CN113295788A - Extracting agent for extracting various B vitamins and preparation method and application thereof - Google Patents
Extracting agent for extracting various B vitamins and preparation method and application thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention provides a preparation method of an extracting agent for extracting various B vitamins, which mainly comprises the following steps: (1) weighing an EDTA solution, glacial acetic acid and triethylamine according to a proportion, and uniformly mixing to obtain a mixed solution; (2) adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution; (3) and mixing the diluted solution with methanol in proportion, and performing ultrasonic degassing to obtain the extracting agent for extracting various B vitamins. The preparation method of the extractant for extracting the B vitamins is simple to operate and low in cost, and the extractant prepared by the preparation method can extract various B vitamins at one time, so that various vitamins can be simultaneously detected in a sample in one liquid phase sample injection, the extraction steps are simplified, the time is saved, and the requirements on detection instruments are reduced.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to an extracting agent for extracting various B vitamins, a preparation method and application thereof.
Background
The liquid chromatography detection technology can effectively detect the vitamin substances contained in the vitamin substances. The liquid chromatography detection technology is applied, vitamins in food need to be preferentially extracted, the specific content of the vitamins in the food is analyzed through detecting extracted substances, whether the content of the vitamins in the food is proper or not is judged, and the problem of imbalance of the vitamins in the food is solved.
At present, the liquid phase method for detecting B vitamins mostly adopts a national standard method, taking the national standard method of vitamin B2 as an example, the extraction steps of the method are as follows: taking about 500g of sample, fully stirring uniformly by using a tissue triturator, subpackaging in a clean brown ground bottle, sealing, marking, and storing in a dark place for later use. Weighing 2 g-10 g (accurate to 0.01g) of homogenized sample (the content of vitamin B2 in the sample is more than 5 mu g) into a 100mL conical flask with a plug, adding 60mL of 0.1mol/L hydrochloric acid solution, fully shaking uniformly, and plugging the plug. Placing the conical flask into an autoclave, maintaining at 121 deg.C for 30min, cooling to room temperature, and taking out. Adjusting the pH value to 6.0-6.5 by using 1mol/L sodium hydroxide solution, adding 2mL mixed enzyme solution, shaking uniformly, and placing in an incubator or a constant-temperature water bath kettle at 37 ℃ for overnight enzymolysis. Transferring the enzymolysis solution into a 100mL volumetric flask, adding water to a constant volume to reach a scale, filtering or centrifuging by using filter paper, taking filtrate or supernatant, and passing through a 0.45-micrometer water-phase filter membrane to serve as a solution to be detected. The method can only extract one vitamin at one time at present, the extraction steps are complex to operate, a large amount of mobile phase and mixed enzyme extractant are required to be prepared, the detection cost is greatly increased, in addition, the sample is required to be added with enzymolysis liquid and then is subjected to enzymolysis at 37 ℃ overnight, and the detection efficiency is reduced.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of an extractant for extracting a plurality of B vitamins, the preparation method is simple to operate and low in cost, and the extractant prepared by the preparation method can extract a plurality of B vitamins at one time, so that a sample can be simultaneously detected with a plurality of vitamins in one liquid phase sample injection, the extraction steps are simplified, the time is saved, and the detection cost is reduced.
The second purpose of the invention is to provide an extracting agent for extracting a plurality of B vitamins, which is prepared by the preparation method.
The third purpose of the invention is to provide a method for simultaneously measuring the amount of B vitamins, wherein the extraction agent prepared by the extraction method is used for simultaneously extracting a plurality of B vitamins.
In order to solve the technical problems, the invention provides a preparation method of an extracting agent for extracting a plurality of B vitamins, which is characterized by comprising the following steps:
(2) weighing an EDTA solution, glacial acetic acid and triethylamine according to a proportion, and uniformly mixing to obtain a mixed solution;
(2) adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution;
(3) and mixing the diluted solution with methanol in proportion, and performing ultrasonic degassing to obtain the extracting agent for extracting various B vitamins.
The vitamin B complex has low general stability, is easy to generate oxidation reaction and is easy to decompose under visible light, EDTA is used as a stabilizer in the formula to prevent the vitamin extracted from the solution from being degraded, glacial acetic acid is used for adjusting the pH of the solution to make the solution acidic, so that the vitamin is easier to separate and dissolve from a sample, triethylamine is used as a solvent to effectively extract the vitamin from the sample, and finally, methanol is added to improve the solubility of the vitamin B complex in the mixed solution.
Preferably, the volume ratio of the EDTA solution to the glacial acetic acid to the triethylamine is (20-60): (3-6): (0.8-1.2).
Preferably, the concentration of the EDTA solution is 0.1% -0.3%.
Preferably, the volume ratio of the mixed solution and the water in the step (2) is 1: (2-3).
Preferably, the volume ratio of the diluted solution to the methanol in the step (3) is (3-5): 1.
in addition, the invention also provides an extracting agent for extracting various B vitamins, which is prepared according to the preparation method. The extraction agent can extract various B vitamins at one time, so that various vitamins can be detected simultaneously in one-time liquid phase sample injection, the extraction steps are simplified, the time is saved, and the requirements on detection instruments are reduced.
In addition, the invention also provides a method for simultaneously measuring the contents of various B vitamins, which comprises the following steps:
(1) adding an extracting agent prepared by the preparation method of any one of claims 1-5 into a sample to be tested to obtain an extraction solution, wherein the volume ratio of the extracting agent to the sample to be tested is (5-10): 1;
(2) extracting, centrifuging and filtering the extraction solution obtained in the step (1) to obtain a sample solution;
(3) preparing a standard solution of multiple vitamins;
(4) and detecting and analyzing the sample solution and the standard solution by adopting a liquid chromatography, drawing a standard curve according to the result of the standard solution, substituting sample peak result data into a standard curve formula to calculate the vitamin concentration of the sample solution, and further calculating the content of the vitamin in the sample to be detected.
Preferably, the chromatographic conditions of the liquid chromatography are: c18 chromatographic column with column length of 250mm, inner diameter of 4.6mm and particle size of 5 μm; column temperature: 30 ℃; the flow rate is 1 mL/min; sample introduction amount: 10 mu L of the solution; setting the wavelength of the ultraviolet detector to be 280 nm; the mobile phase is sodium heptanesulfonate: methanol: acetonitrile (70: 15: 15).
Sodium sulfonate mobile phase: methanol: the proportion of acetonitrile can greatly influence the peak retention time of each component substance, although the organic phase with high proportion can effectively improve the peak height, the separation effect is also reduced, and the optimal sodium sulfonate mobile phase can be obtained from the comparison of peak diagrams: methanol: the acetonitrile ratio should be 70: 15: 15. under the proportion, the complete separation of the peaks of all the components can be ensured, and the peak emergence time can be improved as much as possible, so that the detection time is shortened, and the detection efficiency is improved.
Preferably, the preparation of the standard solution comprises the following steps: weighing vitamin B6, vitamin B2 and vitamin B1 standard substances, preparing standard stock solutions with mass concentration of 100mg/mL, absorbing the standard stock solutions with different volumes according to test requirements, and diluting the standard stock solutions into standard solutions step by using 0.01mol/L hydrochloric acid solution.
Preferably, ultrasonic wave is adopted for extraction at normal temperature in the step (2), and the extraction time is 30-40 min; and (3) filtering by adopting a 0.45 mu m filter membrane in the step (2).
Compared with the prior art, the invention has the beneficial effects that:
firstly, the extracting agent for extracting various B vitamins prepared by the preparation method can extract various B vitamins at one time, so that various vitamins can be simultaneously detected in a sample in one liquid phase sample injection, the extraction method is simple and rapid, and compared with the traditional method of adding enzymatic hydrolysate, the steps of adjusting pH and then carrying out enzymolysis overnight are carried out, the flow is simplified; in addition, the extracting agent only uses no enzyme of any kind, and greatly reduces the detection cost in large-scale detection.
Secondly, the extractant of the invention can simultaneously determine the content of a plurality of B vitamins without enzymolysis overnight, saves more than ten hours compared with the traditional method, and reduces the condition requirements on detection equipment, in addition, the invention also adopts mixed mobile phase sodium heptane sulfonate: methanol: after the acetonitrile (70: 15: 15) is prepared according to the optimal proportion, the complete separation of the peaks of all components is ensured, and the peak emergence time can be improved as much as possible, so that the detection time is shortened, and the detection efficiency is improved.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Simultaneously determining the contents of vitamins B1, B2 and B6 in the lily:
firstly, weighing 0.2% EDTA solution, glacial acetic acid and triethylamine according to the volume ratio of 40:5:1, and uniformly mixing to obtain a mixed solution; adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution, wherein the volume ratio of the mixed solution to the water is 1: 2.5; mixing the diluted solution with methanol according to a volume ratio of 4: 1, and then ultrasonically degassing to obtain the extractant for extracting various B vitamins for later use;
and then adding the prepared extractant into a lily sample to be detected to obtain an extraction solution, wherein the volume ratio of the extractant to the sample to be detected is 7:1, extracting the extraction solution at normal temperature by adopting ultrasonic waves for 35min, taking out the extraction solution during which several times of shaking can be carried out to prevent the sample from caking, centrifuging for 15min by using a high-speed centrifuge 4000r/min after the ultrasonic waves are finished, and filtering by using a 0.45-micron filter membrane to obtain the sample solution to be detected.
Weighing vitamin B6, vitamin B2 and vitamin B1 standard substances, preparing standard stock solutions with mass concentration of 100mg/mL, absorbing the standard stock solutions with different volumes according to test requirements, and diluting the standard stock solutions into standard solutions step by using 0.01mol/L hydrochloric acid solution.
And (3) carrying out liquid chromatography detection on the sample solution and the standard solution, wherein the chromatographic conditions of the liquid chromatography are as follows: c18 chromatographic column with column length of 250mm, inner diameter of 4.6mm and particle size of 5 μm; column temperature: 30 ℃; the flow rate is 1 mL/min; sample introduction amount: 10 mu L of the solution; setting the wavelength of the ultraviolet detector to be 280 nm; the mobile phase is sodium heptanesulfonate: methanol: acetonitrile (70: 15: 15). And drawing a standard curve according to the result of the standard solution, substituting the sample peak result data into a standard curve formula to calculate the vitamin concentration of the sample solution, and further calculating the content of the vitamin in the sample to be detected.
Example 2
Simultaneously determining the contents of vitamins B1, B2 and B6 in the lily:
firstly, weighing 0.1% EDTA solution, glacial acetic acid and triethylamine according to the volume ratio of 20:3:0.8, and uniformly mixing to obtain a mixed solution; adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution, wherein the volume ratio of the mixed solution to the water is 1: 2; mixing the diluted solution and methanol according to the volume ratio of 3:1, and then carrying out ultrasonic degassing to obtain the extracting agent for extracting various B vitamins for later use;
and then adding the prepared extractant into a lily sample to be detected to obtain an extraction solution, wherein the volume ratio of the extractant to the sample to be detected is 5:1, extracting the extraction solution at normal temperature by adopting ultrasonic waves for 30min, taking out the extraction solution during the extraction for several times, shaking the extraction solution to prevent the sample from caking, centrifuging the extraction solution for 8min at 10000r/min of a high-speed centrifuge after the ultrasonic waves are finished, and filtering the extraction solution by adopting a 0.45-micron filter membrane to obtain a sample solution to be detected.
Weighing vitamin B6, vitamin B2 and vitamin B1 standard substances, preparing standard stock solutions with mass concentration of 100mg/mL, absorbing the standard stock solutions with different volumes according to test requirements, and diluting the standard stock solutions into standard solutions step by using 0.01mol/L hydrochloric acid solution.
And (3) carrying out liquid chromatography detection on the sample solution and the standard solution, wherein the chromatographic conditions of the liquid chromatography are as follows: c18 chromatographic column with column length of 250mm, inner diameter of 4.6mm and particle size of 5 μm; column temperature: 30 ℃; the flow rate is 1 mL/min; sample introduction amount: 10 mu L of the solution; setting the wavelength of the ultraviolet detector to be 280 nm; the mobile phase is sodium heptanesulfonate: methanol: acetonitrile (70: 15: 15). And drawing a standard curve according to the result of the standard solution, substituting the sample peak result data into a standard curve formula to calculate the vitamin concentration of the sample solution, and further calculating the content of the vitamin in the sample to be detected.
Example 3
Simultaneously determining the contents of vitamins B1, B2 and B6 in the lily:
firstly, weighing 0.3% EDTA solution, glacial acetic acid and triethylamine according to the volume ratio of 60:6:1.2, and uniformly mixing to obtain a mixed solution; adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution, wherein the volume ratio of the mixed solution to the water is 1: 3; mixing the diluted solution with methanol according to a volume ratio of 5:1, and then ultrasonically degassing to obtain the extractant for extracting various B vitamins for later use;
and then adding the prepared extractant into a lily sample to be detected to obtain an extraction solution, wherein the volume ratio of the extractant to the sample to be detected is 10:1, extracting the extraction solution at normal temperature by adopting ultrasonic waves for 40min, taking out the extraction solution during the extraction for several times, shaking the extraction solution to prevent the sample from caking, centrifuging the extraction solution for 15min by using a high-speed centrifuge 4000r/min after the ultrasonic waves are finished, and filtering the extraction solution by using a 0.45-micron filter membrane to obtain a sample solution to be detected.
Weighing vitamin B6, vitamin B2 and vitamin B1 standard substances, preparing standard stock solutions with mass concentration of 100mg/mL, absorbing the standard stock solutions with different volumes according to test requirements, and diluting the standard stock solutions into standard solutions step by using 0.01mol/L hydrochloric acid solution.
And (3) carrying out liquid chromatography detection on the sample solution and the standard solution, wherein the chromatographic conditions of the liquid chromatography are as follows: c18 chromatographic column with column length of 250mm, inner diameter of 4.6mm and particle size of 5 μm; column temperature: 30 ℃; the flow rate is 1 mL/min; sample introduction amount: 10 mu L of the solution; setting the wavelength of the ultraviolet detector to be 280 nm; the mobile phase is sodium heptanesulfonate: methanol: acetonitrile (70: 15: 15). And drawing a standard curve according to the result of the standard solution, substituting the sample peak result data into a standard curve formula to calculate the vitamin concentration of the sample solution, and further calculating the content of the vitamin in the sample to be detected.
Application example 1
Pure methanol extractant and EDTA extractant which are commonly used in the traditional method are selected for comparison, and orthogonal experiment is carried out by using two traditional mobile phase ratios (heptane mobile phase and hexane mobile phase) and the mixed mobile phase ratio of the method. The test results are shown in Table 1, using vitamin B1 as an example.
TABLE 1
As can be seen from Table 1, the extractant prepared by the invention has the best extraction effect under any kind of mobile phase proportion, and the mixed mobile phase proportion adopted by the invention has the best peak shape and peak height under any kind of same extractant, so that under the proportion, the complete separation of the peaks of the components can be ensured, and the peak emergence time can be advanced as much as possible, thereby shortening the detection time and improving the detection efficiency.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or alterations do not depart from the essence of the corresponding technical solution.
Claims (10)
1. A preparation method of an extracting agent for extracting a plurality of B vitamins is characterized by comprising the following steps:
(1) weighing an EDTA solution, glacial acetic acid and triethylamine according to a proportion, and uniformly mixing to obtain a mixed solution;
(2) adding water into the mixed solution for dilution and volume fixing to obtain a diluted solution;
(3) and mixing the diluted solution with methanol in proportion, and performing ultrasonic degassing to obtain the extracting agent for extracting various B vitamins.
2. The method of claim 1, wherein the volume ratio of the EDTA solution to the glacial acetic acid to the triethylamine is (20-60): (3-6): (0.8-1.2).
3. The method of claim 1, wherein the concentration of the EDTA solution is 0.1% -0.3%.
4. The method of claim 1, wherein the volume ratio of the mixture to water in the step (2) is 1: (2-3).
5. The method of claim 1, wherein the volume ratio of the diluted solution to the methanol in the step (3) is (3-5): 1.
6. an extraction agent for extracting a plurality of B vitamins, which is prepared according to the preparation method of any one of claims 1 to 5.
7. A method for simultaneously measuring the content of a plurality of B vitamins is characterized by comprising the following steps:
(1) adding an extracting agent prepared by the preparation method of any one of claims 1-5 into a sample to be tested to obtain an extraction solution, wherein the volume ratio of the extracting agent to the sample to be tested is (5-10): 1;
(2) extracting, centrifuging and filtering the extraction solution obtained in the step (1) to obtain a sample solution;
(3) preparing a standard solution of multiple vitamins;
(4) and detecting and analyzing the sample solution and the standard solution by adopting a liquid chromatography, drawing a standard curve according to the result of the standard solution, substituting sample peak result data into a standard curve formula to calculate the vitamin concentration of the sample solution, and further calculating the content of the vitamin in the sample to be detected.
8. The method for determining the content of a plurality of B vitamins according to claim 7, wherein the chromatographic conditions of the liquid chromatography are as follows: c18 chromatographic column with column length of 250mm, inner diameter of 4.6mm and particle size of 5 μm; column temperature: 30 ℃; the flow rate is 1 mL/min; sample introduction amount: 10 mu L of the solution; setting the wavelength of the ultraviolet detector to be 280 nm; the mobile phase is sodium heptanesulfonate: methanol: acetonitrile (70: 15: 15).
9. The method for determining the content of a plurality of B vitamins according to claim 7, wherein the preparation of the standard solution comprises the following steps: weighing vitamin B6, vitamin B2 and vitamin B1 standard substances, preparing standard stock solutions with mass concentration of 100mg/mL, absorbing the standard stock solutions with different volumes according to test requirements, and diluting the standard stock solutions into standard solutions step by using 0.01mol/L hydrochloric acid solution.
10. The method for determining the contents of various B vitamins according to claim 7, wherein ultrasonic wave is adopted for extraction at normal temperature in the step (2), and the extraction time is 30-40 min; and (3) filtering by adopting a 0.45 mu m filter membrane in the step (2).
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| WO2009100229A1 (en) * | 2008-02-07 | 2009-08-13 | Covance Laboratories, Inc. | Methods for analysis of vitamins |
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Application publication date: 20210824 |