CN113267575A - Folic acid detection method - Google Patents
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a folic acid detection method, which relates to the technical field of detection, wherein the folic acid is dissolved by hot water and subjected to ultrasonic oscillation, so that a folic acid sample is dissolved sufficiently, protein and fat in the folic acid sample can be removed sufficiently by a perchloric acid solution and dichloromethane, a sample solution is filtered by a composite filter membrane, and impurities in the sample solution are further removed, so that a pure folic acid product with high purity and few impurities is obtained; in the process of detecting folic acid, a composite filter membrane is prepared, and a hydrophilic multi-arm compound containing benzene rings and amide groups in molecules and an amino group at the tail end is synthesized to form a polyamide functional layer film with terephthaloyl chloride at a two-phase interface, so that the hydrophilicity of the surface of the composite filter membrane is improved, the separation efficiency of the composite filter membrane is effectively improved, impurities in a sample liquid can be effectively removed, and the purity of folic acid is improved.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a folic acid detection method.
Background
Folic acid, also known as vitamin M, vitamin Bc, etc., is named as pteroylglutamic acid. Folic acid is yellow or orange yellow thin slice or needle crystal, has no odor and odor, melting point of 250 deg.C, and can be immediately decomposed when heated to 250 deg.C, and is dissolved in hot water and alkaline solution such as potassium carbonate, potassium hydroxide, sodium hydroxide, etc., slightly dissolved in cold water and ethanol, and insoluble in organic solvent such as chloroform, diethyl ether, benzene, etc. Folic acid is mainly distributed in animal liver, green leaf vegetables and microorganisms, and yeast cells contain more folic acid. It is a group of organic substances essential for the maintenance of the normal life processes of organisms, and although the amount required is small, it is important for the maintenance of health. Biochemical and medical researchers have demonstrated that folic acid, as a cofactor, plays a particularly important role in purine, nucleic acid, protein synthesis, and in assisting cells in rapid cell division, growth, and repair and methylation of DNA, so folic acid is required to renew healthy red blood cells and prevent anemia in both children and adults. Folic acid has a very close relationship with human health, and is mainly reflected in the influence of folic acid on pregnancy, fertility, heart diseases, cancer and the like. For example, folic acid supplementation during pregnancy can reduce the risk of congenital heart defects, lip cracks, limb defects and urinary tract abnormalities of infants; administration of folic acid can reduce homocysteine content, and thus can prevent heart disease and cancer.
Since most quantitative analysis methods are based on direct or indirect comparison with known values, it is essential for quantitative analysis to provide a reference sample (i.e. standard substance) of known composition and properties for accurate calibration, which standard substance usually requires high purity, with a composition that is highly consistent with the chemical formula and chemically stable, and due to this stringent requirement for standard substances, the preparation of standard substances generally requires manufacture under highly controlled conditions to achieve such high purity and to avoid as much as possible the influence of the impurities contained on the subsequent measurement. However, at present, due to the thermal instability and the photo-instability of folic acid, the technology for extracting and purifying folic acid still stays at the primary stage of large energy consumption, long time consumption and low extraction rate, and the purity of the obtained folic acid cannot meet the requirements of standard substances.
Therefore, how to solve the problems that the existing folic acid reference sample has low purity and still contains a large amount of impurities, and the impurities can cause adverse effects on subsequent measurement is needed to be solved by the invention.
Disclosure of Invention
In order to overcome the technical problems, the invention aims to provide a method for detecting folic acid, which comprises the following steps: through using hot water to dissolve, the ultrasonic oscillation helps folic acid sample fully to dissolve, later can fully remove protein and fat in the folic acid sample through perchloric acid solution and dichloromethane, later with compound filter membrane filtration for the sample liquid, further get rid of the impurity in the sample liquid, it is low to have solved among the current folic acid reference sample purity, still has a large amount of impurities, and the impurity that contains can cause the problem of adverse effect to follow-up survey.
The purpose of the invention can be realized by the following technical scheme:
a method for detecting folic acid comprises the following steps:
the method comprises the following steps: adding a folic acid sample into a centrifuge tube, dissolving with ultrapure water of 50 ℃, uniformly mixing, ultrasonically oscillating for 5min, then adding a perchloric acid solution and dichloromethane, ultrasonically oscillating for 5min, and then centrifuging for 5 min;
step two: after the centrifugal separation is finished, taking out supernatant, adding 50 ℃ ultrapure water and perchloric acid solution into the lower-layer residue, repeatedly extracting once, and combining the two supernatants to obtain sample solution;
step three: taking a polyether sulfone filter membrane as a base membrane, laying the water phase solution on the upper surface of the polyether sulfone filter membrane, immersing for 1-2 hours, removing the redundant water phase solution, airing at room temperature, laying the oil phase solution on the upper surface of the polyether sulfone filter membrane, immersing for 1-2 hours, removing the redundant oil phase solution, airing at room temperature, putting the polyether sulfone filter membrane into a vacuum drying oven, and carrying out heat treatment for 10-20min at the temperature of 55-65 ℃ to obtain a composite filter membrane;
step four: eluting the sample solution with ammonia water methanol solution, collecting eluent, blowing the eluent at 50 ℃ with nitrogen till the eluent is nearly dry, redissolving the eluent with redissolution, shaking and uniformly mixing the redissolution and the eluent, and then filtering the redissolution with a composite filter membrane to obtain filtrate;
step five: and (4) detecting and quantifying the filtrate on a computer by using an LC-MS/MS instrument to complete the detection of the folic acid.
As a further scheme of the invention: in the first step, the consumption ratio of the folic acid sample, the ultrapure water, the perchloric acid solution and the dichloromethane is 0.5-2 g: 15mL of: 0.5 mL: 5mL, wherein the mass fraction of the perchloric acid solution is 60%; the ultrasonic frequency during ultrasonic oscillation is 90-100kHz, and the centrifugal speed is 6000 rpm.
As a further scheme of the invention: the adding amount of the ultrapure water in the step two is the same as that in the step one, and the adding amount of the perchloric acid solution in the step two is the same as that in the step one.
As a further scheme of the invention: in the fourth step, the ammonia water-methanol solution is a mixture formed by mixing methanol, water and ammonia water according to a volume ratio of 76:19:5, the mass fraction of the ammonia water is 0.5%, the complex solution is a mixed solution of deionized water and an internal standard solution according to a volume ratio of 9:1, and the preparation process of the internal standard solution is as follows: accurately weighing 0.5mg folic acid sample in a 50mL volumetric flask, dissolving with 0.5% ammonia water solution by mass fraction, fixing the volume to scale, preparing into standard stock solution with concentration of 10 mug/mL, and storing at 4 deg.C in dark place.
As a further scheme of the invention: the preparation process of the oil phase solution in the third step comprises the following steps: adding paraphthaloyl chloride into n-hexane, and performing ultrasonic dispersion until paraphthaloyl chloride is completely dissolved to obtain an oil phase solution, wherein the dosage ratio of paraphthaloyl chloride to n-hexane is 0.3 g: 100 mL.
As a further scheme of the invention: the preparation process of the aqueous phase solution in the third step comprises the following steps:
s1: adding p-phenylenediamine into a three-neck flask, adding glacial acetic acid, stirring at the temperature of 20-30 ℃ and the stirring speed of 100-200r/min until the p-phenylenediamine is completely dissolved, adding 2-ethylhexyl acrylate, controlling the adding time to be 10min, heating the reaction system to 80 ℃ under the conditions of introducing nitrogen and keeping the tinfoil out of the sun, cooling to below 50 ℃ after condensing and refluxing for 2-3h, carrying out reduced pressure rotary evaporation to remove the glacial acetic acid and unreacted 2-ethylhexyl acrylate, and then carrying out reduced pressure drying to constant weight to obtain an intermediate 1;
the reaction principle is as follows:
s2: adding the intermediate 1 into a three-neck flask, adding methanol, performing ultrasonic treatment under the ultrasonic frequency of 45-65kHz until the intermediate 1 is completely dissolved, then adding 1, 3-cyclohexyldimethylamine, heating a reaction system to 35 ℃ under the conditions of introducing nitrogen for protection and keeping the tinfoil away from light, reacting for 10-12h, stopping the reaction, cooling the reaction to below 50 ℃, performing reduced pressure rotary evaporation to remove the methanol and unreacted 1, 3-cyclohexyldimethylamine, settling and washing a rotary evaporation product with diethyl ether for three times, and finally performing reduced pressure drying to constant weight to obtain an intermediate 2;
the reaction principle is as follows:
s3: and adding the intermediate 2 into distilled water, then adding sodium dodecyl sulfate and triethylamine, stirring and carrying out ultrasonic treatment until the intermediate is completely dissolved to obtain an aqueous phase solution.
As a further scheme of the invention: in the step S1, the dosage ratio of p-phenylenediamine, glacial acetic acid and 2-ethylhexyl acrylate is 4 g: 40mL of: 136.381 g.
As a further scheme of the invention: in the step S2, the dosage ratio of the intermediate 1, methanol and 1, 3-cyclohexyldimethylamine is 2 g: 5mL of: 30.176 g.
As a further scheme of the invention: in the step S3, the dosage ratio of the intermediate 2, the distilled water, the sodium dodecyl sulfate and the triethylamine is 2 g: 100mL of: 0.1 g: 0.1 g.
The invention has the beneficial effects that:
adding a folic acid sample into a centrifuge tube, dissolving the folic acid sample with ultrapure water of 50 ℃, uniformly mixing, ultrasonically oscillating for 5min, then adding perchloric acid solution and dichloromethane, ultrasonically oscillating for 5min, then centrifuging for 5min, taking out supernatant after centrifugal separation is finished, adding ultrapure water of 50 ℃ and perchloric acid solution into lower-layer residue, repeatedly extracting for one time, combining the two supernatants to obtain sample liquid, taking a polyether sulfone filter membrane as a base membrane, laying the aqueous phase solution on the upper surface of the polyether sulfone filter membrane, removing the redundant aqueous phase solution after immersing for 1-2h, drying at room temperature, laying an oil phase solution on the upper surface of the polyether sulfone filter membrane, removing the redundant oil phase solution after immersing for 1-2h, drying at room temperature, then putting the polyether sulfone filter membrane into a vacuum drying oven, and carrying out heat treatment at the temperature of 55-65 ℃ for 10-20min, obtaining a composite filter membrane, eluting a sample solution by using an ammonia water methanol solution, collecting an eluent, blowing the eluent to be nearly dry at 50 ℃, redissolving the eluent by using a redissolution, shaking and uniformly mixing the redissolution, filtering the mixture by using the composite filter membrane to obtain a filtrate, detecting and quantifying the filtrate on a computer by using an LC-MS/MS instrument, and drying the filtrate to obtain a pure folic acid product; according to the detection method, the folic acid sample is dissolved by using hot water and subjected to ultrasonic oscillation, the folic acid sample is favorably and fully dissolved, then the protein and fat in the folic acid sample can be fully removed by using a perchloric acid solution and dichloromethane, then the sample liquid is filtered by using a composite filter membrane, and the impurities in the sample liquid are further removed, so that the pure folic acid product with high purity and less impurities is obtained.
In the process of detecting folic acid, a composite filter membrane is prepared to further improve the purity of a folic acid sample, p-phenylenediamine is reacted with 2-ethylhexyl acrylate in glacial acetic acid, hydrogen atoms of the 2-ethylhexyl acrylate are replaced by hydrogen atoms of the 2-ethylhexyl acrylate so as to be connected to the p-phenylenediamine, an intermediate 1 is generated, the intermediate 1 is reacted with 1, 3-cyclohexyldimethylamine in methanol so that the 1, 3-cyclohexyldimethylamine replaces the hydrogen atoms of the 1, 3-cyclohexyldimethylamine and is connected to the intermediate 1, an intermediate 2 is generated, the intermediate 2 is prepared into a water phase solution, terephthaloyl chloride is added into n-hexane to prepare an oil phase solution, a polyether sulfone filter membrane is used as a base membrane, the water phase solution is laid on the upper surface of the polyether sulfone filter membrane and dried at room temperature, the oil phase solution is laid on the upper surface of the polyether sulfone filter membrane and dried at room temperature, and then heat treatment is carried out, obtaining a composite filter membrane; the hydrophilic multi-arm compound containing benzene rings and amide groups in molecules and having amino groups at the tail ends is synthesized, and a polyamide functional layer film is formed at a two-phase interface with terephthaloyl chloride, so that the hydrophilicity of the surface of the composite filter membrane is improved, the pollution resistance of the composite filter membrane is improved, the possibility of reduction of the filtering effect caused by pollution is avoided, the separation efficiency of the composite filter membrane is effectively improved, impurities in a sample liquid can be effectively removed, the purity of folic acid is improved, and the accuracy of purity data in the detection process is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
this example is a process for preparing an oil phase solution comprising the steps of: adding paraphthaloyl chloride into n-hexane, and performing ultrasonic dispersion until paraphthaloyl chloride is completely dissolved to obtain an oil phase solution, wherein the dosage ratio of paraphthaloyl chloride to n-hexane is 0.3 g: 100 mL.
Example 2:
this example is a process for the preparation of an aqueous solution comprising the following steps:
s1: adding p-phenylenediamine into a three-neck flask, then adding glacial acetic acid, stirring at the temperature of 20 ℃ and the stirring speed of 100r/min until the p-phenylenediamine is completely dissolved, then adding 2-ethylhexyl acrylate, controlling the adding time to be 10min, heating a reaction system to 80 ℃ under the conditions of introducing nitrogen for protection and keeping the tinfoil out of the sun, condensing and refluxing for 2h, then cooling to below 50 ℃, carrying out reduced pressure rotary evaporation to remove the glacial acetic acid and unreacted 2-ethylhexyl acrylate, and then carrying out reduced pressure drying to constant weight to obtain an intermediate 1; controlling the dosage ratio of p-phenylenediamine, glacial acetic acid and 2-ethylhexyl acrylate to be 4 g: 40mL of: 136.381 g;
s2: adding the intermediate 1 into a three-neck flask, adding methanol, performing ultrasonic treatment under the ultrasonic frequency of 45kHz until the intermediate 1 is completely dissolved, then adding 1, 3-cyclohexyldimethylamine, heating a reaction system to 35 ℃ under the conditions of introducing nitrogen for protection and keeping the tinfoil away from light, stopping the reaction after 10h of reaction, cooling the reaction to below 50 ℃, performing reduced pressure rotary evaporation to remove the methanol and unreacted 1, 3-cyclohexyldimethylamine, settling and washing a rotary evaporation product with diethyl ether for three times, and finally performing reduced pressure drying to constant weight to obtain an intermediate 2; controlling the dosage ratio of the intermediate 1, the methanol and the 1, 3-cyclohexyldimethylamine to be 2 g: 5mL of: 30.176 g;
s3: adding the intermediate 2 into distilled water, then adding sodium dodecyl sulfate and triethylamine, stirring and carrying out ultrasonic treatment until the mixture is completely dissolved to obtain an aqueous phase solution; controlling the dosage ratio of the intermediate 2, distilled water, sodium dodecyl sulfate and triethylamine to be 2 g: 100mL of: 0.1 g: 0.1 g.
Example 3:
this example is a process for the preparation of an aqueous solution comprising the following steps:
s1: adding p-phenylenediamine into a three-neck flask, then adding glacial acetic acid, stirring at the temperature of 30 ℃ and the stirring speed of 200r/min until the p-phenylenediamine is completely dissolved, then adding 2-ethylhexyl acrylate, controlling the adding time to be 10min, heating a reaction system to 80 ℃ under the conditions of introducing nitrogen for protection and keeping the tinfoil out of the sun, condensing and refluxing for 3h, then cooling to below 50 ℃, carrying out reduced pressure rotary evaporation to remove the glacial acetic acid and unreacted 2-ethylhexyl acrylate, and then carrying out reduced pressure drying to constant weight to obtain an intermediate 1; controlling the dosage ratio of p-phenylenediamine, glacial acetic acid and 2-ethylhexyl acrylate to be 4 g: 40mL of: 136.381 g;
s2: adding the intermediate 1 into a three-neck flask, adding methanol, performing ultrasonic treatment under the ultrasonic frequency of 65kHz until the intermediate 1 is completely dissolved, then adding 1, 3-cyclohexyldimethylamine, heating a reaction system to 35 ℃ under the conditions of introducing nitrogen for protection and keeping the tinfoil away from light, stopping the reaction after 12h of reaction, cooling the reaction to below 50 ℃, performing reduced pressure rotary evaporation to remove the methanol and unreacted 1, 3-cyclohexyldimethylamine, settling and washing a rotary evaporation product with diethyl ether for three times, and finally performing reduced pressure drying to constant weight to obtain an intermediate 2; controlling the dosage ratio of the intermediate 1, the methanol and the 1, 3-cyclohexyldimethylamine to be 2 g: 5mL of: 30.176 g;
s3: adding the intermediate 2 into distilled water, then adding sodium dodecyl sulfate and triethylamine, stirring and carrying out ultrasonic treatment until the mixture is completely dissolved to obtain an aqueous phase solution; controlling the dosage ratio of the intermediate 2, distilled water, sodium dodecyl sulfate and triethylamine to be 2 g: 100mL of: 0.1 g: 0.1 g.
Example 4:
the embodiment is a method for detecting folic acid, which comprises the following steps:
the method comprises the following steps: adding a folic acid sample into a centrifuge tube, dissolving with ultrapure water of 50 ℃, uniformly mixing, ultrasonically oscillating for 5min, then adding a perchloric acid solution and dichloromethane, ultrasonically oscillating for 5min, and then centrifuging for 5 min; controlling the dosage ratio of the folic acid sample, the ultrapure water, the perchloric acid solution and the dichloromethane to be 0.5 g: 15mL of: 0.5 mL: 5mL, and the mass fraction of the perchloric acid solution is 60%; the ultrasonic frequency during ultrasonic oscillation is 90kHz, and the centrifugal speed is 6000 rpm;
step two: after the centrifugal separation is finished, taking out supernatant, adding 50 ℃ ultrapure water and perchloric acid solution into the lower-layer residue, repeatedly extracting once, and combining the two supernatants to obtain sample solution; controlling the adding amount of ultrapure water to be the same as that in the step one, and controlling the adding amount of perchloric acid solution to be the same as that in the step one;
step three: taking a polyether sulfone filter membrane as a base membrane, laying the water phase solution from the example 2 on the upper surface of the polyether sulfone filter membrane, immersing for 1h, removing the redundant water phase solution, airing at room temperature, laying the oil phase solution from the example 1 on the upper surface of the polyether sulfone filter membrane, immersing for 1h, removing the redundant oil phase solution, airing at room temperature, putting the polyether sulfone filter membrane into a vacuum drying oven, and carrying out heat treatment at the temperature of 55 ℃ for 10min to obtain a composite filter membrane;
step four: eluting the sample solution with ammonia water methanol solution, collecting eluent, blowing the eluent at 50 ℃ with nitrogen till the eluent is nearly dry, redissolving the eluent with redissolution, shaking and uniformly mixing the redissolution and the eluent, and then filtering the redissolution with a composite filter membrane to obtain filtrate;
step five: and (4) detecting and quantifying the filtrate on a computer by using an LC-MS/MS instrument to complete the detection of the folic acid.
Example 5:
the embodiment is a method for detecting folic acid, which comprises the following steps:
the method comprises the following steps: adding a folic acid sample into a centrifuge tube, dissolving with ultrapure water of 50 ℃, uniformly mixing, ultrasonically oscillating for 5min, then adding a perchloric acid solution and dichloromethane, ultrasonically oscillating for 5min, and then centrifuging for 5 min; controlling the dosage ratio of the folic acid sample, the ultrapure water, the perchloric acid solution and the dichloromethane to be 2 g: 15mL of: 0.5 mL: 5mL, and the mass fraction of the perchloric acid solution is 60%; the ultrasonic frequency during ultrasonic oscillation is 100kHz, and the centrifugal speed is 6000 rpm;
step two: after the centrifugal separation is finished, taking out supernatant, adding 50 ℃ ultrapure water and perchloric acid solution into the lower-layer residue, repeatedly extracting once, and combining the two supernatants to obtain sample solution; controlling the adding amount of ultrapure water to be the same as that in the step one, and controlling the adding amount of perchloric acid solution to be the same as that in the step one;
step three: taking a polyether sulfone filter membrane as a base membrane, laying the water phase solution from the example 3 on the upper surface of the polyether sulfone filter membrane, immersing for 2 hours, removing the redundant water phase solution, airing at room temperature, laying the oil phase solution from the example 1 on the upper surface of the polyether sulfone filter membrane, immersing for 2 hours, removing the redundant oil phase solution, airing at room temperature, putting the polyether sulfone filter membrane into a vacuum drying oven, and carrying out heat treatment at the temperature of 65 ℃ for 20min to obtain a composite filter membrane;
step four: eluting the sample solution with ammonia water methanol solution, collecting eluent, blowing the eluent at 50 ℃ with nitrogen till the eluent is nearly dry, redissolving the eluent with redissolution, shaking and uniformly mixing the redissolution and the eluent, and then filtering the redissolution with a composite filter membrane to obtain filtrate;
step five: and (4) detecting and quantifying the filtrate on a computer by using an LC-MS/MS instrument to complete the detection of the folic acid.
Comparative example 1:
comparative example 1 differs from examples 4 and 5 in that the pure folic acid product is obtained by directly placing the sample solution into a plastic centrifuge tube and freeze-drying the sample solution.
Comparative example 2:
comparative example 2 is different from examples 4 and 5 in that the sample solution is filtered by common filter paper in a laboratory, then is put into a plastic centrifuge tube, and is freeze-dried to form pure folic acid.
Comparative example 3:
the difference between the comparative example 3 and the examples 4 and 5 is that the pure folic acid is formed by filtering the polyethersulfone filter membrane used as the basement membrane, putting the filter membrane into a plastic centrifuge tube and freeze-drying the filter membrane.
The purity of the pure folic acid products of examples 4 to 5 and comparative examples 1 to 3 was measured by an LC-MS/MS instrument, and the results are shown in the following table:
| sample (I) | Folate sample purity/%) | Pure folic acid purity/%) |
| Example 4 | 90.16 | 97.55 |
| Example 5 | 90.16 | 97.78 |
| Comparative example 1 | 90.16 | 93.23 |
| Comparative example 2 | 90.16 | 96.54 |
| Comparative example 3 | 90.16 | 97.17 |
As shown in the table, the purity of the composite filter membrane is only improved to a small extent without filtration in the comparative example 1, the purity of the composite filter membrane is greatly improved after filtration in the comparative examples 2 and 3 and the examples 4 and 5, and impurities are fully removed.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.
Claims (4)
1. A method for detecting folic acid is characterized by comprising the following steps:
the method comprises the following steps: adding a folic acid sample into a centrifuge tube, dissolving with ultrapure water of 50 ℃, uniformly mixing, ultrasonically oscillating for 5min, then adding a perchloric acid solution and dichloromethane, ultrasonically oscillating for 5min, and then centrifuging for 5 min;
step two: after the centrifugal separation is finished, taking out supernatant, adding 50 ℃ ultrapure water and perchloric acid solution into the lower-layer residue, repeatedly extracting once, and combining the two supernatants to obtain sample solution;
step three: taking a polyether sulfone filter membrane as a base membrane, laying the water phase solution on the upper surface of the polyether sulfone filter membrane, immersing for 1-2 hours, removing the redundant water phase solution, airing at room temperature, laying the oil phase solution on the upper surface of the polyether sulfone filter membrane, immersing for 1-2 hours, removing the redundant oil phase solution, airing at room temperature, putting the polyether sulfone filter membrane into a vacuum drying oven, and carrying out heat treatment for 10-20min at the temperature of 55-65 ℃ to obtain a composite filter membrane;
step four: eluting the sample solution with ammonia water methanol solution, collecting eluent, blowing the eluent at 50 ℃ with nitrogen till the eluent is nearly dry, redissolving the eluent with redissolution, shaking and uniformly mixing the redissolution and the eluent, and then filtering the redissolution with a composite filter membrane to obtain filtrate;
step five: and (4) detecting and quantifying the filtrate on a computer by using an LC-MS/MS instrument to complete the detection of the folic acid.
2. The method for detecting folic acid according to claim 1, wherein the ratio of the folic acid sample, the ultrapure water, the perchloric acid solution and the dichloromethane in the step one is 0.5-2 g: 15mL of: 0.5 mL: 5mL, wherein the mass fraction of the perchloric acid solution is 60%; the ultrasonic frequency during ultrasonic oscillation is 90-100kHz, and the centrifugal speed is 6000 rpm.
3. The method according to claim 1, wherein the amount of ultrapure water added in the second step is the same as the amount of ultrapure water added in the first step, and the amount of perchloric acid solution added in the second step is the same as the amount of perchloric acid solution added in the first step.
4. The method for detecting folic acid according to claim 1, characterized in that the ammonia water methanol solution in step four is a mixture of methanol, water and ammonia water in a volume ratio of 76:19:5, the mass fraction of the ammonia water is 0.5%, the complex solution is a mixed solution of deionized water and an internal standard solution in a volume ratio of 9:1, and the preparation process of the internal standard solution is as follows: accurately weighing 0.5mg folic acid sample in a 50mL volumetric flask, dissolving with 0.5% ammonia water solution by mass fraction, fixing the volume to scale, preparing into standard stock solution with concentration of 10 mug/mL, and storing at 4 deg.C in dark place.
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Cited By (2)
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| CN114019083A (en) * | 2021-09-23 | 2022-02-08 | 杭州美康盛德医学检验实验室有限公司 | Folic acid detection kit and preparation method thereof |
| CN115436514A (en) * | 2022-09-05 | 2022-12-06 | 常州市新鸿医药化工技术有限公司 | Folic acid content detection device |
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| CN115436514A (en) * | 2022-09-05 | 2022-12-06 | 常州市新鸿医药化工技术有限公司 | Folic acid content detection device |
| CN115436514B (en) * | 2022-09-05 | 2023-11-07 | 常州市新鸿医药化工技术有限公司 | Folic acid content detection device |
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