CN112179996A - High performance liquid chromatography analysis method for determining vitamin A in complex components - Google Patents
High performance liquid chromatography analysis method for determining vitamin A in complex components Download PDFInfo
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Abstract
The invention provides a liquid chromatography analysis method for efficiently measuring vitamin A in complex components, which uses methanol-water as a mobile phase to perform isocratic elution, can avoid the interference of other components and accurately and sensitively detect the content of the vitamin A within 6 minutes. The method has the advantages of strong specificity, high precision, good stability, high accuracy, simple operation, and cheap and easily-obtained reagents and consumables. The method is suitable for detecting the content of the vitamin A in complex components of dosage forms such as tablets, powder and capsules containing the vitamin A and controlling the detection quality, and can effectively save the detection cost and time.
Description
Technical Field
The invention relates to a liquid chromatography analysis method for efficiently and rapidly determining the content of vitamin A in complex components. Particularly, the pretreatment of the sample and the optimization of chromatographic conditions are adopted, so that the experiment is simple, and the content of the vitamin A in the complex components can be conveniently, accurately and rapidly detected.
Background
Vitamin a (vitamine a), also known as retinol (its aldehyde derivative, retinal) or anti-dry eye factor, is an unsaturated monohydric alcohol having an alicyclic ring, which plays an important role in the human body. For example, not only can maintain normal visual function, maintain the health of epithelial tissue cells and promote the synthesis of immunoglobulin, maintain the normal growth and development of bones, promote growth and reproduction, but also can inhibit the growth of tumors; therefore, the health food containing vitamin A is beneficial for maintaining human health. Vitamin a has the following structural formula:
vitamin A is insoluble in water and soluble in organic solvents. Most of the pretreatment adopted for detecting vitamin A at the present stage is high-temperature saponification, and then reagents such as normal hexane, sodium hydroxide and the like are used for multiple extraction, nitrogen blowing and redissolution, so that more reagents are needed, and the operation process is complex and tedious. Therefore, it is necessary to develop a pretreatment method which can sufficiently extract the vitamin a component and can be easily performed.
Since multivitamin mineral tablets in health foods contain multiple ingredients, some of the ingredients may interfere with or degrade vitamin a to some extent. Therefore, it is necessary to develop a high performance liquid chromatography method for detecting vitamin a, which has strong specificity and can be well separated from other components or impurity peaks.
Disclosure of Invention
In order to overcome the problems, the invention provides a high performance liquid chromatography method of special vitamin A, which has high extraction rate, good stability, high accuracy and good repeatability, and the method specifically comprises the following steps:
a high performance liquid chromatography method for determining vitamin A content in complex components such as tablet, powder, capsule, etc. comprises extracting sample with low concentration ammonia water solution and/or organic solvent by water bath ultrasound or water bath oscillation; the mobile phase A is methanol, the mobile phase B is water, and the mixing ratio of the mobile phase A to the mobile phase B is 100-90: 0-10, isocratic elution; the ultraviolet detection wavelength is set to 300-350 nm.
In the present invention, the extraction solvent for the sample is a low-concentration aqueous ammonia solution, and the concentration of the extraction solvent is 0.01 to 0.05%, preferably 0.02 to 0.03%, and more preferably 0.025%.
In the present invention, the organic solvent used for the extraction of the test sample is selected from one of acetonitrile, methanol or ethanol, preferably ethanol. Wherein the concentration of the organic solvent is selected from 50% to 100%, preferably 95% to 100%, more preferably 100%.
In the invention, the temperature of the water bath ultrasound in the extraction method is selected from 25-80 ℃, preferably 30-60 ℃, and more preferably 40-50 ℃.
In the invention, the temperature of the water bath oscillation of the extraction method is selected from 25-80 ℃, preferably 30-60 ℃, and more preferably 40-50 ℃.
In the invention, the flow rate in the high performance liquid chromatography is 0.8-1.5 ml/min.
In the invention, the chromatographic column temperature of the vitamin A solution is 25-30 ℃, and the optimal column temperature is 25 ℃.
In one embodiment, the chromatographic apparatus and conditions for performing the above chromatographic analysis are:
a detection instrument: high performance liquid chromatograph (Thermo Fisher, U3000) equipped with an ultraviolet detector;
chromatographic conditions are as follows:
a chromatographic column: welch-C18150mm × 4.6mm, 5 μm; mobile phase: pure methanol-water (100-90: 0-10); detection wavelength: 300-350 nm; flow rate: 0.8-1.5 ml/min; column temperature: 25-30, optimum column temperature at ° c: 25 ℃; operating time: and 6 min.
In the invention, the high performance liquid chromatography is adopted to carry out quantitative detection on vitamin A in a multi-vitamin mineral tablet solution, and the method needs to be confirmed in aspects of specificity, accuracy and the like, and specifically comprises the following steps:
the specificity is as follows: the sample solution and the blank auxiliary material solution without vitamin A are prepared and respectively injected, and the result shows that the blank solution and the blank auxiliary material solution have no interference on the main peak appearance of the test solution and can be effectively separated from the adjacent peaks, which shows that the method provided by the invention can accurately determine the content of the functional component vitamin A of the multivitamin mineral substance tablets.
Accuracy: the high performance liquid chromatography method provided by the invention is adopted to detect vitamin A in the multi-vitamin mineral tablet solution, and the result shows that the recovery rate is within the qualified standard range and the accuracy is good.
Linearity: the high performance liquid chromatography method provided by the invention is adopted to detect the vitamin A reference substance solution in the multivitamin mineral tablets, the result shows that the linear correlation coefficient of the main component is not less than 0.998 within the linear range of 60-200%, and the detection result has a good linear relation with the concentration of the vitamin A.
The extraction solvents adopted in the sample pretreatment are low-concentration ammonia water and ethanol, the used reagents are low in toxicity, the personal safety of experimenters in the working environment can be guaranteed, the pretreatment operation is simple, the complex processes of multiple extraction, dissolution and the like are avoided, the usage amount of the solvents is small, and the generation of waste liquid is reduced. The method provided by the invention is specially used for detecting the vitamin A in the complex component product, can well separate the vitamin A from other components and impurity peaks, and has the characteristics of high extraction rate, good stability, high accuracy and good repeatability.
Drawings
FIG. 1 shows a chromatogram for HPLC detection of a test solution;
FIGS. 2, 3, 4 show chromatograms of HPLC detection under chromatographic conditions for example 1;
FIG. 5 shows a chromatogram of an HPLC assay under chromatographic conditions of example 2;
FIG. 6 shows a chromatogram of an HPLC assay under chromatographic conditions of example 3;
FIG. 7 shows a chromatogram of an HPLC assay under chromatographic conditions for example 4;
Detailed Description
The present invention will be described in further detail by way of examples, which are intended to exemplify tablets, capsules and soft capsules, respectively. Reagent: methanol (SINENCE, batch No. 1702M0603), ethanol (AR, Anhui Ante food products, Inc., Anhui), ammonia (25-30% of chemical reagents, Inc., national drug group, AR), vitamin A acetate reference (China food and drug testing research institute), sample (provided by Shenzhen Fulaite Nutrition and health, Inc.), soft capsule (provided by Shenzhen Fulaite Nutrition and health, Inc.)
Example 1:
shenzhen Fulaite nutritious and health Limited A1111-A2131-A3333 brand multivitamin mineral tablet (containing vitamin A, vitamin E, vitamin B)1Vitamin B2Nicotinic acid, pantothenic acid and vitamin B6Biotin, vitamin C, vitamin D, folic acid and vitamin B12Iron, zinc, selenium, magnesium, copper, calcium, chromium, potassium and the like) in the sample. The specific detection steps of the first embodiment are as follows:
the first step is as follows: preparing an extraction solvent 0.025% ammonia water solution: 1.0ml of concentrated ammonia water was weighed out, added to 1000ml of ultrapure water, and shaken up.
The second step is that: preparing a reference substance solution: precisely weighing 42.0mg of vitamin A acetate reference substance, placing in a 50ml volumetric flask, adding appropriate amount of anhydrous ethanol, ultrasonically dissolving, diluting with anhydrous ethanol to scale, shaking up, and using as reference substance stock solution I; precisely measuring 1ml of the reference substance storage solution I, placing the reference substance storage solution in a 100ml volumetric flask, diluting the reference substance storage solution to a scale with absolute ethyl alcohol, and shaking up to obtain the product.
The third step: preparing a sample solution: mixing with fresh water and keeping out of the sun, collecting 20 tablets, precisely weighing, grinding, precisely weighing appropriate amount, placing into 100ml measuring flask, adding 10ml 0.025% ammonia water, ultrasonic treating at 45 deg.C for 10min, adding 50ml anhydrous ethanol, shaking in 45 deg.C water bath for 5min, cooling, adding anhydrous ethanol to dilute to scale, shaking, and filtering to obtain the final product. A1111-A2131-A3333 brand multivitamin mineral tablets are prepared in two parts respectively.
The fourth step: the details of the liquid chromatography conditions are as follows:
[ column for chromatography: welch-C18150mm mm, 5 μm
Mobile phase: methanol-water (98: 2)
Detection wavelength: 325nm
Flow rate: 1.2ml/min
Column temperature: 25 deg.C
Sample introduction volume: 20ul of
Operating time: 6min
Detecting chromatogram shown in figures 2, 3 and 4, and detection results shown in Table 1
TABLE 1 test results
Example 2:
and (3) measuring the content of vitamin A in a sample of Shenzhen Fulaite nutrition and health Limited producing vitamin A tablets. The method comprises the following specific steps:
the first step is as follows: preparation of 0.025% ammonia water solution as extraction solvent: 1.0ml of concentrated ammonia water was weighed out, added to 1000ml of ultrapure water, and shaken up.
The second step is that: preparing a reference substance solution: precisely weighing 4.0mg of vitamin A acetate reference substance, placing in a 50ml volumetric flask, adding appropriate amount of 95% ethanol, ultrasonically dissolving, diluting with 95% ethanol to scale, shaking up, and using as reference substance stock solution I; precisely measuring 1ml of the reference substance storage solution I, placing the reference substance storage solution in a 100ml volumetric flask, diluting the reference substance storage solution with 95% ethanol to a scale, and shaking up to obtain the product.
The third step: preparing a sample solution: mixing with fresh water and keeping out of the sun, collecting 20 tablets, precisely weighing, grinding, precisely weighing appropriate amount, placing into 100ml measuring flask, adding 10ml 0.025% ammonia water, performing ultrasonic treatment at 40 deg.C for 10min, adding 50ml 95% ethanol, shaking in 40 deg.C water bath for 5min, cooling, adding 95% ethanol to dilute to desired volume, shaking, and filtering to obtain the final product.
The fourth step: the details of the liquid chromatography conditions are as follows:
a chromatographic column: welch-C18150mm mm, 5 μm
Mobile phase: methanol-water (98: 2)
Detection wavelength: 330nm
Flow rate: 1.2ml/min
Column temperature: 25 deg.C
Sample introduction volume: 20ul of
Operating time: 6min
The sample detection chromatogram is shown in FIG. 5, and the detection results are shown in Table 2.
TABLE 2 test results
Example 3:
shenzhen Fulaite nutrient and health Limited's multivitamin capsules (containing vitamin A, vitamin E, vitamin B)1Vitamin B2Nicotinic acid, pantothenic acid and vitamin B6Biotin, vitamin C, vitamin D, folic acid and vitamin B12Iron, zinc, selenium, magnesium, copper, calcium, chromium, potassium and the like) in the sample. The method comprises the following specific steps:
the first step is as follows: preparation of 0.025% ammonia water solution as extraction solvent: 1.0ml of concentrated ammonia water was weighed out, added to 1000ml of ultrapure water, and shaken up.
The second step is that: preparing a reference substance solution: precisely weighing 42.0mg of vitamin A acetate reference substance, placing in a 50ml volumetric flask, adding appropriate amount of anhydrous ethanol, ultrasonically dissolving, diluting with anhydrous ethanol to scale, shaking up, and using as reference substance stock solution I; precisely measuring 1ml of the reference substance storage solution I, placing the reference substance storage solution in a 100ml volumetric flask, diluting the reference substance storage solution to a scale with absolute ethyl alcohol, and shaking up to obtain the product.
The third step: preparing a sample solution, and carrying out fresh preparation and operation in a dark place. Taking a plurality of vitamin A capsules, cutting capsule skins, putting the contents in a beaker, precisely weighing a proper amount, putting the beaker in a 100ml measuring flask, adding 10ml of 0.025% ammonia water, carrying out ultrasonic treatment at 50 ℃ for 10min, adding 50ml of absolute ethyl alcohol, shaking in a water bath at 50 ℃ for 5min, cooling, adding absolute ethyl alcohol to dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the vitamin A capsule.
The fourth step: the details of the liquid chromatography conditions are as follows:
a chromatographic column: welch-C18150mm mm, 5 μm
Mobile phase: methanol-water (98: 2)
Detection wavelength: 325nm
Flow rate: 1.2ml/min
Column temperature: 25 deg.C
Sample introduction volume: 20ul of
Operating time: 6min
The sample detection chromatogram is shown in FIG. 6, and the detection results are shown in Table 3.
TABLE 3 test results
Example 4:
shenzhen Fulaite Nutrition and vitamin A content determination of vitamin A soft capsule produced by health Limited company. The method comprises the following specific steps:
the first step is as follows: preparing a reference solution, (i.e. operating in a dark place), precisely weighing 25.85mg of a vitamin A acetate reference (which is about equal to 7.5mg of vitamin A), placing the reference solution into a 20ml measuring flask, adding a proper amount of absolute ethyl alcohol, dissolving by ultrasonic waves, cooling to room temperature, adding absolute ethyl alcohol to dilute to a scale, and shaking up to obtain a vitamin A acetate reference stock solution; precisely transferring 3.0ml of vitamin A acetate stock solution, placing in a 200ml measuring flask, adding anhydrous ethanol for diluting to scale, shaking, and filtering to obtain final product
The second step is that: preparing a sample solution, (operating in a dark place), taking 10 vitamin A soft capsules, shearing capsule skins, taking the contents in a 25ml beaker, uniformly stirring with a glass rod, taking a proper amount (approximately 267 mu g of vitamin A), precisely weighing, putting into a 50ml measuring flask, adding a proper amount of absolute ethyl alcohol, shaking up to dissolve, carrying out ultrasonic treatment for 10min, cooling to room temperature, diluting to a scale with absolute ethyl alcohol, shaking up, standing for 10min, and filtering with a 0.45 mu m filter membrane.
The third step: the details of the liquid chromatography conditions are as follows:
a chromatographic column: welch-C18150mm mm, 5 μm
Mobile phase: methanol
Detection wavelength: 325nm
Flow rate: 1.0ml/min
Column temperature: 25 deg.C
Sample introduction volume: 20ul of
Operating time: for 10min
The sample detection chromatogram is shown in FIG. 7, and the detection results are shown in Table 4.
TABLE 4 test results
Examples 1-3 show that vitamin A can peak in about 4min when samples containing multivitamin mineral tablets, vitamin A tablets and multivitamin mineral capsules are detected; example 4 shows that the soft capsule sample is measured by slightly changing the original detection method for measuring vitamin A on the basis, and the result shows that the vitamin A in the soybean oil can peak in about 6 min; and the peak shape is good, and the separation degree with other components is large, which indicates that the method is suitable for detecting vitamin A in complex components of different dosage forms.
The above-mentioned embodiments provide a series of detailed descriptions for solving the technical problems, technical solutions and beneficial effects of the present invention. However, the description is only for the specific embodiment of the present invention, and the equivalent embodiments or modifications without departing from the technical basis, principle and spirit of the present invention are included in the protection scope of the present invention.
Claims (7)
1. A high performance liquid chromatography analysis method for high-efficiently determining the content of vitamin A in complex components is characterized in that the extraction solvent of a sample is low-concentration ammonia water solution and organic solvent, and the extraction method is water bath ultrasound or water bath oscillation; the mobile phase A is methanol, the mobile phase B is water, and the mixing ratio of the mobile phase A to the mobile phase B is 100-90: 0-10, isocratic elution; the ultraviolet detection wavelength is 300-350 nm.
2. The method according to claim 1, wherein the low concentration aqueous ammonia solution has a concentration of 0.01 to 0.05%, preferably 0.02 to 0.03%, more preferably 0.025%.
3. The method according to claim 1, wherein the organic solvent is selected from one of methanol, acetonitrile or ethanol, preferably ethanol, and the concentration of the organic solvent is 50-100%, preferably 90-100%, more preferably 100%.
4. The method according to claim 1, wherein the temperature of the water bath ultrasound is 25 to 80 ℃, preferably 30 to 60 ℃, more preferably 40 to 50 ℃.
5. The method according to claim 1, wherein the temperature of the water bath oscillation is 25 to 80 ℃, preferably 30 to 60 ℃, more preferably 40 to 50 ℃.
6. The method according to claim 1, wherein the flow rate in the HPLC is 0.8-1.5 ml/min.
7. The method according to claim 1, wherein the column temperature is 25 ℃ to 30 ℃, preferably 25 ℃.
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| CN106855545A (en) * | 2016-12-26 | 2017-06-16 | 新希望六和股份有限公司 | Liposoluble vitamin simultaneously in detection feed and the method for water soluble vitamin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106855545A (en) * | 2016-12-26 | 2017-06-16 | 新希望六和股份有限公司 | Liposoluble vitamin simultaneously in detection feed and the method for water soluble vitamin |
Non-Patent Citations (2)
| Title |
|---|
| 张利锋 等: "HPLC法测定保健食品中维生素A含量", 《中国卫生工程学》 * |
| 张连龙 等: "HPLC法测定黄金搭档组合维生素片中VitA、VitE的含量", 《安徽医药》 * |
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Application publication date: 20210105 |