CN109100456A - Method that is a kind of while measuring 3 kinds of liposoluble vitamin contents in multivitamin injection - Google Patents
Method that is a kind of while measuring 3 kinds of liposoluble vitamin contents in multivitamin injection Download PDFInfo
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- CN109100456A CN109100456A CN201811252956.5A CN201811252956A CN109100456A CN 109100456 A CN109100456 A CN 109100456A CN 201811252956 A CN201811252956 A CN 201811252956A CN 109100456 A CN109100456 A CN 109100456A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention discloses a kind of methods for measuring 3 kinds of liposoluble vitamin contents in multivitamin injection simultaneously, are related to analytical chemistry field.The present invention using octadecylsilane chemically bonded silica as stationary phase the following steps are included: be filled into the chromatographic column of liquid chromatograph；Methanol-acetonitrile-ethyl acetate is prepared as mobile phase；Precision weighs vitamin A, vitamin D3It is appropriate with vitamin E reference substance, add isopropanol that contrast solution is made；Precision measures multivitamin injection to be measured and is configured to test solution with isopropanol；Reference substance solution and test solution are taken respectively, and injection liquid chromatograph carries out isocratic elution, records chromatogram；By by external standard method with the concentration of calculated by peak area test solution.The present invention solves the prior art using three different Detection wavelengths, and gradient elution, mobile phase are complicated, the cumbersome time-consuming problem of sample handling processes.
Description
Technical field
The present invention relates to analytical chemistry field, especially it is a kind of measure in multivitamin injection simultaneously 3 kinds it is fat-soluble
The method of vitamin content.
Background technique
Vitamin is maintenance human health, enhancing development and adjusts one kind organic compound necessary to physiological function
Object.With the development of society, occurring more and more multivitamin products in the market.So, in product vitamin survey
The method of determining is particularly important.
Vitamin A, vitamin D3It is liposoluble vitamin with vitamin E, is easy to accumulate in vivo, excess intake will lead to
Poisoning, this method for just needing a kind of pair of liposoluble vitamin to be used for quickly detecting, what use was more now is high-efficient liquid phase color
Spectrometry.Detection of the vitamin A. D. E in food, dairy products, serum and injection has been reported that, but but deposits in technical aspect
In numerous problems: using three different Detection wavelengths, gradient elution, mobile phase are complicated, and sample handling processes are cumbersome time-consuming etc.,
The result measured in this way is possible to cause the content of liposoluble vitamin in sample to decline, and reduces the accuracy of detection.
Summary of the invention
Technical problem to be solved by the invention is to provide it is a kind of measure in multivitamin injection simultaneously 3 kinds it is fat-soluble
The method of vitamin content, the present invention solve the prior art using three different Detection wavelengths, and gradient elution, mobile phase are multiple
It is miscellaneous, the cumbersome time-consuming problem of sample handling processes.
In order to solve the above technical problems, the technical solution used in the present invention is: a kind of measure multivitamin note simultaneously
The method for penetrating 3 kinds of liposoluble vitamin contents in liquid, which comprises the following steps:
A. it is filled into octadecylsilane chemically bonded silica as stationary phase in the chromatographic column of liquid chromatograph；
B. methanol-acetonitrile-ethyl acetate is prepared as mobile phase；
C precision weighs vitamin A, vitamin D3It is appropriate with vitamin E reference substance, add isopropanol that every 1ml is made and contains
5000 unit of vitamin A, vitamin D3The reference substance solution of 2500 units, vitamin E 0.4mg；
D. accurate measurement multivitamin injection to be measured is configured to test solution with isopropanol；
E. reference substance solution and test solution are taken respectively, and injection liquid chromatograph carries out isocratic elution, records chromatogram；
F. it presses by external standard method with the concentration of calculated by peak area test solution.
In step B, methanol, acetonitrile, ethyl acetate volume ratio be 46.5:46.5:7.
A further technical solution lies in the liquid chromatograph ultraviolet detector detects vitamin A, vitamin D3With
Detection wavelength when vitamin E is 265nm.
Further technical solution also resides in, and the column temperature of the chromatographic column is maintained at 30 DEG C.
Further technical solution also resides in, and flow velocity of the mobile phase in chromatographic column is 1.0ml/min.
Further technical solution also resides in, the specific preparation process of the step D are as follows: precision measures a variety of dimensions to be measured
Raw element injection 5ml, sets in 50ml measuring bottle, with isopropanol to scale, shakes up, as test solution.
The beneficial effects of adopting the technical scheme are that mobile phase uses methanol-acetonitrile-second in the present invention
Acetoacetic ester, and ratio is adjusted to 46.5:46.5:7, compared with existing literature data, effectively improves and tie up life in liquid chromatogram
The peak shape of plain A, vitamine D3 and vitamin E keep the peak area of each vitamin obtained in liquid chromatogram more accurate, more have
Conducive to the accurate measurement to liposoluble vitamin content；Using high performance liquid chromatograph, when detecting 3 kinds of liposoluble vitamins, with
Existing literature data is compared, and the present invention can meet following condition simultaneously: using same ultraviolet detection wavelength and meanwhile measure, etc.
Degree elution, sample pre-treatments are easy, and test consumable is saved while guaranteeing that acquired results meet testing requirements；Use isopropanol
Reference substance and test sample are dissolved, shortens time and operating procedure without complicated processes, direct injection analysis such as extraction processings,
Improve detection efficiency.Therefore the method for the present invention is suitable for quickly continuously detecting the assay of multiple samples, saves experiment
The labor intensity of personnel.The method of the present invention has good specificity, is 3 kinds of liposoluble vitamins in multivitamin injection
The specific method of assay, is verified in actual production, becomes the important technology text for determining whether product is qualified
Part and foundation are conducive to promote the use of a large area suitable for using in quality control standard.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is reference substance solution chromatogram；
Fig. 2 is test solution chromatogram；
Fig. 3 is vitamin A canonical plotting；
Fig. 4 is vitamin D3Canonical plotting；
Fig. 5 is vitamin E canonical plotting.
Specific embodiment
With reference to the attached drawing in the embodiment of the present invention, technical solution in the embodiment of the present invention carries out clear, complete
Ground description, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by the specific embodiments disclosed below.
The present embodiment belongs to high performance liquid chromatography, chromatographic condition are as follows:
Chromatographic column selects octadecylsilane chemically bonded silica column；
Using methanol-acetonitrile-ethyl acetate as mobile phase, ratio is 46.5:46.5:7 (V/V)；
The Detection wavelength 254nm of detector；
Column temperature: 30 DEG C；
Flow velocity: 1.0ml/min；
Sampling volume: 20 μ l；
Selection for mobile phase, We conducted grope to test for several times, finally determine while using methanol-acetonitrile-second
Acetoacetic ester is tested, and ratio is 46.5:46.5:7 (V/V), and the peak shape of vitamin A, vitamine D3 and vitamin E is good, dimension
The retention time of raw element E is 7.295min, the retention time of vitamine D3 is 9.939min, and the retention time of vitamin A is
25.066min test result is ideal.
The content detection of 3 kinds of liposoluble vitamins in sample
(1) preparation of reference substance solution: precision weighs vitamin A (palmitate), vitamine D3 and vitamin E reference substance
In right amount, add isopropanol be made every 1ml containing vitamin A (palmitate), vitamine D3 and vitamin E be respectively 3.0mg (about
5000 units), the contrast solution of 0.6mg (about 2500 unit), 0.4mg.
(2) preparation of test solution: precision measures multivitamin injection 5ml to be measured, sets in 50ml measuring bottle, uses
Isopropanol shakes up, to scale as test solution.
(3) reference substance solution and test solution are measured into 20 μ L respectively, inject high performance liquid chromatograph, records chromatogram
(see attached drawing 1, attached drawing 2) obtains containing for vitamin A in sample (palmitate), vitamine D3 and vitamin E through analytical calculation
Amount, then be calculated the mark content of Retinol Palmitate, vitamine D3 and vitamin E be respectively 97.17%,
98.08%, 101.42%.
Test result shows this method to vitamin A (palmitate), vitamine D3 and dimension in multivitamin injection
Raw element E separation is good, and method is easy to operate, analysis real result, accurately.
The range of linearity investigates precision and weighs vitamin A (palmitate), vitamine D3 and appropriate vitamin E, sets 100ml amount
In bottle, add isopropanol to scale, every 1ml, which is made, containing vitamin A (palmitate), vitamine D3 and vitamin E is respectively
The standard solution of 8.0mg (about 13600 unit), 1.6mg (about 6400 unit), 1.2mg.Precision measurement standard solution is appropriate, adds
Isopropanol is diluted to respectively containing vitamin A (palmitate) 2.0mg/ml, 2.5mg/ml, 3.0mg/ml, 3.5mg/ml, 4.0mg/
ml；Vitamine D3 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml；Vitamin E 0.2mg/ml,
The contrast solution of 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml.Liquid phase is injected after 0.22 μm of filtering with microporous membrane
Chromatograph, the method measures according to the present invention, records chromatogram, with reference substance concentration C (mg/ml) for abscissa, peak area A
It is returned for ordinate, obtains equation of linear regression A=6E+06C+357.49, the r=0.9999 of vitamin A (palmitate),
Vitamin A (palmitate) linear relationship within the scope of 2.0mg/ml~4.0mg/ml is good；The linear regression side of vitamine D3
Journey A=897793C+2024.6, r=0.9991, vitamine D3 linear relationship within the scope of 0.4mg/ml~0.8mg/ml are good；
The equation of linear regression A=4E+06C+8745.2 of vitamin E, r=0.9999, vitamin E is in 0.2mg/ml~0.6mg/ml model
It is good (see attached drawing 3, attached drawing 4, attached drawing 5) to enclose interior linear relationship.
Precision investigates the preparation method according to test solution, prepares 6 parts of test solutions in parallel, investigates the method
Intermediate precision.To investigate influence of the random fluctuation factor to precision, another analyst independently establishes system, reconfigures
6 parts of test solutions are detected, and need to be used different instruments, be carried out on the different dates.The Intermediate precision of vitamin A tries
The RSD=0.26% tested, repetitive test RSD=0.28%, the RSD=0.82% of the Intermediate precision test of vitamine D3,
Repetitive test RSD=1.16%, the RSD=0.14%, repetitive test RSD=of the Intermediate precision test of vitamin E
0.20%, show the content precision of the method for the invention measurement vitamin A (palmitate), vitamine D3 and vitamin E
Height, favorable reproducibility, relative standard deviation are small.
The Precision test result of vitamin A (palmitate)
The Precision test result of vitamine D3
The Precision test result of vitamin E
The rate of recovery is investigated
The test liquid of basic, normal, high three kinds of various concentrations is prepared, the method measures according to the present invention, obtains the flat of each component
Equal rate of recovery difference is as shown in the table.
Vitamin A (palmitate) recovery test result
Vitamine D3 recovery test result
Vitamin E recovery test result
The study on the stability of solution
Test solution is prepared according to the method, is placed, is measured respectively at 0h, 2h, 4h, 6h, 8h, 10h sample introduction.Root
It is measured according to the method for the invention, records chromatogram.The result shows that: test solution of the present invention is placed 10 hours at room temperature and is tieed up
The peak area of raw element A, vitamine D3 and vitamin E have no significant change, will not impact to routine testing.
Vitamin A (palmitate) stability test result in sample
Vitamine D3 stability test result in sample
Stability of Vitamin E test result in sample
The present invention passes through and reference substance solution sample introduction Comparative result, retention time, separating degree, tailing factor, symmetrical factor, reason
Meet States Pharmacopoeia specifications requirement by number of plates items evaluation index, ties up life in the multivitamin injection that measurement result can reflect
Plain A (palmitate), vitamine D3, vitamin E content.The method of the present invention is simple, and assay is accurate and reliable, testing result
Well, it can be used in the inspection control of the content project.
Claims (5)
1. a kind of method for measuring 3 kinds of liposoluble vitamin contents in multivitamin injection simultaneously, which is characterized in that including
Following steps:
A. it is filled into octadecylsilane chemically bonded silica as stationary phase in the chromatographic column of liquid chromatograph；
B. methanol-acetonitrile-ethyl acetate is prepared as mobile phase；
C precision weighs vitamin A, vitamin D3It is appropriate with vitamin E reference substance, add isopropanol that every 1ml is made containing vitamin
5000 unit of A, vitamin D3The reference substance solution of 2500 units, vitamin E 0.4mg；
D. accurate measurement multivitamin injection to be measured is configured to test solution with isopropanol；
E. reference substance solution and test solution are taken respectively, and injection liquid chromatograph carries out isocratic elution, records chromatogram；
F. it presses by external standard method with the concentration of calculated by peak area test solution.
In step B, methanol, acetonitrile, ethyl acetate volume ratio be 46.5:46.5:7.
2. according to claim 1 a kind of 3 kinds of liposoluble vitamin contents are measured in multivitamin injection simultaneously
Method, it is characterised in that: the liquid chromatograph ultraviolet detector detects vitamin A, vitamin D3With inspection when vitamin E
Surveying wavelength is 265nm.
3. according to claim 1 a kind of 3 kinds of liposoluble vitamin contents are measured in multivitamin injection simultaneously
Method, it is characterised in that: the column temperature of the chromatographic column is maintained at 30 DEG C.
4. according to claim 1 a kind of 3 kinds of liposoluble vitamin contents are measured in multivitamin injection simultaneously
Method, it is characterised in that: flow velocity of the mobile phase in chromatographic column is 1.0ml/min.
5. according to claim 1 a kind of 3 kinds of liposoluble vitamin contents are measured in multivitamin injection simultaneously
Method, which is characterized in that the specific preparation process of the step D are as follows: precision measures multivitamin injection 5ml to be measured,
It is placed in 50ml measuring bottle, with isopropanol to scale, shakes up, as test solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113960211A (en) * | 2021-11-04 | 2022-01-21 | 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 |
| CN114965742A (en) * | 2022-04-21 | 2022-08-30 | 广西铭磊维生制药有限公司 | Method for determining vitamin K1 drop-related substances |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113960211A (en) * | 2021-11-04 | 2022-01-21 | 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 |
| CN114965742A (en) * | 2022-04-21 | 2022-08-30 | 广西铭磊维生制药有限公司 | Method for determining vitamin K1 drop-related substances |
| 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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