CN117269375B - Method for determining related substances in vitamin A acetate powder - Google Patents

Method for determining related substances in vitamin A acetate powder Download PDF

Info

Publication number
CN117269375B
CN117269375B CN202311561089.4A CN202311561089A CN117269375B CN 117269375 B CN117269375 B CN 117269375B CN 202311561089 A CN202311561089 A CN 202311561089A CN 117269375 B CN117269375 B CN 117269375B
Authority
CN
China
Prior art keywords
vitamin
acetate powder
mobile phase
related substance
substances
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202311561089.4A
Other languages
Chinese (zh)
Other versions
CN117269375A (en
Inventor
罗瑶
唐燕
梁丰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan Kerusi Pharmaceutical Technology Co ltd
Original Assignee
Hunan Kerusi Pharmaceutical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan Kerusi Pharmaceutical Technology Co ltd filed Critical Hunan Kerusi Pharmaceutical Technology Co ltd
Priority to CN202311561089.4A priority Critical patent/CN117269375B/en
Publication of CN117269375A publication Critical patent/CN117269375A/en
Application granted granted Critical
Publication of CN117269375B publication Critical patent/CN117269375B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Engineering & Computer Science (AREA)
  • Library & Information Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to a method for determining related substances in vitamin A acetate powder, which is a high performance liquid chromatography method, wherein the method is used for analyzing the vitamin A acetate powder so as to obtain a chromatogram, and determining the related substances in the vitamin A acetate powder based on the chromatogram.

Description

Method for determining related substances in vitamin A acetate powder
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for measuring related substances in vitamin A acetate powder.
Background
Vitamin A belongs to vitamin medicines, is necessary for growth and development, can maintain normal functions of skin, cornea and various mucous membranes, participates in synthesis of rhodopsin, and can enhance the photosensitive performance of retina; can be used for treating vitamin A deficiency, and can be added into various vitamin supplements.
Meanwhile, vitamin A is a fat-soluble vitamin with a chemical formula of C 20 H 30 O has the following structural formula:the method comprises the steps of carrying out a first treatment on the surface of the Is insoluble in water and glycerin, and can be dissolved in most organic solvents such as alcohol, ether, hydrocarbon, halogenated hydrocarbon and the like; it is stable to heat, acid and alkali, is easy to oxidize and is not easy to store for a long time. Because vitamin A in Chinese pharmacopoeia is an oil solution prepared by adding refined vegetable oil into vitamin A acetate crystals, however, all pharmacopoeia standards do not have detection methods of related substances of vitamin A bulk drugs, vitamin A acetate powder and related preparations thereof; in other legal standards, the vitamin A acetate powder import registration standard JX20170163 receives a method of carrying related substances of the vitamin A acetate; however, only three isomers of vitamin a acetate related substance 1 were controlled, and from the structural analysis, the related substance 1 should have four isomers; therefore, a proper method capable of accurately detecting related substances in the vitamin A acetate powder is required to be developed for researching related substance changes of the vitamin A in the process of placing stability, so that the quality of products is estimated.
Disclosure of Invention
The invention aims to provide a method for measuring various related substances in vitamin A acetate powder, which has the advantages of low detection cost, strong specificity, good reproducibility, sensitivity and short analysis time.
The invention provides a method for determining related substances in vitamin A acetate powder, which is a high performance liquid chromatography, wherein the high performance liquid chromatography adopts the following conditions:
chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as filler,
and (3) a monitor: the DAD is used to determine the position of the DAD,
detection wavelength: a wavelength of 286nm, and a wavelength of at least one of the three,
detection wavelength of control solution: at a wavelength of 325nm,
mobile phase a: an acetonitrile-water solution of the components,
mobile phase B: methanol-isopropyl alcohol (methanol-isopropyl alcohol) is used as a solvent,
mobile phase C: the concentration of acetonitrile in the aqueous solution is,
flow rate: 0.9ml/min-1.1ml/min,
column temperature: 30-40 ℃,
the elution gradient is:
；
wherein the related substance structure is as follows:
。
it can be understood that, the related substances 1 have four isomers, the related substances 2 and the related substances 3 are added, the polarity difference among the related substances is large, part of the related substances have large polarities, the related substances remain weak or almost have no retention in a chromatographic system, and part of the related substances have small polarities and remain extremely strong in the chromatographic system and are not easy to elute.
In some embodiments, the chromatography column is Hypersil BDS C18, the specification of the chromatography column is 250mm x 4.6mm,5 μm.
In some embodiments, the volume fraction of acetonitrile in mobile phase a is 48% -52%; the volume fraction of methanol in the mobile phase B is 88% -92%.
Preferably, the volume fraction of acetonitrile in the mobile phase a is 49%, 50% or 51%; the volume fraction of methanol in mobile phase B was 89%, 90% or 91%.
In some embodiments, the flow rate is 1ml/min and the column temperature is 35 ℃.
In some embodiments, the vitamin a acetate powder is provided in the form of a test solution, wherein the test solution is a vitamin a acetate powder methanol solution, and wherein the concentration of vitamin a acetate is 0.02-0.03mg/ml per milliliter of test solution.
Preferably, the vitamin A acetate concentration is 0.023mg/ml, 0.025mg/ml, 0.026mg/ml, or 0.029mg/ml.
In some embodiments, the preparation method of the test solution comprises breaking the wall of the starch layer of the vitamin A acetate powder with propylene glycol and extracting with methanol.
In some embodiments, the vitamin a acetate powder related substance content is determined by the following formula:wherein: a is that s Peak area of single related substances in the sample at 286nm wavelength; a is that r The main peak area in the control solution at the wavelength of 286 nm; v (V) r Dilution fold for control solution; f is self-correction factor, wherein vitamin a acetate powder related substance self-correction factor = main peak area of control solution at 286nm wavelength/main peak area of control solution at 325nm wavelength.
The detection method provided by the invention can be used for determining various related substances in the vitamin A acetate powder, realizes effective separation and content determination of each related substance, and has the advantages of low detection cost, strong specificity, good reproducibility, sensitivity and short analysis time.
Drawings
FIG. 1 is a chromatogram of vitamin A acetate powder under chromatographic conditions as described in example 1;
FIG. 2 is an ultraviolet absorption curve of vitamin A acetate;
FIG. 3 is an ultraviolet absorption curve of isomer 1 of substance 1 of example 4;
FIG. 4 is an ultraviolet absorption curve of isomer 2 of substance 1 of example 4;
FIG. 5 is a graph showing the ultraviolet absorption curve of isomer 3 of substance 1 of example 4;
FIG. 6 is an ultraviolet absorption curve of isomer 4 of substance 1 of example 4;
FIG. 7 is an ultraviolet absorption curve of the substance 2 of example 4;
FIG. 8 is an ultraviolet absorption curve of the substance 3 of example 4;
FIG. 9 is a chromatogram of vitamin A acetate powder under the chromatographic conditions of the mobile phase of Table 2 described in example 5;
FIG. 10 is a chromatogram of vitamin A acetate powder under the chromatographic conditions of the mobile phase of Table 3 described in example 5;
FIG. 11 is a chromatogram of vitamin A acetate powder under chromatographic conditions for the mobile phase of Table 4 described in example 5;
FIG. 12 is a superimposed chromatogram under chromatographic conditions for the mobile phase of Table 4 described in example 5; wherein (a) is a blank solution chromatogram, (b) is a blank auxiliary material chromatogram, (c) is a related substance positioning solution chromatogram, and (d) is a sample solution chromatogram;
FIG. 13 is a chromatogram of vitamin A acetate powder under chromatographic conditions for the mobile phase of Table 5 described in example 5;
FIG. 14 is a graph of vitamin A acetate powder at different wavelengths, wherein (a) is a graph of vitamin A acetate powder at a wavelength of 286nm and (b) is a graph of vitamin A acetate powder at a wavelength of 325 nm;
FIG. 15 is a chromatogram of a blank solution under chromatographic conditions described in example 8;
FIG. 16 is a chromatogram of a test solution under chromatographic conditions described in example 8;
FIG. 17 is a chromatogram of the control solution under 286nm wavelength conditions of the chromatographic conditions described in example 8;
FIG. 18 is a chromatogram of the control solution under the chromatographic conditions described in example 8 at a wavelength of 325 nm;
reference numerals: 1: the related substance 1 isomer 1 peaks; 2: the related substance 1 isomer 2 peaks; 3: the related substance 1 isomer 3 peaks; 4: the related substance 1 isomer 4 peaks; 5: vitamin a acetate peaks; 6: peak of related substance 2; 7: peak of related substance 3; 8: the dibutylhydroxytoluene peaked.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
The indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.
In the present invention, a feature defining "first" or "second" may explicitly or implicitly include one or more of such feature for distinguishing between the descriptive features, and not sequentially or lightly. In the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
Example 1 method reproduction in the Prior Art
In the prior art, only the imported registration standard JX20170163 discloses a detection method of related substances of vitamin A acetate powder, the method is reproduced, and as the column temperature is not noted in the standard, the column temperature is set to 30 ℃ temporarily, and chromatographic conditions are as follows:
chromatographic column: waters Sunfire C18 (4.6. Times.150 mm,3.5 μm),
mobile phase a/mobile phase B: methanol/water (97:3),
elution mode: the elution is carried out in an isocratic way,
detection wavelength: 307nm of the wavelength of the light,
sample injection volume: 10. Mu.l of the total volume of the solution,
flow rate: 1.3ml/min of the total volume of the mixture,
column temperature: 30 ℃,
acquisition time: for 40 minutes, the temperature of the mixture is controlled,
the obtained chromatogram is shown in fig. 1, wherein vitamin A acetate peaks at about 2.850min, there is a risk that the related substances remain in the solvent peak, and the theory of isomers of related substance 1 should be four, but only three are detected, and the method is not applicable.
Example 2 selection of elution mode
The choice of isocratic elution may present a risk of the relevant material remaining in the solvent peaks. In addition, the vitamin A acetate powder has a large number of related substances, the molecular weight and the hydrophobicity of each related substance have a large difference, and the isocratic method is difficult to detect all related substance peaks, so that gradient elution is selected, and related substances with different hydrophobicity can be better separated and retained.
Example 3 selection of sample injection amount
The sample injection amount is increased to 30 mu l, the sample injection volume is increased, and the sensitivity of each related substance in the sample solution can be improved.
Example 4 screening for detection wavelengths
Comparing the ultraviolet absorption curves of the main component and each related substance, wherein the characteristic absorption wavelength of each component is shown in the following table 3, the ultraviolet absorption curves of each component are shown in fig. 2-8, and the maximum absorption wavelength of the known related substance is mostly closer to 286nm, and the detection capability of the unknown impurity of 286nm in the sample solution is higher than 325nm, so that the detection wavelength is finally selected as the detection wavelength of 286 nm; since the substance 2 of interest is not absorbed at 286nm, 325nm alone was selected to detect the substance 2 of interest.
Table 1 characteristic absorption wavelength table of substances
。
EXAMPLE 5 screening of mobile phases
When water and methanol are selected as the mobile phase, only hydrogen bonding and weaker hydrophobic effect are adopted, acetonitrile and isopropanol are determined to be added into a water and methanol system after screening, the acetonitrile can introduce dipole effect, the isopropanol can increase the hydrogen bonding and the hydrophobic effect, and the two reagents can both increase the mobile phase selectivity, so that the separation of each peak is better. As shown in FIG. 9, the chromatogram obtained by using the mobile phase in Table 2 shows poor separation of the main peak and the adjacent front peak, and the baseline separation is not achieved; as shown in fig. 10, using the mobile phase chromatograms of table 3, the separation of the main peak from the adjacent front peaks was improved, but the baseline separation was not achieved; the chromatograms obtained using the mobile phases of table 4 are shown in fig. 11 and 12; the main peak and the adjacent peaks can basically achieve baseline separation, the peak types of all related substances are good, but the peak of the related substance 1 isomer 4 is similar to the peak of the related substance 1 isomer 4, and interference is caused; thus, by prolonging the elution procedure, the main peak and the adjacent peaks can basically achieve baseline separation, the peak types of the related substances are good, and the separation degree of the peak of the related substance 1 isomer 4 and the gradient change peak is increased by adopting the chromatogram obtained by the mobile phase in the table 5 as shown in fig. 13; thus the mobile phase conditions described in table 5 were employed.
TABLE 2 Mobile phase conditions
；
TABLE 3 mobile phase conditions
；
TABLE 4 mobile phase conditions
；
TABLE 5 mobile phase conditions
。
Example 6 screening of test sample preparation method
The vitamin A acetate powder is prepared from vitamin A acetate raw material medicines through a spray drying process, and is prepared into oil-in-water emulsion through granulation and then dried to obtain particles which are flowable fine particles. Therefore, the preparation of the test solution requires demulsification of the vitamin A acetate powder and extraction of the vitamin A acetate. In comparison with the following two preparation methods, in the method 2, propylene glycol is adopted to break the wall of the starch layer of the vitamin A acetate powder, and then methanol is used for extraction. The two solutions were formulated as follows and the results are shown in table 6 below:
the preparation method 1 comprises the following steps: 3.7mg of vitamin A acetate powder (which is approximately equivalent to 1850IU of vitamin A and 0.636mg of vitamin A acetate) is precisely weighed, placed in a 120ml brown sample bottle, added with 20ml of dimethyl sulfoxide, uniformly shaken, ultrasonically shaken at 40 ℃ for 40 minutes (periodically shaken), cooled to room temperature, precisely added with 50ml of n-hexane, shaken at 300rpm for 50 minutes, measured and 6ml of supernatant liquid, blown dry by nitrogen, added with 3ml of isopropanol, and vortexed for 2 minutes to dissolve residues, thus obtaining the vitamin A acetate. (vitamin A acetate about 26. Mu.g/ml);
the preparation method 2 comprises the following steps: precisely weighing about 3.7mg (about corresponding to 1850IU vitamin A acetate and 0.636mg vitamin A acetate), placing into a 30ml brown sample bottle, precisely adding 3ml propylene glycol to completely moisten the powder, ultrasonically treating at room temperature for 5min (shaking at all times), adding 22ml methanol, ultrasonically treating at room temperature for 10min (shaking at all times), centrifuging for 10min at 4000rpm, and collecting supernatant. (vitamin A acetate about 26. Mu.g/ml);
TABLE 6 peak area and standard deviation of vitamin A acetate under different test sample solutions formulation methods
；
From the above table, the sample solution of preparation method 2 was selected as the preparation method of the vitamin a acetate powder sample solution because the peak content and the main peak area reproducibility of each relevant substance were the best in both methods.
Example 7 selection of a calculation mode of the content of substances
Because the related substances of the vitamin A acetate powder are extremely difficult to prepare, the content of each related substance is calculated by adopting a main component self-comparison method. As shown in fig. 14, the response of the vitamin a acetate peak at a wavelength of 286nm was about 1/3 of the wavelength of 325nm using the above-mentioned preferable chromatographic conditions, so that when the content of the relevant substance was calculated by the main component self-correlation method, the main peak area in the control solution was corrected by the response of 325nm, and the self-correction factor=the main peak area at 286 nm/the main peak area at 325 nm.
Example 8 confirmation of the procedure
1.1 Solution preparation
(1) Mobile phase
Mobile phase a: weighing 500ml of water and 500ml of acetonitrile, uniformly mixing, and performing ultrasonic treatment for 10min to obtain the water-based emulsion;
mobile phase B: weighing 900ml of methanol and 100ml of isopropanol, placing in the same mobile phase bottle, uniformly mixing, and performing ultrasonic treatment for 10min to obtain the final product;
mobile phase C: acetonitrile;
(2) Blank solution (thinner)
Respectively weighing 3ml of propylene glycol and 22ml of methanol, placing in the same 120ml brown sample bottle, and shaking uniformly to obtain the final product;
(3) Vitamin A acetate powder test solution
Precisely weighing 11.1mg of vitamin A acetate powder (about corresponding to 5550 units of vitamin A and 1.908mg of vitamin A acetate), placing into a 120ml brown sample bottle, precisely adding 9ml of propylene glycol, performing ultrasonic treatment for 5min (shaking at time), precisely adding 66ml of methanol, performing ultrasonic treatment for 10min (shaking at time), taking about 10ml of the solution, centrifuging at 4000rpm for 10min, and taking supernatant to obtain the vitamin A acetate;
(4) Control solution
Precisely measuring 1ml of vitamin A acetate powder sample solution, placing in a 100ml measuring flask, adding methanol to dilute to scale, and shaking to obtain the final product.
1.2 Chromatographic conditions
Chromatographic column: hypersil BDS C18, 250 mm. Times.4.6 mm,5 μm,
flow rate: 1.0ml/min of the total volume of the mixture,
column temperature: 35 c,
detection wavelength: the vitamin A acetate powder is 286nm, the control solution is 286nm and 325nm,
sample injection amount: 30 μl of the glass fiber reinforced plastic (W) is processed,
detection time: the time period of the reaction is 65 minutes,
mobile phase: mobile phase a:50% acetonitrile in water; mobile phase B: methanol-isopropanol (90:10); mobile phase C: the concentration of acetonitrile in the aqueous solution is,
elution mode: the gradient elution is carried out,
TABLE 7 elution gradient
。
1.3 Sample injection
Taking 30 μl of blank solution for sample injection, wherein the impurities are required to not interfere with detection, and the obtained chromatogram is shown in FIG. 15;
taking 30 μl of vitamin A acetate powder sample solution, and collecting the chromatogram shown in figure 16;
30 μl of the control solution was sampled and the resulting chromatograms were as shown in FIGS. 17 and 18.
1.4 Content calculation formula
(1) Knowing the relative retention time of the substances of interest
TABLE 8 relative retention time of substances of interest
；
(2) Vitamin A acetate powder
The method comprises the steps of carrying out a first treatment on the surface of the Wherein: a is that s Peak area of single related substances in the sample at 286nm wavelength; a is that r The main peak area in the control solution at the wavelength of 286 nm; v (V) r Dilution fold for control solution; f is self-correction factor, wherein vitamin a acetate powder related substance self-correction factor = main peak area of control solution at 286nm wavelength/main peak area of control solution at 325nm wavelength.
1.5 Content calculation result
TABLE 9 calculation of content
；
Description: ND is undetected and N/A is not involved.
The method is used for detecting the substances related to the vitamin A acetate powder, and the blank solution does not interfere with the determination of the substances related to the vitamin A acetate powder.

Claims (7)

1. A method for determining related substances in vitamin a acetate powder, which is characterized by comprising the following steps of:
chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as filler,
a detector: the DAD is used to determine the position of the DAD,
detection wavelength: the detection wavelength of the related substances 2 in the sample solution is 325nm, the detection wavelength of other related substances is 286nm,
detection wavelength of control solution: 286nm and 325nm, respectively,
mobile phase a: an acetonitrile-water solution of the components,
mobile phase B: methanol-isopropyl alcohol (methanol-isopropyl alcohol) is used as a solvent,
mobile phase C: the concentration of acetonitrile in the aqueous solution is,
flow rate: 0.9ml/min-1.1ml/min,
column temperature: 30-40 ℃,
the elution gradient is:
；
the related substances comprise related substance 1, related substance 2 and related substance 3, wherein the related substance 1 comprises related substance 1 isomer 1, related substance 1 isomer 2, related substance 1 isomer 3 and related substance 1 isomer 4, and the related substance 1 has the structure ofThe structure of the related substance 2 is thatThe structure of the substance 3 concerned is +.>The method comprises the steps of carrying out a first treatment on the surface of the Wherein the volume fraction of acetonitrile in the mobile phase A is 48% -52%; the volume fraction of methanol in the mobile phase B is 88% -92%.
2. The method for determining related substances in vitamin a acetate powder according to claim 1, wherein the chromatographic column is Hypersil BDS C18, and the specification of the chromatographic column is 250mm x 4.6mm,5 μm.
3. The method for determining relevant substances in vitamin a acetate powder according to claim 1, wherein the volume fraction of acetonitrile in mobile phase a is 50%; the volume fraction of methanol in mobile phase B was 90%.
4. The method for determining substances of interest in vitamin a acetate powder according to claim 1, wherein the flow rate is 1ml/min and the column temperature is 35 ℃.
5. The method for determining substances of interest in vitamin a acetate powder according to claim 1, wherein the vitamin a acetate powder is provided in the form of a sample solution, wherein the sample solution is a methanol solution of vitamin a acetate powder and wherein the concentration of vitamin a acetate is 0.02-0.03mg/ml per ml of sample solution.
6. The method for measuring related substances in vitamin A acetate powder according to claim 5, wherein the preparation method of the sample solution is to use propylene glycol to break the wall of the starch layer of the vitamin A acetate powder and then extract the starch layer with methanol.
7. The method for determining the content of related substances in vitamin a acetate powder according to claim 1, wherein the content of related substances in vitamin a acetate powder is determined by the following formula:wherein: as is the peak area of a single related substance in the sample under the corresponding detection wavelength; a is that r The main peak area in the control solution at the wavelength of 286 nm; v (V) r Dilution fold for control solution; f is self-correction factor, wherein vitamin a acetate powder related substance self-correction factor = main peak area of control solution at 286nm wavelength/main peak area of control solution at 325nm wavelength.
CN202311561089.4A 2023-11-22 2023-11-22 Method for determining related substances in vitamin A acetate powder Active CN117269375B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311561089.4A CN117269375B (en) 2023-11-22 2023-11-22 Method for determining related substances in vitamin A acetate powder

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311561089.4A CN117269375B (en) 2023-11-22 2023-11-22 Method for determining related substances in vitamin A acetate powder

Publications (2)

Publication Number Publication Date
CN117269375A CN117269375A (en) 2023-12-22
CN117269375B true CN117269375B (en) 2024-02-13

Family

ID=89212828

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311561089.4A Active CN117269375B (en) 2023-11-22 2023-11-22 Method for determining related substances in vitamin A acetate powder

Country Status (1)

Country Link
CN (1) CN117269375B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532166A (en) * 1994-04-18 1996-07-02 Ma; Yinfa Quantitative retinol assay for serum and dried blood spots
JP2002257808A (en) * 2001-03-06 2002-09-11 Keio Gijuku Method for separating and analyzing retinoid
WO2013176454A1 (en) * 2012-05-21 2013-11-28 한국콜마주식회사 Method of simultaneous analysis of vitamins using liquid chromatography
CN108445098A (en) * 2018-02-26 2018-08-24 济南康和医药科技有限公司 The analysis method of impurity in a kind of detection Retinol Palmitate
WO2022225241A1 (en) * 2021-04-23 2022-10-27 이화여자대학교 산학협력단 Method for early prediction of preterm birth, using retinoid metabolites

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6861512B2 (en) * 2000-03-01 2005-03-01 Eisai Co., Ltd. Separation of olefinic isomers
CN107754776B (en) * 2012-05-15 2021-09-21 沃特世科技公司 Chromatography material
ITMI20130995A1 (en) * 2013-06-17 2014-12-18 Chemelectiva S R L DERIVATIVES OF RETINOIC ACID AND PROCESS FOR THEIR PREPARATION
CN111141840B (en) * 2019-12-18 2021-06-29 西安交通大学 Method for simultaneously measuring fat-soluble vitamins and carotenoids in serum
EP4271990A1 (en) * 2020-12-31 2023-11-08 Waters Technologies Corporation Method for determining b vitamins and their vitamers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532166A (en) * 1994-04-18 1996-07-02 Ma; Yinfa Quantitative retinol assay for serum and dried blood spots
JP2002257808A (en) * 2001-03-06 2002-09-11 Keio Gijuku Method for separating and analyzing retinoid
WO2013176454A1 (en) * 2012-05-21 2013-11-28 한국콜마주식회사 Method of simultaneous analysis of vitamins using liquid chromatography
CN108445098A (en) * 2018-02-26 2018-08-24 济南康和医药科技有限公司 The analysis method of impurity in a kind of detection Retinol Palmitate
WO2022225241A1 (en) * 2021-04-23 2022-10-27 이화여자대학교 산학협력단 Method for early prediction of preterm birth, using retinoid metabolites

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Simultaneous determination of six vitamin A substances by multi-wavelength HPLC and application to different marine shellfishes;Long Xing et al;《Journal of food composition and analysis》;全文 *
Single-run analysis of retinal isomers, retinol and photooxidation products by high-performance liquid chromatography;Ceugniet C et al;《Journal of chromatography A》;全文 *
大鼠肝脏视黄醇含量的反相高效液相色谱检测;许青, 李石, 贺平, 刘彦君, 王皓, 谢天培;中华预防医学杂志(02);全文 *
超高效液相色谱法测定化妆品中6 种维生素A 类物质的含量;章欣 等;《香料香精化妆品》;全文 *
高效液相色谱法测定原料及化妆品中7种视黄醇及其衍生物的含量;闫秀芳 等;《日用化学工业》;全文 *

Also Published As

Publication number Publication date
CN117269375A (en) 2023-12-22

Similar Documents

Publication Publication Date Title
CN109738565B (en) Method for determining illegally added compounds in health food
CN104749269A (en) Method for determining enantiomer impurity in alogliptin crude drug and preparation by virtue of HPLC
Lopes et al. Quantification of carbamazepine and its active metabolite by direct injection of human milk serum using liquid chromatography tandem ion trap mass spectrometry
CN110231412B (en) Detection method for saponin content in gynostemma pentaphylla
CN105467021B (en) A kind of method in relation to substance in HPLC method separation determination paricalcitol bulk pharmaceutical chemicals and preparation
Jeong et al. A sensitive UPLC–ESI–MS/MS method for the quantification of cinnamic acid in vivo and in vitro: Application to pharmacokinetic and protein binding study in human plasma
Bleyzac et al. Rapid and sensitive high-performance liquid chromatographic method for busulfan assay in plasma
Weng et al. Determination of licochalcone A in rat plasma by UPLC–MS/MS and its pharmacokinetics
Prasad et al. Simultaneous determination of prednisolone acetate, prednisolone, prednisone, cortisone and hydrocortisone in swine plasma using solid-phase and liquid—liquid extraction techniques
CN107449846B (en) Method for measuring effective components in infantile nerve-soothing and brain-nourishing granules by HPLC-MS (high Performance liquid chromatography-Mass Spectrometry)
Yang et al. Simultaneous quantitation of the diastereoisomers of scholarisine and 19‐epischolarisine, vallesamine, and picrinine in rat plasma by supercritical fluid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study
CN117269375B (en) Method for determining related substances in vitamin A acetate powder
US20070172958A1 (en) Methods for detecting 14-hydroxy codeinone and codeinone
Noula et al. Solid-phase extraction for purification of alkannin/shikonin samples and isolation of monomeric and dimeric fractions
Takahashi et al. Determination of atropine in pharmaceutical preparations by liquid chromatography with fluorescence detection
Dong et al. Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume
Yoshida et al. A simple, sensitive determination of ganciclovir in infant plasma by high-performance liquid chromatography with fluorescence detection
CN104833753B (en) HPLC-ELSD detection method for EDC residue
CN113759048B (en) Inspection method of mono-tert-butyl octadecanedioate
Burrows Determination of oxpentifylline and three metabolites in plasma by automated capillary gas chromatography using nitrogen-selective detection
Pilkington et al. The impact of impurities in various crude A. annua extracts on the analysis of artemisinin by liquid chromatographic methods
CN100491999C (en) Method for detecting glossy ganoderma spore oil content in glossy ganoderma oil preparation
CN111474270B (en) Method for preparing related detection and test solution in misoprostol solid preparation
CN106290596A (en) The method that separation analysis succinum love song Ge Lieting and preparation thereof have related substance
Ferguson et al. Application of SFC for the characterization of formulated drug products

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant