阅读说明：本技术 一种三丁酸甘油酯含量的测定方法 (Method for measuring content of tributyrin ) 是由 尹玉秀 李宜鹏 邬本成 王计伟 于 2020-12-17 设计创作，主要内容包括：本发明属于物质含量测定技术领域。本发明提供了一种三丁酸甘油酯含量的测定方法,包括如下步骤：将三丁酸甘油酯标准品和有机溶剂混合,得到标准工作液；采用气相色谱仪对标准工作液进行检测,根据外标法得到标准曲线方程；将三丁酸甘油酯粉末样品和有机溶剂混合后经过超声、过滤处理,得到样品待测液；采用气相色谱仪对样品待测液进行检测,根据标准曲线方程和测定的峰面积得到样品待测液中三丁酸甘油酯含量。本发明的测定方法具有操作简便、重复性好、精确等优点；三丁酸甘油酯的回收率在98.8～100.2％之间。(The invention belongs to the technical field of substance content determination. The invention provides a method for measuring the content of tributyrin, which comprises the following steps: mixing a tributyrin standard substance and an organic solvent to obtain a standard working solution; detecting the standard working solution by using a gas chromatograph, and obtaining a standard curve equation according to an external standard method; mixing a tributyrin powder sample and an organic solvent, and then carrying out ultrasonic treatment and filtration treatment to obtain a sample to-be-detected liquid; and detecting the sample solution to be detected by adopting a gas chromatograph, and obtaining the content of the tributyrin in the sample solution to be detected according to a standard curve equation and the determined peak area. The determination method has the advantages of simple and convenient operation, good repeatability, accuracy and the like; the recovery rate of the tributyrin is 98.8-100.2%.)

1. A method for measuring the content of tributyrin is characterized by comprising the following steps:

1) mixing a tributyrin standard substance and an organic solvent to obtain a standard working solution;

2) detecting the standard working solution by using a gas chromatograph, and obtaining a standard curve equation according to an external standard method;

3) mixing a tributyrin powder sample and an organic solvent, and then carrying out ultrasonic treatment and filtration treatment to obtain a sample to-be-detected liquid;

4) and detecting the sample solution to be detected by adopting a gas chromatograph, and obtaining the content of the tributyrin in the sample solution to be detected according to a standard curve equation and the determined peak area.

2. The method according to claim 1, wherein the concentration of the standard working solution in step 1) is 0.098 to 0.147g/100 mL; the purity of the tributyrin standard substance is more than or equal to 98 percent.

3. The method according to claim 2, wherein the standard working solution contains 5 concentrations of 0.0098 to 0.0147g/100mL, 0.0294 to 0.0441g/100mL, 0.049 to 0.0735g/100mL, 0.0686 to 0.1029g/100mL, and 0.098 to 0.147g/100mL, respectively.

4. The method according to claim 2 or 3, wherein the organic solvent in step 1) and step 3) is acetone or n-hexane.

5. The assay method according to claim 4, wherein in the detection in step 2) and step 4), the chromatographic column is a Wonda CAP WAX capillary chromatographic column; the detector is a hydrogen flame ionization detector; the sample injection amount is 0.8-1.2 muL.

6. The method according to claim 5, wherein in the detection in step 2) and step 4), the programmed temperature is increased from 90 to 110 ℃ to 220 to 240 ℃, and the temperature increase rate of the temperature increase to 220 to 240 ℃ is 15 to 25 ℃/min; the temperature of the sample inlet is 250-270 ℃, and the temperature of the detector is 270-290 ℃.

7. The determination method according to claim 6, wherein in the detection process of the step 2) and the step 4), the nitrogen flow rate is 8.5-9.5 mL/min, and the hydrogen flow rate is 35-45 mL/min; the air flow rate is 350-450 mL/min; the flow dividing ratio is 18-22: 1.

8. The method of measuring according to any one of claims 5 to 7,

step 2) the standard curve equation is that y is 716.0864x-11051.6839, R²＝0.9999；

Wherein x is the concentration of the tributyrin standard solution, and y is the measured peak area.

9. The method according to claim 8, wherein the filtration treatment in step 3) is carried out using a microporous filter membrane.

10. The determination method according to claim 9, wherein in the step 4), after the concentration of tributyrin in the solution to be determined of the sample is obtained according to the standard curve equation and the determined peak area, the content of tributyrin in the sample is obtained from the concentration of tributyrin.

Technical Field

The invention relates to the technical field of substance content determination, in particular to a method for determining the content of tributyrin.

Background

Research shows that the tributyrin can regulate the structure and the function of animal intestinal tracts, repair intestinal mucosa injury, promote the growth of intestinal villi and realize the effect of preventing and treating digestive disorder and diarrhea of piglets after weaning. The tributyrin has the effects of improving the digestibility of the feed, reducing the incidence rate of feed excrement and water excrement and improving the weight gain of the livestock and the utilization rate of the feed in the livestock and poultry breeding.

With the comprehensive banning of the feed industry, the tributyrin product has wide application in the feed industry due to the unique functionality. The tributyrin product fundamentally replaces antibiotics in the feed, and eliminates the residue of the antibiotics in the feed, thereby producing high-quality pollution-free animal feed products. The content of tributyrin determines the efficacy of tributyrin products, and is the most key index for evaluating the products.

At present, no detection methods such as national standards and industrial standards are available for measuring the content of tributyrin. Therefore, the method has very important significance for accurately measuring the content of the tributyrin in the related products.

Disclosure of Invention

The invention aims to provide a method for measuring the content of tributyrin, which is accurate in analysis and simple and convenient to operate, aiming at the defects of the prior art and realizes the accurate measurement of the content of tributyrin.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for measuring the content of tributyrin, which comprises the following steps:

1) mixing a tributyrin standard substance and an organic solvent to obtain a standard working solution;

2) detecting the standard working solution by using a gas chromatograph, and obtaining a standard curve equation according to an external standard method;

3) mixing a tributyrin powder sample and an organic solvent, and then carrying out ultrasonic treatment and filtration treatment to obtain a sample to-be-detected liquid;

4) and detecting the sample solution to be detected by adopting a gas chromatograph, and obtaining the content of the tributyrin in the sample solution to be detected according to a standard curve equation and the determined peak area.

Preferably, the concentration of the standard working solution in the step 1) is 0.098-0.147 g/100 mL; the purity of the tributyrin standard substance is more than or equal to 98 percent.

Preferably, the standard working solution contains 5 concentrations, and the concentrations of the standard working solution are respectively 0.0098-0.0147 g/100mL, 0.0294-0.0441 g/100mL, 0.049-0.0735 g/100mL, 0.0686-0.1029 g/100mL and 0.098-0.147 g/100 mL.

Preferably, the organic solvent in step 1) and step 3) is acetone or n-hexane.

Preferably, during the detection in step 2) and step 4), the chromatographic column is a wonda CAP WAX capillary chromatographic column; the detector is a hydrogen flame ionization detector; the sample injection amount is 0.8-1.2 muL.

Preferably, in the detection processes of the step 2) and the step 4), the temperature of the program is increased from 90-110 ℃ to 220-240 ℃, and the temperature increasing rate of the temperature increased to 220-240 ℃ is 15-25 ℃/min; the temperature of the sample inlet is 250-270 ℃, and the temperature of the detector is 270-290 ℃.

Preferably, in the detection processes of the step 2) and the step 4), the nitrogen flow rate is 8.5-9.5 mL/min, and the hydrogen flow rate is 35-45 mL/min; the air flow rate is 350-450 mL/min; the flow dividing ratio is 18-22: 1.

Preferably, the standard curve equation in the step 2) is that y is 716.0864x-11051.6839, and R is²0.9999; wherein x is the concentration of the tributyrin standard solution, and y is the measured peak area.

Preferably, the filtration treatment in step 3) is performed by using a microporous membrane.

Preferably, in the step 4), after the concentration of the tributyrin in the liquid to be detected of the sample is obtained according to the standard curve equation and the determined peak area, the content of the tributyrin in the sample is obtained according to the concentration of the tributyrin.

The beneficial effects of the invention include the following:

1) the determination method has the advantages of simple and convenient operation, good repeatability, accuracy and the like.

2) The determination method has good repeatability, and repeated determination is carried out for multiple times under the same condition, wherein the relative deviation of the two parallel determination results is not more than 5%.

Drawings

FIG. 1 is a standard graph of tributyrin of example 1;

FIG. 2 is a chromatogram of a standard working solution of example 1 at a concentration of 112.612 ug/mL;

FIG. 3 is a chromatogram of a standard working solution of example 1 at a concentration of 337.835 ug/mL;

FIG. 4 is a chromatogram of a standard working solution of example 1 at a concentration of 563.059 ug/mL;

FIG. 5 is a chromatogram of a standard working solution of example 1 at a concentration of 788.283 ug/mL;

FIG. 6 is a chromatogram of a standard working solution of example 1 at a concentration of 1126.118 ug/mL;

FIG. 7 is a chromatogram of a sample of tributyrin powder of example 1;

fig. 8 is a chromatogram of a sample of tributyrin powder of example 2.

Detailed Description

The invention provides a method for measuring the content of tributyrin, which comprises the following steps:

1) mixing a tributyrin standard substance and an organic solvent to obtain a standard working solution;

2) detecting the standard working solution by using a gas chromatograph, and obtaining a standard curve equation according to an external standard method;

3) mixing a tributyrin powder sample and an organic solvent, and then carrying out ultrasonic treatment and filtration treatment to obtain a sample to-be-detected liquid;

4) and detecting the sample solution to be detected by adopting a gas chromatograph, and obtaining the content of the tributyrin in the sample solution to be detected according to a standard curve equation and the determined peak area.

The purity of the tributyrin standard substance is preferably more than or equal to 98 percent; the concentration A of the standard stock solution is equal to the mass of the tributyrin standard substance m multiplied by the purity of the tributyrin standard substance/100V multiplied by 10⁶(ii) a Wherein, the unit of A is mu g/mL, the unit of m is g, and the unit of V is mL.

The concentration of the standard working solution in the step 1) is preferably 0.098-0.147 g/100 mL; the standard working solution preferably contains 5 concentrations, and the concentrations of the standard working solution are preferably 0.0098-0.0147 g/100mL, 0.0294-0.0441 g/100mL, 0.049-0.0735 g/100mL, 0.0686-0.1029 g/100mL and 0.098-0.147 g/100mL respectively; further preferred are 0.0112612g/100mL, 0.0337835g/100mL, 0.0563059g/100mL, 0.0788283g/100mL and 0.1126118g/100 mL.

After being mixed with an organic solvent, the tributyrin standard substance is preferably prepared into a standard stock solution, and the stock solution is diluted by the organic solvent to obtain the first four standard working solutions; the concentration of the standard storage solution is preferably 0.098-0.147 g/100 mL; when the standard stock solution is diluted to obtain the standard working solution, the volume ratio of the standard stock solution to the organic solvent is preferably 1:9, 3:7, 5:5 and 7:3 respectively.

The organic solvent in step 1) and step 3) of the present invention is preferably acetone or n-hexane, and the acetone or n-hexane is preferably chromatographically pure.

In the detection process of the step 2) and the step 4), the chromatographic column is preferably a Wonda CAP WAX capillary chromatographic column; the type of the chromatographic column is preferably 30m multiplied by 0.25mm multiplied by 0.25 um; the detector is preferably a hydrogen flame ionization detector; the amount of the sample is preferably 0.8 to 1.2. mu.L, and more preferably 1. mu.L.

In the detection process of the step 2) and the step 4), the temperature of the program is preferably increased from 90-110 ℃ to 220-240 ℃, and is further preferably increased from 100 ℃ to 230 ℃; when the program temperature is 90-110 ℃, preferably preserving heat for 1-3 min, and further preferably for 2 min; when the program temperature is 220-240 ℃, preferably preserving heat for 8-12 min, and further preferably for 10 min; the heating rate of the temperature to 220-240 ℃ is preferably 15-25 ℃/min, more preferably 18-22 ℃/min, and even more preferably 20 ℃/min; the injection port temperature is preferably 250-270 ℃, and is further preferably 255-265 ℃; more preferably 260 ℃; the temperature of the detector is preferably 270-290 ℃, more preferably 275-285 ℃, and more preferably 280 ℃.

In the detection process of the steps 2) and 4), the nitrogen flow rate is preferably 8.5-9.5 mL/min, and more preferably 9.0 mL/min; the hydrogen flow rate is preferably 35-45 mL/min, and more preferably 40 mL/min; the air flow rate is preferably 350-450 mL/min, more preferably 380-420 mL/min, and even more preferably 400 mL/min; the split ratio is preferably 18-22: 1, and more preferably 20: 1.

The standard curve equation in the step 2) of the invention is preferably 716.0864x-11051.6839, R²0.9999; wherein x is the concentration of the tributyrin standard solution, and y is the measured peak area.

The time of ultrasonic treatment in the step 3) is preferably 25-35 min, and more preferably 30 min; the filtration treatment preferably adopts a microporous filter membrane, and the pore diameter of the microporous filter membrane is preferably 0.4-0.5 μm, and is further preferably 0.45 μm.

In the mixing of the step 3) of the invention, preferably, a tributyrin powder sample is uniformly dissolved by using a small amount of organic solvent; preferably, an organic solvent is adopted to perform constant volume, shaking up and standing treatment on the solution after the ultrasonic treatment and before the filtration treatment; the time of the standing treatment is preferably 8-12 min, and more preferably 10 min.

In the step 4), preferably, after the concentration of tributyrin in the liquid to be detected of the sample is obtained according to a standard curve equation and the peak area of the detection, the content of tributyrin in the sample is obtained according to the concentration of tributyrin; the formula for obtaining the content of the tributyrin in the sample from the concentration of the tributyrin in the sample to be detected is that the concentration A of the tributyrin in the sample to be detected is multiplied by the constant volume V/mass m/10⁴(ii) a Wherein A is in units of μ g/mL, V is in units of mL, and m is in units of g.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

0.11491g of 98% pure tributyrin standard substance is put into a 100mL volumetric flask, and after being fully dissolved by normal hexane, the tributyrin standard substance is diluted to a scale mark and fully shaken up to obtain a standard stock solution with the concentration of 1126.118 mug/mL. 0.1mL, 0.3mL, 0.5mL, 0.7mL and 1mL of standard stock solutions were taken, and 0.9mL, 0.7mL, 0.5mL, 0.3mL and 0mL of n-hexane were added to the standard stock solutions 0.1mL, 0.3mL, 0.7mL and 1mL, respectively, to dilute the solutions to obtain standard working solutions with concentrations of 112.612ug/mL, 337.835ug/mL, 563.059ug/mL, 788.283ug/mL and 1126.118ug/mL, respectively.

Injecting the standard working solution with different concentrations into a gas chromatograph for testing, wherein the chromatographic column is a wonda CAP WAX capillary chromatographic column (30m × 0.25mm × 0.25um), the detector is an FID detector, the program temperature is kept for 2min at an initial temperature of 100 ℃, and then the temperature is raised to 230 ℃ at a rate of 20 ℃/min and kept for 10 min; the injection port temperature was 260 ℃ and the detector temperature was 280 ℃. The nitrogen flow rate is 9.0mL/min, the hydrogen flow rate is 40mL/min, the air flow rate is 400mL/min, the split ratio is 20:1, and the sample injection amount is 1 muL. The peak areas measured for standard working solutions at concentrations of 112.612ug/mL, 337.835ug/mL, 563.059ug/mL, 788.283ug/mL, and 1126.118ug/mL were 71803, 229098, 392166, 550880, and 797429, respectively.

Performing regression analysis according to the concentration of the tributyrin standard working solution and the peak area corresponding to the tributyrin standard working solution to obtain a standard curve equation: 716.0864x-11051.6839, R2 0.9999; wherein x is the concentration of the tributyrin standard solution, and y is the measured peak area.

0.1228g of tributyrin powder sample with the purity of 98% is dissolved by a small amount of normal hexane and transferred into a volumetric flask of 100mL, after ultrasonic treatment for 30min, the normal hexane is used for constant volume to scale, shaking up is carried out, standing is carried out for 10min, and after filtration by a 0.45 mu m microporous filter membrane, the sample solution to be detected is obtained. And injecting the sample solution to be detected into the gas chromatograph, and detecting the sample solution to be detected by using the gas chromatograph to obtain 259180 peak area of the sample solution to be detected. Substituting the measured peak area 259180 into a standard curve equation to obtain the tributyrin concentration of 377.373 mu g/mL of the sample to-be-measured liquid, and obtaining the tributyrin content of 30.731% according to the content and concentration formula of the tributyrin.

The tributyrin standard curve chart is shown in FIG. 1, the chromatogram of a standard working solution with a concentration of 112.612ug/mL is shown in FIG. 2, the chromatogram of a standard working solution with a concentration of 337.835ug/mL is shown in FIG. 3, the chromatogram of a standard working solution with a concentration of 563.059ug/mL is shown in FIG. 4, the chromatogram of a standard working solution with a concentration of 788.283ug/mL is shown in FIG. 5, the chromatogram of a standard working solution with a concentration of 1126.118ug/mL is shown in FIG. 6, and the chromatogram of the tributyrin powder sample of example 1 is shown in FIG. 7.

Example 2

The mass of the tributyrin powder sample was 0.1324g, and the sample was dissolved in acetone and the volume was constant, and the other conditions were the same as in example 1. The peak area measured by a gas chromatograph is 278169, and 278169 is substituted into a standard curve equation to obtain the tributyrin concentration of the sample to be measured, which is 403.891 mug/mL. The content of tributyrin is 30.505% as obtained from the formula of the content and concentration of tributyrin.

The chromatogram of the tributyrin powder sample of example 2 is shown in fig. 8.

Example 3

The same sample of tributyrin powder was tested in duplicate according to example 1 and the results are shown in table 1.

Table 1 repeatability test results (n ═ 7)

As can be seen from table 1, the RSD of the same tributyrin powder sample was determined repeatedly 7 times, and the results showed that the method of the present invention had good reproducibility.

Comparative example 1

Preparing standard working solution with the concentration of 100 mug/mL, 50 mug/mL, 33 mug/mL, 25 mug/mL and 20 mug/mL, preparing tributyrin sample solution to be tested with the concentration of 400 mug/mL, and performing the same other conditions as in the step 1). The sample solution to be tested with the concentration of 400 mug/mL is tested repeatedly for 7 times, and the content of tributyrin is determined.

Comparative example 1 the relative standard deviation RSD of 7 tests was 3.5%.

Example 4

To three tributyrin samples each having a mass (m) of 0.1163g and a content of 30.62%, tributyrin standards of different masses (m') were added, and sample treatment and testing were performed in the same manner as in example 1 to calculate the average recovery rate of tributyrin, and the results are shown in table 2.

Table 2 recovery rate experiment (n ═ 3)

As can be seen from Table 2, 0.02002g, 0.04003g and 0.06005g of tributyrin standard substances are respectively added to tributyrin samples with the mass of 0.1163g and the content of tributyrin of 30.62%, and n-hexane is added to respectively fix the volume to 100mL for testing, so that the peak area is obtained. And calculating the concentration of the tributyrin through a standard curve equation, and calculating the standard-added determined content through the concentration of the tributyrin. The peak area, tributyrin concentration and spiked assay content are shown in table 2.

Normalized theoretical content (%) ═ normalized measured content/normalized theoretical content × 100 (sample content × m + m '× 98%)/(m + m') × 100; the addition standard theoretical content and the recovery rate of the serial numbers 1, 2 and 3 are respectively calculated by the formula. The result shows that the recovery rate of the tributyrin is between 98.8 and 100.2 percent, and the measurement result is accurate and reliable.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.