuracy and precision, and the detection limit and the quantification limit are low, indicating that it can accurately analyze samples with less short-chain fatty acid content, and it satisfies the need for rapid and accurate determination of short-chain fatty acids.
  • Example 2 Determination of the content of short-chain fatty acids in the colon contents of SD rats
  • Sample dissolution Mix about 50 mg of sample with 500 ⁇ L saturated sodium chloride solution, add 0.1 times the volume of saturated sodium chloride 10% sulfuric acid solution to acidify, shake to dissolve the sample;
  • Heating program After holding at 100°C for 1 min, increase the temperature to 150°C at a heating rate of 5°C/min, and keep it at 5°C for a total of 16 minutes.
  • the concentration of each short-chain fatty acid in the colon content of the SD rat was measured: acetic acid 45.224 ⁇ 1.111mmol/g; propionic acid 24.199 ⁇ 3.253mmol/g; isobutyl Acid 1.198 ⁇ 0.127mmol/g; n-butyric acid 14.975 ⁇ 2.331mmol/g; isovaleric acid 1.710 ⁇ 0.009mmol/g; n-valeric acid 1.774 ⁇ 0.104mmol/g.
  • the experimental precision results are shown in Table 2.
  • Example 3 Determination of the content of short-chain fatty acids in fresh feces of SD rats
  • Sample dissolution Mix about 50 mg of sample with 500 ⁇ L saturated sodium chloride solution, add 0.05 times the volume of saturated sodium chloride 10% sulfuric acid solution to acidify, and shake to make the sample fully dissolved;
  • Analysis conditions FID detector, high purity N 2 carrier gas, flow rate 2.0 mL/min, sample injection volume 2 ⁇ L, split ratio 1:20, inlet temperature 270°C, detector temperature 280°C;
  • Heating program After holding at 100°C for 1 min, increase the temperature to 150°C at a heating rate of 5°C/min, and keep it at 5°C for a total measurement time of 16 minutes.
  • the concentration of each short-chain fatty acid in the colon content of the SD rat was measured: acetic acid 39.825 ⁇ 2.556 mmol/g; propionic acid 24.459 ⁇ 4.684 mmol/g; isobutyl Acid 1.211 ⁇ 0.175mmol/g; n-butyric acid 14.116 ⁇ 0.774mmol/g; isovaleric acid 1.358 ⁇ 0.186mmol/g; n-valeric acid 1.734 ⁇ 0.195mmol/g.
  • the experimental precision results are shown in Table 3.
  • Example 2 The method of Example 2 was used to determine the mixed standard product with a concentration of 0.015 ⁇ L/mL of short-chain fatty acids. The difference is: (4) Gas chromatography determination: Change the heating program to: the initial temperature is 150 °C for 1 min, press 2 The temperature rise rate is °C/min to 180°C, keep for 0min, and the measurement time is 16min.
  • Example 2 The method of Example 2 was used to determine the content of short-chain fatty acids in the contents of the intestinal tract. The differences are: (4) Gas chromatography determination: Change the chromatographic column to TR-FAME capillary chromatographic column (column parameter: 60m ⁇ 0.25 ⁇ m ⁇ 0.25mm id).
  • Heating program After holding at 100°C for 1 min, increase the temperature to 150°C at a heating rate of 5°C/min, and keep it at 5°C for a total of 16 minutes.
  • the measured sample chromatogram is shown in Figure 5. Comparing Figure 2 and Figure 5, it can be seen that after replacing the chromatographic column, even if the gas chromatographic analysis conditions and heating program are unchanged, the peak diagram of the test sample is still not separated, and there is adhesion between the peaks of the target substance, and the peak shape is not sharp. , Is not discrete, and the peak pattern is not beautiful. This result cannot be used for the quantitative analysis of short-chain fatty acids in the sample.
  • the packing type of the chromatographic column has different affinity for short-chain fatty acids. Short-chain fatty acids have strong polarity and should be analyzed by a strong-polarity column.
  • TR-FAME chromatographic column has relatively weak polarity and its ability to separate short-chain fatty acids is not as good. Strongly polar columns, such as DP-FFAP, this is why the situation in Figure 5 appears. That is, after replacing the TR-FAME chromatographic column with weaker polarity, even if the chromatographic analysis conditions and heating program remain unchanged, the separation purpose cannot be achieved.
  • Example 2 The method of Example 2 was used to determine the concentration of each short-chain fatty acid in the colon content of SD rats. The difference is that the sample preparation temperature in Example 2 was changed to room temperature (25°C) in the example, and other conditions were the same as in Example 2.
  • the concentration of short-chain fatty acids in the colon contents of SD rats was measured: acetic acid 10.454 ⁇ 0.380mmol/g; propionic acid 7.456 ⁇ 1.965mmol/g; isobutyric acid 0.949 ⁇ 0.212mmol/g; n-butyric acid 5.630 ⁇ 0.285 mmol/g; isovaleric acid 1.157 ⁇ 0.284mmol/g; n-valeric acid 1.138 ⁇ 0.461mmol/g.
  • Example 2 Comparing Example 2 with the results, it shows that pre-processing samples under normal temperature conditions will cause serious loss of volatility of short-chain fatty acids, affect the final measurement results, and make the measurement results low.
  • the existing short-chain fatty acid pretreatment method (refer to the document Zhang Mengjie et al. "Pretreatment Method for Detecting Short-chain Fatty Acids in Feces by Gas Chromatography", which contains the pretreatment steps of compound acid solution) is used to process the SD rat colon contents sample, and then determine The specific steps for the concentration of each short-chain fatty acid are as follows:
  • Heating program After holding at 100°C for 1 min, increase the temperature to 150°C at a heating rate of 5°C/min, and keep it at 5°C for a total of 16 minutes.
  • the sample is the same as in Example 2.
  • the sample pretreatment time is about 25 minutes, the machine detection time is 16 minutes, and the total analysis time is about 41 minutes.
  • the concentration of each short-chain fatty acid in the colon content of SD rats was measured: acetic acid 28.218 ⁇ 1.792mmol/g; propionic acid 17.476 ⁇ 4.171mmol/g; isobutyric acid 1.603 ⁇ 0.147mmol/g; n-butyric acid 16.190 ⁇ 3.805mmol /g; isovaleric acid 1.707 ⁇ 0.018mmol/g; n-valeric acid 2.238 ⁇ 0.472mmol/g.
  • this method uses a mixed dilute acid aqueous solution to extract short-chain fatty acids at room temperature, and stabilizes the short-chain fatty acids.
  • the extraction efficiency is reduced. Therefore, the measured short-chain fatty acid content is relatively low.
  • the sample pretreatment time in Comparative Example 4 exceeded 20 minutes, which was much higher than the method of the present invention.
  • the acid diluent for extracting short-chain fatty acids was directly passed into the gas chromatograph after passing the membrane. Long-term analysis of water-containing samples will cause damage to the chromatograph and the chromatographic column, and increase the maintenance cost of the equipment.
  • the steps are simpler, the time used is shorter, and the loss of the gas chromatograph in the test process is lower.

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Abstract

一种测定肠道内容物或粪便中短链脂肪酸含量的方法，采用乙醚对样品进行预处理，然后用气相色谱法测定样品中的短链脂肪酸含量；预处理温度为2～8℃。该方法不需要其他衍生化前处理步骤，前处理时间短于15min，总测试时长短于20min；低温可有效降低短链脂肪酸的挥发损失，回收率高为98.10±0.29％～101.30±0.27％；采用该方法检测限能低至0.0036μL/mL，定量限能低至0.0041μL/mL，对于短链脂肪酸的检测更为准确和高效。

Description

一种测定肠道内容物或粪便中短链脂肪酸含量的方法 技术领域
本发明涉及一种测定肠道内容物或粪便中短链脂肪酸含量的方法，属于生物样品检测领域。
背景技术
肠道不仅是人体消化吸收的重要场所，同时也是最大的免疫器官，在维持正常免疫防御功能中发挥着极其重要的作用。人体肠道为微生物提供了良好的栖息环境，居住在人体胃肠道中的微生物群体可达数十万亿，是人体细胞数量的10倍。有超过1000个菌种已经被鉴定确认，所有个体中均存在的微生物大约有160种。肠道微生物的基因组中包括约300万个基因，是人类基因组的150倍，具有人体自身不具备的代谢功能。肠道微生态是指肠道正常菌群与其宿主之间相互作用、相互影响的统一整体。目前，越来越多的研究表明，肠道微生态与宿主健康存在着直接或者间接的联系，肠道微生态功能的失衡，影响着宿主健康，促使炎症性肠病、心脑血管疾病、肾脏疾病和抑郁类疾病等诸多疾病的发生。作为人体内最庞大、最复杂的微生态系统，肠道微生物不仅能调节营养物质代谢，其代谢产物，即短链脂肪酸，还在膳食和宿主健康之间起到重要的纽带作用，近年来受到了广泛关注。
短链脂肪酸又称挥发性有机酸，是碳链为1～6的有机脂肪酸，按碳数多少可分为甲酸、乙酸、丙酸、丁酸、戊酸等，而生物体内的短链脂肪酸主要为乙酸、丙酸和丁酸，约占短链脂肪酸总量的90％～95％，由碳水化合物经肠道细菌降解生成，其余短链脂肪酸只占很小部分(约为5％～10％)，其中异丁酸和异戊酸等支链短链脂肪酸来源于蛋白质的分解。短链脂肪酸是肠腔内重要的有机酸阴离子，通过离子或非离子形式被结肠粘膜吸收，是结肠和小肠上皮细胞的主要供能物质。研究发现，短链脂肪酸可以促进钠离子吸收，维持肠上皮细胞的内外渗透压，促进结肠细胞增殖和粘膜生长，提供代谢能源，增加肠道血流，刺激胃肠激素生成，是结肠粘膜的重要营养素。此外，某些短链脂肪酸对肠道黏 膜和固有层的免疫细胞也有重要调控效应，其对于维持肠道正常生理功能和肠上皮细胞形态、功能等具有极其重要的作用。
短链脂肪酸含量可以反映体内菌群的活性和机体整体的健康状况，通过测定肠道内容物或粪便中短链脂肪酸含量水平可以整体诊断体内厌氧菌的变化，成为评价和帮助判断多种慢性疾病的初筛指标之一，研究发现，短链脂肪酸合成的减少与多种疾病的产生有关，例如，溃疡性结肠炎症、克罗恩病、风湿性关节炎和过敏等疾病。因此，建立一种简单、高效和准确测定肠道内容物或粪便中短链脂肪酸的方法，对于在大量样本中进行慢性疾病(如炎症性肠病)的筛查，显得尤为重要。目前测定短链脂肪酸的方法主要有气相色谱质谱联用法、气相色谱法、离子色谱法和液相色谱法，但是之前的这些方法均存在一定缺点。气相色谱质谱联用法仪器成本较高，而且数据结果处理比较繁琐；之前的气相色谱法和液相色谱法，或者是涉及到短链脂肪酸的衍生化步骤，或者是前处理温度较高，这些均导致了样品中短链脂肪酸的挥发损失，造成测定结果不准确。
王彩等人(王彩，高培鑫，许竞男，et al.小鼠粪便中短链脂肪酸提取与检测方法的建立及应用[J].现代生物医学进展，2017(06)：21-24.)建立了采用高效液相色谱法测定粪便中短链脂肪酸的方法，其同样直接采用乙醚萃取短链脂肪酸，采用等度洗脱程序(磷酸和乙腈作为流动相)进行测定目标物，但是其处理过程在室温完成，并涉及到用氢氧化钠水溶液中和和萃取的短链脂肪酸这一过程，因而，前处理时间较长，造成短链脂肪酸损失，导致加标回收率范围偏大(86.6％～105.8％)。此外，液相洗脱程序超过25min，而且仅测定出乙酸、丁酸和异戊酸三种目标产物。
耿梅梅等人(耿梅梅，许丽卫，袁红朝，et al.气相色谱法测定猪结肠内容物中短链脂肪酸含量[J].现代生物医学进展，2015，15(6)：1010-1014.)采用气相色谱法测定猪结肠内容物中的短链脂肪酸含量，最终确定出乙酸、丙酸、异丁酸、丁酸、异戊酸和戊酸5中目标物，虽然测定时间更短(12min左右)，但是，其方法直接采用超纯水萃取短链脂肪酸，用偏磷酸处理后直接进入气相色谱仪。首先，超纯水萃取短链脂肪酸效率不高，这会对样品的检测值低于实际值；其次， 该方法中偏磷酸作用是减少短链脂肪酸损失，但是偏磷酸处理时间长(大于2h)，大大降低了实验效率；最后，其样品中含有大量的水分，直接进样，不仅会导致色谱柱寿命严重降低，还会严重损害检测器。张梦洁等人也采用气相色谱法定量粪便中短链脂肪酸含量，其同样采用稀酸溶液(2-乙基丁酸+盐酸)萃取短链脂肪酸，过膜直接进气相检测，该方法同样存在短链脂肪酸提取率低(加标回收率83.75％～94.95％)，色谱分析时间长(约40min)，易损害色谱柱和仪器等诸多缺点。
发明内容
针对上述问题，本发明只需要将测试样品溶解后，用溶剂(如乙醚)提取、在较低温度下离心，取上清用无水硫酸钠干燥后，即可直接进行气相进样分析，不需要其他衍生化前处理步骤，前处理时间大大缩短短，只需15min左右时间，简化了的前处理步骤，节省时间；此外，低温处理和短时的前处理过程，可有效降低短链脂肪酸的挥发损失，使分析方法更为准确和高效，即，本发明开发了一种简单、高效和准确的测定肠道内容物或粪便中短链脂肪酸含量的方法。
本发明的第一个目的是提供一种测定短链脂肪酸含量的预处理方法，采用乙醚直接萃取样品中的短链脂肪酸，在低温下进行预处理，所述预处理温度为2～8℃。
本发明的第二个目的是提供一种上述预处理方法在检测短链脂肪酸含量方面的应用。
本发明的第三个目的是提供一种测定短链脂肪酸含量的方法，所述方式是采用上述预处理方法处理待测样品，然后用气相色谱法测定样品中的短链脂肪酸含量。
在本发明一种实施方式中，所述方法包括以下步骤：
(1)样品预处理：将样品用饱和氯化钠溶液混匀后，加入酸溶液酸化，使样品充分溶解，再加入乙醚萃取样品中的短链脂肪酸；将萃取后的提取液在2～8℃下离心，取上清液加入无水硫酸钠干燥、离心、过膜；
(2)气相色谱法测定：采用升温程序进行测定。
在本发明一种实施方式中，步骤(1)中样品溶解和酸化步骤条件为：饱和氯 化钠加入体积为样品的5-10倍；酸化是采用硫酸或盐酸溶液，浓度为5～15％(V/V)，加入量为0.05～0.1倍饱和氯化钠体积。
在本发明一种实施方式中，步骤(1)中用乙醚萃取短链脂肪酸，乙醚用量为10-20μL/mg样品。
在本发明一种实施方式中，步骤(1)中萃取后离心条件为：转速8000～15 000r/min，离心时间5～10min，温度2～8℃。
在本发明一种实施方式中，步骤(1)中加入无水硫酸钠干燥、离心条件为：无水硫酸钠加入量为测试样品质量的3～5倍；离心转速4000～6000r/min，离心时间3～5min，温度2～8℃。
在本发明一种实施方式中，步骤(2)中所述气相色谱仪升温程序为：色谱柱初始温度为80～120℃，按5～10℃/min升温速率升温至150～200℃。
在本发明一种实施方式中，步骤(2)气相色谱法条件为：流速1.8～2.5mL/min，进样量1～2μL，分流比1∶(10～50)，进样口温度250～270℃，检测器温度为260～280℃。
在本发明一种实施方式中，所述方法包括以下步骤：
(1)样品预处理：将样品用饱和氯化钠溶液混匀后，加入酸溶液酸化，使样品充分溶解，再加入乙醚萃取样品中的短链脂肪酸；将萃取后的提取液在2～8℃下离心，取上清液加入无水硫酸钠干燥、离心、过膜；
(2)气相色谱法测定：采用升温程序进行测定；所述气相色谱条件为：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱；气相色谱仪升温程序为：色谱柱初始温度为80～120℃，按5～10℃/min升温速率升温至150～200℃。
在本发明一种实施方式中，所述方法包括以下步骤：
(1)样品溶解：将约50mg样品用500μL饱和氯化钠溶液混匀，加入0.1倍饱和氯化钠体积的10％硫酸溶液酸化，震荡使样品充分溶解；
(2)短链脂肪酸提取：加入800μL乙醚，震荡30s，充分萃取样品中的短链脂肪酸；
(3)样品制备：提取液在12 000r/min，4℃下，离心10min，取上清，加入0.25g无水硫酸钠固体，震荡30s后，4500r/min，4 ℃，离心3min，去除痕量水分，过0.22μm有机微孔滤膜，进色谱仪分析；
(4)气相色谱法测定：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱。每次每样进行3次独立重复测定后，对实验数据进行分析。
分析条件：FID检测器，载气为高纯N 2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃，测定时间共16min。
在本发明一种实施方式中，所述样品为肠道内容物或粪便。
在本发明一种实施方式中，所述短链脂肪酸包括乙酸、丙酸、异丁酸、正丁酸、异戊酸、正戊酸。
在本发明一种实施方式中，采用外标法检测短链脂肪酸的含量。
在本发明一种实施方式中，乙酸标准曲线为y＝509204.16x-852.08，y为峰面积，x为乙酸浓度，μL/mL。
在本发明一种实施方式中，丙酸标准曲线为y＝826031.70x-1147.79，y为峰面积，x为丙酸浓度，μL/mL。
在本发明一种实施方式中，异丁酸标准曲线为y＝1020753.28x-1119.04，y为峰面积，x为异丁酸浓度，μL/mL。
在本发明一种实施方式中，正丁酸标准曲线为y＝1037614.63x-632.86，y为峰面积，x为正丁酸浓度，μL/mL。
在本发明一种实施方式中，异戊酸标准曲线为y＝1122548.15x-1222.30，y为峰面积，x为异戊酸浓度，μL/mL。
在本发明一种实施方式中，正戊酸标准曲线为y＝1113530.09x-1433.43，y为峰面积，x为正戊酸浓度，μL/mL。
本发明的第四个目的是提供所述方法在生物样品检测方面的应用。
发明概述
发明的有益效果
有益效果
本发明通过采用乙醚提取、低温离心后，即可进行气相进样分析，不需要其他 衍生化前处理步骤，前处理时间约15min，样品上机分析时间约15min，总共约30min(小于40min)，更适合同时短时间、大量的结肠内容物或粪便样品中短链脂肪酸含量的定量分析；低温可有效降低短链脂肪酸的挥发损失，回收率高为98.10±0.29％～101.30±0.27％；采用本发明方法检测限能低至0.0036μL/mL，定量限能低至0.0041μL/mL，更为准确和高效。
对附图的简要说明
附图说明
图1为短链脂肪酸混合标准品气相色谱图。
图2为SD大鼠结肠内容物样品中短链脂肪酸气相色谱图。
图3为SD大鼠新鲜粪便样品中短链脂肪酸气相色谱图。
图4为改变升温程序后测定短链脂肪酸混合标准品气相色谱图。
图5为更换色谱柱后测定肠道内容物中短链脂肪酸样品气相色谱图。
发明实施例
本发明的实施方式
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。
实施例1：测定短链脂肪酸混合标准品标准曲线
所述样品为短链脂肪酸的混合标准溶液，每种短链脂肪酸标品在标准曲线中的浓度范围设定为：0.015～0.200μL/mL(短链脂肪酸标准品：溶剂；V/V)；相应的标准品中各短链脂肪酸的质量体积浓度范围为：乙酸15.738～209.840μg/mL，丙酸14.850～198.000μg/mL，异丁酸14.235～189.800μg/mL，丁酸14.460～192.800μg/mL，异戊酸13.962～186.160μg/mL，戊酸14.085～187.800μg/mL。每个标准分析物质设定7浓度(例如，分别为0.015，0.025，0.050，0.075，0.100，0.150，0.200μL/mL)；每个浓度标准分析物质平行测定3次。取峰面积标准差的3倍为每种短链脂肪酸的最低检测限(LOD)；取峰面积标准差的10倍为每种短链脂肪酸的最低定量限(LOQ)。
绘制标准曲线，为了便于比较不同生物样品中短链脂肪酸含量的差异，由标准曲线计算出测试样品中短链脂肪酸的浓度为C 0(单位：μL/mL)，可以通过如 下公式换算成为C(单位：μmol/g)
Figure PCTCN2019113213-appb-000001
其中，C为换算后测试样品中短链脂肪酸的浓度(μmol/g)，C 0为从标准曲线计算得到的测试样品中短链脂肪酸的浓度(μL/mL)，ρ表示短链脂肪酸的浓度(g/mL)，V表示萃取样品中短链脂肪酸加入溶剂的体积(μL)，m表示测试样品的湿重(g)，M表示短链脂肪酸的摩尔质量(g/mol)。
通过测定样品的回收率确定测定方法的准确度，回收率(RR％)通过如下公式计算：
Figure PCTCN2019113213-appb-000002
其中，c 1表示测得的浓度，c 2表示加入初始样品中的标准物质的浓度。平行独立测定5次，回收率表示为平均值±标准差(SD)。
表1标准溶液中短链脂肪酸的保留时间、标准曲线、线性范围、最低检测限、最低定量限和回收率
[Table 1]
Figure PCTCN2019113213-appb-000003
Figure PCTCN2019113213-appb-000004
注：a.标准偏差通过5次测定结果计算得出；b.LOD为最低检测限(μL/mL)，取峰面积标准差的3倍；LOQ最低定量限(μL/mL)，取峰面积标准差的10倍；1为乙酸；2为丙酸；3为异丁酸；4为正丁酸；5为异戊酸；6为正戊酸。
由图1可知，岛津GC-2030 Nexis气相色谱仪，配合Agilent DB-FFAP强极性柱毛细管色谱柱，对于六种短链脂肪酸(乙酸、丙酸、异丁酸、丁酸、异戊酸和戊酸)表现出较好的分离效果，每种短链脂肪酸对应的峰尖而离散。由表1可知，该方法对不同的短链脂肪酸具有很好的分析能力，响应时间稳定，标准曲线相关系数高(R 2＞0.9975)，检测限为0.0036～0.0054μL/mL(换算为质量体积浓度为3.351～5.666μg/mL)；定量限为0.0041～0.0066μL/mL(换算质量体积浓度为3.816～6.925μg/mL)，回收率高(98.10±0.29％～101.30±0.27％)，说明该方法具有良好的准确度和精密度，并且检测限和定量限低，说明其可以准确分析短链脂肪酸含量较少的样品，其很好地满足快速及准确测定短链脂肪酸的需要。
实施例2：测定SD大鼠结肠内容物中短链脂肪酸的含量
(1)样品溶解：将约50mg样品用500μL饱和氯化钠溶液混匀，加入0.1倍饱和氯化钠体积的10％硫酸溶液酸化，震荡使样品充分溶解；
(2)短链脂肪酸提取：加入800μL乙醚，震荡30s，充分萃取样品中的短链脂肪酸；
(3)样品制备：提取液在12000r/min，4℃下，离心10min，取上清，加入0.25g无水硫酸钠固体，震荡30s后，4500r/min，4℃，离心3min，去除痕量水分，过0.22μm有机微孔滤膜，进色谱仪分析；
(4)气相色谱法测定：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱。每次每样进行3次独立重复测定后，对实验数据进行分析。
分析条件：FID检测器，载气为高纯N 2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃，测定时间共16min。
测得的样品色谱图如图2所示。
根据与混标中各短链脂肪酸标准曲线比对，测得该SD大鼠结肠内容物中各短链脂肪酸的浓度：乙酸45.224±1.111mmol/g；丙酸24.199±3.253mmol/g；异丁酸1.198±0.127mmol/g；正丁酸14.975±2.331mmol/g；异戊酸1.710±0.009mmol/g；正戊酸1.774±0.104mmol/g。实验精密度结果见表2。
表2大鼠结肠内容物中短链脂肪酸测定精密度实验结果
Figure PCTCN2019113213-appb-000005
实施例3：测定SD大鼠新鲜粪便中短链脂肪酸的含量
(1)样品溶解：将约50mg样品用500μL饱和氯化钠溶液混匀，加入0.05倍饱和氯化钠体积的10％硫酸溶液酸化，震荡使样品充分溶解；
(2)短链脂肪酸提取：加入800μL乙醚，震荡30s，充分萃取样品中的短链脂肪酸；
(3)样品制备：提取液在12000r/min，4℃下，离心10min，取上清，加入0.25g无水硫酸钠固体，震荡30s后，4500r/min，4℃，离心3min，去除痕量水分，过0.22μm有机微孔滤膜，进色谱仪分析；
(4)气相色谱法测定：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱(色谱柱参数：30m×0.25μm×0.25mm i.d.)。每次每样进行3次独立重复测定后，对实验数据进行分析。
分析条件：FID检测器，载气为高纯N 2，流速2.0mL/min，进样量2μL，分流比1∶20，进样口温度270℃，检测器温度280℃；
升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃， 测定时间共16min。
测得的样品色谱图如图3所示。
根据与混标中各短链脂肪酸标准曲线比对，测得该SD大鼠结肠内容物中各短链脂肪酸的浓度：乙酸39.825±2.556mmol/g；丙酸24.459±4.684mmol/g；异丁酸1.211±0.175mmol/g；正丁酸14.116±0.774mmol/g；异戊酸1.358±0.186mmol/g；正戊酸1.734±0.195mmol/g。实验精密度结果见表3。
表3大鼠新鲜粪便中短链脂肪酸测定精密度实验结果
Figure PCTCN2019113213-appb-000006
对比例1：改变升温程序
采用实施例2的方法测定短链脂肪酸浓度为0.015μL/mL的混合标准品，其区别在于：(4)气相色谱法测定：将升温程序改为：初始温度为150℃保持1min后，按2℃/min升温速率升温至180℃，保持0min，测定时间共16min。
其他条件如下：
色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱；
分析条件：FID检测器，载气为高纯N 2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
测得的样品色谱图如图4所示。对比图1和4可知，改变气相色谱检测条件后，虽然检测时间仍然为16分钟，但是由于升温速率降低，各短链脂肪酸出峰时间出峰时间严重滞后；6种短链脂肪酸出峰顺序不变，但是分离效果极差，各个目标短链脂肪酸峰型加宽，峰与峰之间出现较为严重的黏连现象，表明该方法不能用于定量。当气相色谱仪分析条件改变的时候，峰型和保留时间均会受到影 响，当升温速率降低，样品的保留时间会延迟，而且峰型会加宽，这也导致了图4中的结果，峰与峰之间出现黏连，峰图不尖锐，因此，也不能用于样品定量。发明人经过多次实验证明，初始温度和升温速率过高或过低时，峰型和保留时间均会受到影响。
对比例2：改变色谱柱
采用实施例2的方法测定肠道内容物中短链脂肪酸含量，其区别在于：(4)气相色谱法测定：将色谱柱改为TR-FAME毛细管色谱柱(色谱柱参数：60m×0.25μm×0.25mm i.d.)。
其他条件不变，设置如下：
分析条件：FID检测器，载气为高纯N2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃，测定时间共16min。
测得的样品色谱图如图5所示。对比图2和图5可知，更换色谱柱之后，即使气相色谱分析条件和升温程序不变，但是出来的测试样品的峰图，仍然没有分开，目标物质峰之间出现黏连，而且峰型不尖锐，不离散，峰图不美观，该结果不能用于样品中短链脂肪酸定量分析。色谱柱的填料类型对短链脂肪酸的亲和力不同，短链脂肪酸极性强，应该用强极性的色谱柱分析，TR-FAME色谱柱极性相对较弱，其对于短链脂肪酸的分离能力不如强极性色谱柱，如DP-FFAP，这也是为什么出现图5中的情况。即更换极性较弱的TR-FAME色谱柱后，即使色谱分析条件和升温程序不变，也不能达到分离目的。
对比例3：改变预处理提取温度
采用实施例2的方法测定SD大鼠结肠内容物中各短链脂肪酸的浓度，其区别在于：将实施例将实施例2中样品制备温度改为室温(25℃)，其他条件同实施例2，测得SD大鼠结肠内容物中各短链脂肪酸的浓度：乙酸10.454±0.380mmol/g；丙酸7.456±1.965mmol/g；异丁酸0.949±0.212mmol/g；正丁酸5.630±0.285mmol/g；异戊酸1.157±0.284mmol/g；正戊酸1.138±0.461mmol/g。
对比实施例2与该结果，说明常温条件下前处理样品，会导严重导致短链脂肪酸的挥发性损失，影响最终测定结果，使得测定结果偏低。
对比例4：与现有技术进行对比
采用现有的短链脂肪酸前处理方法(参照文献张梦洁等《气相色谱法检测粪便中短链脂肪酸的前处理方法》，含有复合酸溶液前处理步骤)处理SD大鼠结肠内容物样品，进而测定各短链脂肪酸的浓度，具体步骤如下：
(1)取0.50g结肠内容物样品于20mL一次性离心管中，加入8mL混合酸稀释水溶液(配制方法：15mL，100mmol/L 2-乙基丁酸溶液和50mL，5mmol/L盐酸溶液的混合液)。旋涡混匀2min后，15000r/min室温离心20min。
(2)转移上清液于10mL试管中，取1mL上清液用0.45μm水相纤维滤器过滤入气相进样小瓶中，采用气相色谱仪分析。
(3)气相色谱法测定：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱。每次每样进行3次独立重复测定后，对实验数据进行分析。
分析条件：FID检测器，载气为高纯N2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃，测定时间共16min。
所述样品与实施例2中相同，样品前处理时间约25min，上机检测时间16分钟，总分析时间为约41min。测得SD大鼠结肠内容物中各短链脂肪酸的浓度：乙酸28.218±1.792mmol/g；丙酸17.476±4.171mmol/g；异丁酸1.603±0.147mmol/g；正丁酸16.190±3.805mmol/g；异戊酸1.707±0.018mmol/g；正戊酸2.238±0.472mmol/g。
与实施例2中的数据相比，前处理步骤中，该方法采用混合稀酸水溶液在室温下提取短链脂肪酸，并稳定短链脂肪酸，与用本发明使用有机试剂乙醚在2～8℃下，处理短链脂肪酸相比，萃取效率降低，因此，测定的短链脂肪酸含量相对较低。对比例4中样品前处理时间超过20min，远高于本发明所述方法。此外，对比例4中所述方法，直接将提取短链脂肪酸的酸稀释液过膜后，直接进入气相色谱仪。长期分析含水的样品，对色谱仪和色谱柱会造成损害，增加了设备的 维护费用成本。
总之，采用本发明方法在能保证测试准确性的前提下，步骤更为简单，用时更短，测试过程对气相色谱仪的损耗更低。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (22)

  1. 一种测定短链脂肪酸含量的方法，其特征在于，所述方法包括以下步骤：
    (1)样品预处理：将样品用饱和氯化钠溶液混匀后，加入酸溶液酸化，使样品充分溶解，再加入乙醚萃取样品中的短链脂肪酸；将萃取后的提取液在2～8℃下离心，取上清液加入无水硫酸钠干燥、离心、过膜；
    (2)气相色谱法测定：采用升温程序进行测定；所述气相色谱条件为：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱；气相色谱仪升温程序为：色谱柱初始温度为80～120℃，按5～10℃/min升温速率升温至150～200℃。
  2. 一种测定短链脂肪酸含量的预处理方法，其特征在于，采用乙醚直接萃取样品中的短链脂肪酸，在低温下进行预处理，所述预处理温度为2～8℃。
  3. 根据权利要求2所述的预处理方法，其特征在于，乙醚用量为10-20μL/mg样品
  4. 一种测定短链脂肪酸含量的方法，其特征在于，采用权利要求2或3所述的预处理方法处理待测样品，然后用气相色谱法测定样品中的短链脂肪酸含量。
  5. 根据权利要求4所述的方法，其特征在于，包括以下步骤：
    (1)样品预处理：将样品用饱和氯化钠溶液混匀后，加入酸溶液酸化，使样品充分溶解，再加入乙醚萃取样品中的短链脂肪酸；将萃取后的提取液在2～8℃下离心，取上清液加入无水硫酸钠离心、干燥、过膜；
    (2)气相色谱法测定：采用升温程序进行测定。
  6. 根据权利要求5所述的方法，其特征在于，步骤(1)中样品溶解和酸化步骤条件为：饱和氯化钠加入体积为样品的5-10倍；酸化是采用硫酸或盐酸溶液，浓度为5～15％(V/V)，加入量为0.05～0. 1倍饱和氯化钠体积。
  7. 根据权利要求5所述的方法，其特征在于，步骤(1)中萃取后离心条件为：转速8000～15000r/min，离心时间5～10min，温度2～8℃。
  8. 根据权利要求5所述的方法，其特征在于，步骤(1)中加入无水硫酸钠干燥后离心条件为：转速4000～6000r/min，离心时间3～5min，温度2～8℃。
  9. 根据权利要求5所述的方法，其特征在于，步骤(2)中所述气相色谱仪升温程序为：色谱柱初始温度为80～120℃，按5～10℃/min升温速率升温至150～200℃。
  10. 根据权利要求4或5所述的方法，其特征在于，气相色谱法条件为：色谱柱为Agilent DB-FFAP强极性柱毛细管色谱柱，流速1.8～2.5mL/min，进样量1～2μL，分流比1∶(10～50)，进样口温度250～270℃，检测器温度260～280℃。
  11. 根据权利要求4或5所述的方法，其特征在于，所述方法包括以下步骤：
    (1)样品溶解：将约50mg样品用500μL饱和氯化钠溶液混匀，加入0.1倍饱和氯化钠体积的10％硫酸溶液酸化，震荡使样品充分溶解；
    (2)短链脂肪酸提取：加入800μL乙醚，震荡30s，充分萃取样品中的短链脂肪酸；
    (3)样品制备：提取液在12000r/min，4℃下，离心10min，取上清，加入0.25g无水硫酸钠固体，震荡30s后，4500r/min，4℃，离心3min，去除痕量水分，过0.22μm有机微孔滤膜，进色谱仪分析；
    (4)气相色谱法测定：色谱柱：Agilent DB-FFAP强极性柱毛细管色谱柱。每次每样进行3次独立重复测定后，对实验数据进行分 析；
    分析条件：FID检测器，载气为高纯N 2，流速2.0mL/min，进样量2μL，分流比1∶50，进样口温度260℃，检测器温度270℃；
    升温程序：100℃保持1min后，按5℃/min升温速率升温至150℃，保持5℃，测定时间共16min。
  12. 权利要求1-11任一所述的方法，其特征在于，所述样品为肠道内容物或粪便。
  13. 权利要求1-12任一所述的方法，其特征在于，所述短链脂肪酸包括乙酸、丙酸、异丁酸、正丁酸、异戊酸、正戊酸。
  14. 权利要求13所述的方法，其特征在于，采用外标法检测短链脂肪酸的含量。
  15. 根据权利要求14所述的方法，其特征在于，乙酸标准曲线为y＝509204.16x-852.08，y为峰面积，x为乙酸浓度，μL/mL。
  16. 根据权利要求14所述的方法，其特征在于，丙酸标准曲线为y＝826031.70x-1147.79，y为峰面积，x为丙酸浓度，μL/mL。
  17. 根据权利要求14所述的方法，其特征在于，异丁酸标准曲线为y＝1020753.28x-1119.04，y为峰面积，x为异丁酸浓度，μL/mL。
  18. 根据权利要求14所述的方法，其特征在于，正丁酸标准曲线为y＝1037614.63x-632.86，y为峰面积，x为正丁酸浓度，μL/mL。
  19. 根据权利要求14所述的方法，其特征在于，异戊酸标准曲线为y＝1122548.15x-1222.30，y为峰面积，x为异戊酸浓度，μL/mL。
  20. 根据权利要求14所述的方法，其特征在于，正戊酸标准曲线为y＝1113530.09x-1433.43，y为峰面积，x为正戊酸浓度，μL/mL。
  21. 权利要求2或3所述的预处理方法在检测短链脂肪酸含量方面的应用。
  22. 权利要求4-20任一所述的方法在生物样品检测方面的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110045040A (zh) * 2019-05-17 2019-07-23 江南大学 一种测定肠道内容物或粪便中短链脂肪酸含量的方法
CN112034055A (zh) * 2020-07-23 2020-12-04 华中农业大学 一种定量检测人消化道内短链脂肪酸的方法
CN112285246B (zh) * 2020-12-29 2021-03-26 南京市产品质量监督检验院 一种检测食盐中污染物的方法
CN113138246B (zh) * 2021-04-25 2023-02-10 深圳市中医院 一种靶向测定生物样本中短链脂肪酸的检测方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101665758A (zh) * 2009-09-10 2010-03-10 上海大学 体外条件下重现人体胃肠道及微生态系统的模拟装置
US20120107845A1 (en) * 2009-05-29 2012-05-03 The Brigham & Women's Hospital, Inc. Disrupting FCRN-Albumin Interactions
CN105021726A (zh) * 2015-07-10 2015-11-04 南昌大学 一种快速测定肠道或粪便中短链脂肪酸含量的方法
CN107727783A (zh) * 2017-10-11 2018-02-23 上海中医药大学 一种肠道和粪便中短链脂肪酸的检测方法
CN110045040A (zh) * 2019-05-17 2019-07-23 江南大学 一种测定肠道内容物或粪便中短链脂肪酸含量的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109090033A (zh) * 2018-09-04 2018-12-28 重庆市畜牧科学院 粪便移植实验仔猪模型的构建方法
CN109298115B (zh) * 2018-10-19 2020-07-28 深圳市绘云生物科技有限公司 生物样品中多种代谢物定量检测方法及代谢芯片

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120107845A1 (en) * 2009-05-29 2012-05-03 The Brigham & Women's Hospital, Inc. Disrupting FCRN-Albumin Interactions
CN101665758A (zh) * 2009-09-10 2010-03-10 上海大学 体外条件下重现人体胃肠道及微生态系统的模拟装置
CN105021726A (zh) * 2015-07-10 2015-11-04 南昌大学 一种快速测定肠道或粪便中短链脂肪酸含量的方法
CN107727783A (zh) * 2017-10-11 2018-02-23 上海中医药大学 一种肠道和粪便中短链脂肪酸的检测方法
CN110045040A (zh) * 2019-05-17 2019-07-23 江南大学 一种测定肠道内容物或粪便中短链脂肪酸含量的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENG, MEIMEI ET AL.: "Non-official translation: A Determination Method Based on Gas Chromatography for Analysis of Short-Chain Fatty Acids in Colonic Contents of Piglet", PROGRESS IN MODERN BIOMEDICINE, vol. 15, no. 6, 28 February 2015 (2015-02-28), ISSN: 1673-6273, DOI: 20200110101845Y *

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* Cited by examiner, † Cited by third party
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