CN109100456B - Method for simultaneously determining content of 3 fat-soluble vitamins in multivitamin injection - Google Patents
Method for simultaneously determining content of 3 fat-soluble vitamins in multivitamin injection Download PDFInfo
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- CN109100456B CN109100456B CN201811252956.5A CN201811252956A CN109100456B CN 109100456 B CN109100456 B CN 109100456B CN 201811252956 A CN201811252956 A CN 201811252956A CN 109100456 B CN109100456 B CN 109100456B
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Abstract
The invention discloses a method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection, and relates to the field of analytical chemistry. The invention comprises the following steps: octadecylsilane chemically bonded silica is used as a stationary phase and is filled into a chromatographic column of a liquid chromatograph; preparing methanol-acetonitrile-ethyl acetate as a mobile phase; precisely weighing vitamin A and vitamin D3Adding isopropanol to a proper amount of vitamin E reference substance, and diluting to obtain reference solution; precisely measuring multiple vitamin injection to be tested, diluting with isopropanol, and preparing into test solution; respectively taking the reference solution and the test solution, injecting into a liquid chromatograph for isocratic elution, and recording a chromatogram; the concentration of the test solution was calculated as peak area by external standard method. The invention solves the problems of three different detection wavelengths, gradient elution, complex mobile phase, and complicated and time-consuming sample treatment process in the prior art.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection.
Background
Vitamins are a class of organic compounds essential to maintain human health, promote growth and development, and regulate physiological functions. With the development of society, more and more multivitamin products appear on the market. The determination of the vitamins in the product is of particular importance.
Vitamin A and vitamin D3Vitamin E and vitamin E are fat-soluble vitamins and are easy to accumulate in a body, and excessive intake can cause poisoning, so that a method for quickly detecting the fat-soluble vitamins is needed, and high performance liquid chromatography is mostly adopted at present. The detection of vitamin A, D, E in food, milk, serum and injection is reported, but has a plurality of problems in the technical aspect: three different detection wavelengths are adopted, gradient elution and a mobile phase are complex, the sample processing process is complicated and time-consuming, and the like, so that the content of fat-soluble vitamins in a sample can be reduced due to the measured result, and the detection accuracy is reduced.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection, and solves the problems of three different detection wavelengths, complex gradient elution and mobile phase, and complicated and time-consuming sample treatment process in the prior art.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for simultaneously measuring the content of 3 fat-soluble vitamins in a multivitamin injection is characterized by comprising the following steps:
A. octadecylsilane chemically bonded silica is used as a stationary phase and is filled into a chromatographic column of a liquid chromatograph;
B. preparing methanol-acetonitrile-ethyl acetate as a mobile phase;
c precisely weighing vitamin A and vitamin D3Mixing with vitamin E control, adding isopropanol, and diluting to obtain solution containing 5000 units of vitamin A and vitamin D per 1ml32500 units of a control solution of 0.4mg vitamin E;
D. precisely measuring multiple vitamin injection to be tested, diluting with isopropanol, and preparing into test solution;
E. respectively taking the reference solution and the test solution, injecting into a liquid chromatograph for isocratic elution, and recording a chromatogram;
F. the concentration of the test solution was calculated as peak area by external standard method.
In the step B, the volume ratio of methanol to acetonitrile to ethyl acetate is 46.5: 46.5:7.
The further technical proposal is that the ultraviolet detector of the liquid chromatograph detects vitamin A and vitamin D3The detection wavelength with vitamin E is 265 nm.
The further technical proposal is that the temperature of the chromatographic column is kept at 30 ℃.
The further technical proposal is that the flow rate of the mobile phase in the chromatographic column is 1.0 ml/min.
The further technical scheme is that the specific preparation process of the step D is as follows: precisely measuring 5ml of the multivitamin injection to be detected, placing the multivitamin injection in a 50ml measuring flask, diluting with isopropanol to scale, and shaking up to obtain a test solution.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: in the invention, the mobile phase adopts methanol-acetonitrile-ethyl acetate, and the proportion is adjusted to 46.5:46.5:7, compared with the existing literature data, the peak shapes of vitamin A, vitamin D3 and vitamin E in the liquid chromatogram are effectively improved, so that the peak area of each vitamin obtained in the liquid chromatogram is more accurate, and the accurate determination of the content of fat-soluble vitamin is more facilitated; when a high performance liquid chromatograph is adopted to detect 3 fat-soluble vitamins, compared with the existing literature data, the invention can simultaneously meet the following conditions: the same ultraviolet detection wavelength is adopted for simultaneous determination, isocratic elution and sample pretreatment, so that test consumables are saved while the obtained result is ensured to meet the detection requirement; the isopropanol is used for dissolving the reference substance and the test substance, the complicated processes such as extraction treatment and the like are not needed, the sample injection analysis is directly carried out, the time and the operation steps are shortened, and the detection efficiency is improved. Therefore, the method is suitable for rapidly and continuously detecting the content of a plurality of samples, and the labor intensity of experimenters is saved. The method has good specificity, is a specific method for measuring the content of 3 fat-soluble vitamins in the multivitamin injection, is verified in actual production, becomes an important technical file and basis for judging whether the product is qualified or not, is suitable for being adopted in a quality control standard, and is beneficial to large-area popularization and use.
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The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a chromatogram of a control solution;
FIG. 2 is a chromatogram of a test solution;
FIG. 3 is a graph of vitamin A standards;
FIG. 4 is vitamin D3A standard curve graph;
figure 5 is a graph of vitamin E standard.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in 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.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
This example pertains to high performance liquid chromatography, where the chromatographic conditions are:
the chromatographic column adopts an octadecylsilane chemically bonded silica gel column;
methanol-acetonitrile-ethyl acetate is used as a mobile phase, and the ratio is 46.5:46.5:7 (V/V);
the detection wavelength of the detector is 254 nm;
column temperature: 30 ℃;
flow rate: 1.0 ml/min;
sample introduction volume: 20 mu l of the mixture;
for the choice of mobile phase, we have carried out several trials and finally determined that the test was carried out simultaneously with methanol-acetonitrile-ethyl acetate in a ratio of 46.5:46.5: 7(V/V), the peak shapes of the vitamin A, the vitamin D3 and the vitamin E are good, the retention time of the vitamin E is 7.295min, the retention time of the vitamin D3 is 9.939min, the retention time of the vitamin A is 25.066min, and the test result is ideal.
Content detection of 3 fat-soluble vitamins in sample
(1) Preparation of control solutions: an appropriate amount of vitamin A (palmitate), vitamin D3 and vitamin E control were precisely weighed and diluted with isopropanol to prepare control solutions containing 3.0mg (about 5000 units), 0.6mg (about 2500 units) and 0.4mg of vitamin A (palmitate), vitamin D3 and vitamin E, respectively, per 1 ml.
(2) Preparation of a test solution: precisely measuring 5ml of the multivitamin injection to be detected, placing the multivitamin injection in a 50ml measuring flask, diluting with isopropanol to scale, and shaking up to obtain a test solution.
(3) Measuring 20 μ L of the reference solution and the test solution, respectively, injecting into a high performance liquid chromatograph, recording chromatogram (shown in figure 1 and figure 2), analyzing and calculating to obtain the contents of vitamin A (palmitate), vitamin D3 and vitamin E in the sample, and calculating to obtain the labeled contents of vitamin A palmitate, vitamin D3 and vitamin E of 97.17%, 98.08% and 101.42%, respectively.
Test results show that the method has good separation effect on vitamin A (palmitate), vitamin D3 and vitamin E in the multi-vitamin injection, is simple to operate, and has real and accurate analysis results.
Linear range investigation accurately weighing appropriate amount of vitamin a (palmitate), vitamin D3 and vitamin E, placing in a 100ml measuring flask, adding isopropanol, diluting to scale, and making into standard solution containing vitamin a (palmitate), vitamin D3 and vitamin E8.0 mg (about 13600 unit), 1.6mg (about 6400 unit) and 1.2mg respectively per 1 ml. Precisely measuring a proper amount of standard solution, and adding isopropanol to dilute into 2.0mg/ml, 2.5mg/ml, 3.0mg/ml, 3.5mg/ml and 4.0mg/ml of vitamin A (palmitate); vitamin D30.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8 mg/ml; vitamin E0.2 mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml of control solution. Filtering with a 0.22-micron microporous membrane, injecting into a liquid chromatograph, measuring according to the method of the invention, recording a chromatogram, and performing regression by taking the concentration C (mg/ml) of a reference substance as an abscissa and the peak area A as an ordinate to obtain the vitamin A (palmitate) with a linear regression equation A of 6E +06C +357.49 and r of 0.9999, wherein the vitamin A (palmitate) has a good linear relationship in the range of 2.0-4.0 mg/ml; the linear regression equation A of the vitamin D3 is 897793C +2024.6, r is 0.9991, and the vitamin D3 has good linear relation in the range of 0.4mg/ml to 0.8 mg/ml; the linear regression equation A of vitamin E is 4E +06C +8745.2, r is 0.9999, and vitamin E has good linear relation in the range of 0.2 mg/ml-0.6 mg/ml (see figure 3, figure 4 and figure 5).
Precision examination 6 parts of the test solution were prepared in parallel according to the method for preparing the test solution, and the intermediate precision of this method was examined. In order to examine the influence of random variation factors on precision, another analyst independently establishes a system and reconfigures 6 test sample solutions for detection, wherein different instruments are used and the detection is carried out on different dates. The method for determining the content of the vitamin A (palmitate), the vitamin D3 and the vitamin E has high precision, good reproducibility and small relative standard deviation, wherein the RSD of the vitamin A in the intermediate precision test is 0.26%, the RSD of the vitamin D3 in the intermediate precision test is 0.28%, the RSD of the vitamin D3 in the intermediate precision test is 0.82%, the RSD of the vitamin D1.16%, the RSD of the vitamin E in the intermediate precision test is 0.14% and the RSD of the vitamin E in the repetitive test is 0.20%.
Precision test results for vitamin A (palmitate)
Precision test result of vitamin D3
Results of vitamin E precision test
Investigation of recovery
The average recovery rates of the components are respectively shown in the following table when three test solutions with different concentrations, namely low, medium and high, are prepared and measured by the method of the invention.
Results of vitamin A (palmitate) recovery test
Vitamin D3 recovery test results
Results of vitamin E recovery test
Stability testing of solutions
Preparing a test solution according to the method, standing, and injecting samples for determination at 0h, 2h, 4h, 6h, 8h and 10h respectively. Measured according to the method of the invention, chromatograms are recorded. The results show that: the peak areas of the vitamin A, the vitamin D3 and the vitamin E of the test solution are not obviously changed after the test solution is placed at room temperature for 10 hours, and the common detection is not influenced.
Results of vitamin A (palmitate) stability test in samples
Results of vitamin D3 stability test in samples
Results of vitamin E stability test in samples
Compared with the sample injection result of a reference solution, the evaluation indexes of retention time, separation degree, tailing factor, symmetry factor and theoretical plate number meet the requirements specified by pharmacopoeia, and the measurement result can reflect the contents of vitamin A (palmitate), vitamin D3 and vitamin E in the multivitamin injection. The method is simple, the content measurement is accurate and reliable, the detection result is good, and the method can be used for the inspection control of the content item.
Claims (5)
1. A method for simultaneously measuring the content of 3 fat-soluble vitamins in a multivitamin injection is characterized by comprising the following steps:
A. octadecylsilane chemically bonded silica is used as a stationary phase and is filled into a chromatographic column of a liquid chromatograph;
B. preparing methanol-acetonitrile-ethyl acetate as a mobile phase;
c precisely weighing vitamin A and vitamin D3Mixing with vitamin E control, adding isopropanol, and diluting to obtain solution containing 5000 units of vitamin A and vitamin D per 1ml32500 units of a control solution of 0.4mg vitamin E;
D. precisely measuring multiple vitamin injection to be tested, diluting with isopropanol, and preparing into test solution;
E. respectively taking the reference solution and the test solution, injecting into a liquid chromatograph for isocratic elution, and recording a chromatogram;
F. calculating the concentration of the test solution according to the peak area by an external standard method;
in the step B, the volume ratio of methanol to acetonitrile to ethyl acetate is 46.5: 46.5:7.
2. The method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection according to claim 1, which comprises the following steps: the liquid chromatograph ultraviolet detector detects vitamin A and vitamin D3The detection wavelength with vitamin E is 265 nm.
3. The method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection according to claim 1, which comprises the following steps: the column temperature of the column was maintained at 30 ℃.
4. The method for simultaneously determining the content of 3 fat-soluble vitamins in a multivitamin injection according to claim 1, which comprises the following steps: the flow rate of the mobile phase in the chromatographic column was 1.0 ml/min.
5. The method for simultaneously determining the content of 3 fat-soluble vitamins in a multi-vitamin injection according to claim 1, wherein the specific preparation process of the step D comprises the following steps: precisely measuring 5ml of the multivitamin injection to be detected, placing the multivitamin injection in a 50ml measuring flask, diluting with isopropanol to scale, and shaking up to obtain a test solution.
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| CN113960211B (en) * | 2021-11-04 | 2022-06-14 | 青岛市食品药品检验研究院（青岛市药品不良反应监测中心、青岛市实验动物和动物实验中心） | Method for measuring vitamin K in serum |
| CN114965742B (en) * | 2022-04-21 | 2023-08-18 | 广西铭磊维生制药有限公司 | Method for measuring related substances of vitamin K1 drops |
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