CN109298112B - Method for measuring content of hyaluronic acid - Google Patents
Method for measuring content of hyaluronic acid Download PDFInfo
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- CN109298112B CN109298112B CN201811508968.XA CN201811508968A CN109298112B CN 109298112 B CN109298112 B CN 109298112B CN 201811508968 A CN201811508968 A CN 201811508968A CN 109298112 B CN109298112 B CN 109298112B
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- 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
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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
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Abstract
The invention provides a method for determining the content of hyaluronic acid, which comprises the following steps: carrying out enzymolysis on a sample to be detected containing hyaluronic acid by utilizing hyaluronidase; detecting hyaluronic acid after enzymatic hydrolysis by hyaluronidase using an ion exchange column for analyzing organic acids using a mobile phase not containing salt; and calculating the content of the hyaluronic acid contained in the sample to be detected based on the detection result. The chromatographic column in the method has good durability, and the hyaluronic acid can be effectively detected without adding buffer salt in the mobile phase.
Description
Technical Field
The invention relates to the field of detection of hyaluronic acid, in particular to a method for determining the content of hyaluronic acid.
Background
Hyaluronic Acid (HA), also known as Hyaluronic acid, is a high molecular weight linear mucopolysaccharide composed of (1-3) -2-acetamido-2-deoxy-D-glucose (1-4) -D-glucuronic acid disaccharide repeating units. First extracted from bovine glass eyeball in 1934 by Meyer et al.
Hyaluronic acid exhibits various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of blood vessel walls, regulating proteins, regulating diffusion and operation of aqueous electrolytes, promoting wound healing, and the like. More importantly, hyaluronic acid HAs a special water retention effect, is a substance which is found to have the best moisture retention in nature at present, is called an ideal natural moisture retention factor, and is widely applied to the industrial fields of cosmetics, foods, medicines, clinical treatment and the like because HA HAs good moisture retention, viscoelasticity, permeability and ductility and does not have any immunogenicity and toxicity.
Macromolecular hyaluronic acid can be hydrolyzed into unsaturated hyaluronic acid disaccharide (delta DiHA) by hyaluronidase from bacteria, the delta DiHA has specific ultraviolet absorption at 232nm, and the content can be detected by external standard method after being separated from other components by chromatographic column. At present, the commonly used detection methods of hyaluronic acid include an HPLC method, a colorimetric method, a CTAB turbidimetric method and a carbazole chromogenic method. For example, patent document 1 discloses a method for quantitatively detecting the content of hyaluronic acid in a hyaluronic acid fermentation broth by carbazole coloration, but it is necessary to remove interfering factors such as residual heterosugars and pigments in the fermentation broth before detection. Patent document 2 discloses a method for measuring the degree of crosslinking of crosslinked hyaluronic acid or a salt thereof, which comprises using a molecular sieve column and a KCl-phosphate buffer solution in an amount of 0.1 to 1mol/L as a mobile phase.
Documents of the prior art
Patent document
Patent document 1: CN108362686A
Patent document 2 CN107561179A
Disclosure of Invention
In chromatography, an amino bond and a column are generally used for separating oligosaccharide substances, the column packing is easy to hydrolyze and has poor durability, and a high-concentration buffer salt such as that in patent document 2 needs to be added to a mobile phase (water phase) to elute hyaluronic acid disaccharide, so that a liquid phase pipeline system is damaged by using a high-salt mobile phase for a long time. Therefore, it is required to develop a method capable of efficiently detecting hyaluronic acid without using the above-mentioned buffer salt at a high concentration.
The present inventors have discovered, by chance, in daily analytical work, that a type of ion exchange column for analyzing organic acids is capable of separating hyaluronic acid disaccharide from other small molecular substances, HAs high column efficiency and good durability, and does not require addition of buffer salt in mobile phase, thereby establishing the above method for detecting HA content in health food with complex formulation.
In order to solve the technical problems, the invention adopts the technical scheme that:
1. a method of determining the amount of hyaluronic acid, comprising:
carrying out enzymolysis on a sample to be detected containing hyaluronic acid by utilizing hyaluronidase;
detecting hyaluronic acid after enzymatic hydrolysis by hyaluronidase using an ion exchange column for analyzing organic acids using a mobile phase not containing salt; and
and calculating the content of the hyaluronic acid contained in the sample to be detected based on the detection result.
2. The method of item 1, wherein the ion exchange column for analyzing organic acids is a cation exchange chromatography column.
3. According toThe method of item 1 or 2, wherein the ion exchange column for analyzing organic acids is a strong cation H in which a sulfonated crosslinked styrene divinylbenzene copolymer is a filler+A type exchange column.
4. The method according to any one of items 1 to 3, wherein the ion exchange column for analyzing organic acids is an MCI GEL CK08EH column (8X 300mm, 5 μm) or a SilGreen GH0830078H column.
5. The method according to any one of items 1 to 4, wherein the mobile phase of the high performance liquid chromatography is a weak acid solution.
6. The method according to item 5, wherein the mobile phase of the high performance liquid chromatography is a phosphoric acid solution or an acetic acid solution, preferably a phosphoric acid solution or an acetic acid solution having a concentration of 0.01 to 1.2 wt%.
7. The method according to any one of items 1 to 6, wherein the flow rate of the mobile phase is 0.3 to 1.0ml/min, more preferably 0.4 to 0.8ml/min, for example, 0.6 ml/min.
8. The method according to any one of items 1 to 7, wherein the column temperature of the column used in the high performance liquid chromatography is 25 to 95 ℃, preferably 30 to 80 ℃, and more preferably 40 to 60 ℃.
9. The method according to any one of items 1 to 8, wherein the detection wavelength in high performance liquid chromatography is 220 to 235nm, for example 232 nm.
10. The method according to any one of items 1 to 9, wherein the amount of the sample to be sampled is 5 to 100. mu.L, for example, 20. mu.L, when the sample is analyzed by high performance liquid chromatography.
11. The method of any one of items 1 to 10, wherein the content of citric acid in the detected solution of hyaluronic acid is below the lower detection limit, preferably less than 0.1% o.
12. The method according to any one of items 1 to 11, which is used for measurement of the content of hyaluronic acid in health foods, pharmaceuticals, medical devices, cosmetics, hair care products, oral care products, and paper products.
13. The ion exchange column for analyzing organic acid is used for detecting the content of hyaluronic acid.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the invention, hyaluronidase is used for pretreating a sample, and a liquid chromatography separation technology is combined to obtain the content of the sample, so that the method has high specificity.
According to the method of the present invention, it is possible to prevent the problems that the packing of an analytical column for sugar, such as an amino column, is easily hydrolyzed, has poor durability, and causes damage to a liquid phase piping system due to long-term use of a high-salt mobile phase, and to more effectively detect hyaluronic acid.
Drawings
FIG. 1 is a liquid chromatogram of example 1.
FIG. 2 is a liquid chromatogram of reference example 1.
FIG. 3 is a liquid chromatogram of reference example 2.
Detailed description of the invention
The term "enzymatic method" as used herein means the hydrolysis of a specific substance by an active enzyme, which is used in general biological experiments, and the enzyme (enzyme) is a protein or RNA produced by living cells and having high specificity and high catalytic activity for its substrate. Enzymes are a very important class of biocatalysts (biochatalysts). Due to the action of enzymes, chemical reactions in organisms can be efficiently and specifically carried out under extremely mild conditions. Hyaluronidase (HAase) is a generic term for enzymes that can cause hyaluronic acid to produce low molecular weight action, and the present invention uses hyaluronidase to specifically cleave the glycosidic bond between the two saccharide units of sodium hyaluronate, and the final product is sodium hyaluronate disaccharide with a double bond. The hyaluronidase used in the present invention may be any enzyme known in the art that can cleave the glycosidic bond between sodium hyaluronate disaccharide units, and can be used without any limitation by purchasing commercially available enzymes for degrading hyaluronic acid, but the most preferred enzyme is limited to a lyase of bacterial origin because hyaluronic acid can be completely degraded into disaccharide units with double bonds only in this way. The commercially available enzymes are basically hyaluronidase extracted from animal testis, and the final degradation product is a mixture of tetrasaccharide and hexasaccharide, and the complete degradation effect is not easy to achieve.
Chromatography (also known as "chromatography", or "chromatography") is a separation and analysis method and is widely used in the fields of analytical chemistry, organic chemistry, biochemistry, and the like. The chromatography uses the selective distribution of different substances in different phase states to elute a mixture in a mobile phase and a stationary phase, and different substances in the mixture can move along the stationary phase at different speeds, so that the separation effect is finally achieved.
High Performance Liquid Chromatography (High Performance Liquid Chromatography, HPLC) is also called "High pressure Liquid Chromatography", "High Performance Liquid Chromatography", "High resolution Liquid Chromatography", "modern column Chromatography", and the like. High performance liquid chromatography is an important branch of chromatography, liquid is used as a mobile phase, a high-pressure infusion system is adopted, mobile phases such as single solvents with different polarities or mixed solvents, buffer solutions and the like with different proportions are pumped into a chromatographic column filled with a stationary phase, and after components in the column are separated, the mobile phases enter a detector for detection, so that analysis of a sample is realized.
The stationary phases for HPLC are as follows: (1) bonding or coating various polymers on the surface of the silica gel; (2) other oxide surface coating polymers; (3) non-porous monodisperse filler; (4) an organic polymeric filler; (5) filling a chromatographic packing; and (6) a chiral stationary phase filler. The mobile phase is a key factor influencing liquid chromatography, and the mobile phase in high performance liquid chromatography mainly uses an aqueous solvent, an organic solvent or a mixed solution of the aqueous solvent and the organic solvent.
The ion exchange column refers to a columnar pressure container for performing ion exchange reaction, and the ion exchange reaction refers to a process of reversibly exchanging cations or anions in functional groups of an ion exchanger with isotropic ions in a solution. Ion exchangers are divided into organic and inorganic ion exchangers. Inorganic ion exchangers are divided into natural and man-made materials such as synthetic zeolites. The organic ion exchanger includes ion exchange resins, which are classified into gel type having a pore size of 5nm and macroporous type having a pore size of 20 to 100nm according to physical structure, and styrene type, phenol type, acrylic acid type, epoxy type, vinylpyridine type, etc. according to raw material monomers used for synthesizing the resin. The most commonly used classes of ion exchange resins are based on the classification of the resin ion exchange functionality, including strongly acidic cationic ion exchange resins, weakly acidic cationic ion exchange resins, strongly basic anionic ion exchange resins, and weakly basic anionic ion exchange resins, among others.
In the present invention, an ion exchange column, more preferably a cation exchange chromatography column, which can be used for analysis of organic acids is used.
The present invention will be described in detail below.
According to one aspect of the present invention, there is provided a method for determining the content of hyaluronic acid by an enzymatic method in combination with high performance liquid chromatography, comprising: carrying out enzymolysis on a sample to be detected containing hyaluronic acid by utilizing hyaluronidase; detecting hyaluronic acid after enzymatic hydrolysis by hyaluronidase using an ion exchange column for analyzing organic acids using a mobile phase not containing salt; and calculating the content of the hyaluronic acid contained in the sample to be detected based on the detection result.
The method of the invention is characterized in that: hyaluronic acid after enzymatic hydrolysis by hyaluronidase is detected using an ion exchange column for analysis of organic acids. This can prevent problems that packing for an analytical column for sugars such as an amino-bonded column is easily hydrolyzed, has poor durability, and causes damage to a liquid phase piping system by using a high-salt mobile phase for a long period of time.
According to the invention, hyaluronidase is used for pretreating a sample, and a liquid chromatography separation technology is combined to obtain the content of the sample, so that the method has high specificity.
In a specific embodiment, the ion exchange column for analyzing organic acids may be a cation exchange chromatography column, and particularly may be a strong cationic calcium-type exchange column in which a sulfonated crosslinked styrene divinylbenzene copolymer is used as a filler. For example, MCI GEL CK08EH column (8X 300mm, 5 μm). The MCI GEL CK08EH chromatographic column is cation exchange chromatographic column belonging to CK08E series produced by Mitsubishi chemical, is strong cation hydrogen type chromatographic column with sulfonated cross-linked styrene divinylbenzene copolymer as filler, and can be used for separating saccharides, carboxylic acid, polyalcohol, etc. In addition, the stationary phase in the SilGreen GH0830078H chromatographic column is sulfonated styrene-divinylbenzene resin with 8% crosslinking degree, and dilute acid is used as a mobile phase at normal temperature, so that not only carbohydrates in a sample can be separated, but also organic acid and alcohol can be separated. By using such a column, durability is higher than that of a conventional column for detecting hyaluronic acid.
In one embodiment, the mobile phase of the high performance liquid chromatography is a weak acid solution, preferably a 0.01% to 1.2% by weight phosphoric acid solution or acetic acid solution. Among these, especially high acetic acid concentrations can affect baseline stability. In the invention, as no salt component is added in the mobile phase of the high performance liquid chromatography, the problem that the liquid phase pipeline system is damaged due to long-term use of the high-salt mobile phase can be effectively prevented.
In one embodiment, the flow rate of the mobile phase is 0.3 to 1.0ml/min, and more preferably 0.4 to 0.8 ml/min. The column temperature used in the high performance liquid chromatography is 25 to 95 ℃, preferably 30 to 80 ℃, and more preferably 40 to 60 ℃.
The detection wavelength is 220-235 nm, such as 232nm, and the sample injection amount is 5-100 μ L, such as 20 μ L. Under this condition, hyaluronic acid can be more accurately measured.
In a particular embodiment, the solution of hyaluronic acid detected has a content of citric acid below the lower limit of detection, for example less than 0.1% o. The hyaluronic acid of the solution with the citric acid content lower than the detection lower limit is not easily interfered and is more accurate to detect. Citric acid is required to be added in certain health food formulas to adjust indexes such as flavor, acidity and the like of the products, and the citric acid also has a certain preservative effect. However, under the chromatographic conditions mentioned in the invention, the chromatographic behavior of citric acid is similar to that of degraded sodium hyaluronate, i.e. sodium hyaluronate disaccharide, and if the content of citric acid in the formula is higher, the chromatographic peak of citric acid can interfere with the chromatographic peak of HA disaccharide. Therefore, it is necessary to define the citric acid content of the solution of hyaluronic acid tested according to the invention.
In a specific embodiment, the method of the present invention is used for the determination of the hyaluronic acid content in a health food. The health food is also called functional food, and the health food is food which is claimed to have specific health care function or aims to supplement vitamins and minerals, namely food which is suitable for specific people to eat, has the function of regulating the organism, does not aim to treat diseases and does not cause any acute, subacute or chronic harm to human bodies.
In a specific embodiment, the method of the invention is used for the determination of the hyaluronic acid content in a pharmaceutical or medical device. The hyaluronic acid can be used as raw material or adjuvant of medicine or medical appliance, and can be used in medicinal products such as ophthalmic preparation, intra-articular preparation, postoperative anti-adhesion agent, wound healing external preparation, soft tissue filler, etc.
In a particular embodiment, the method of the invention is used for the determination of the hyaluronic acid content in a cosmetic product. The cosmetic is a chemical industrial product or a fine chemical product which is applied to any part of the surface of a human body, such as skin, hair, nails, lips and teeth, by smearing, spraying or the like, so as to achieve the purposes of cleaning, maintaining, beautifying, decorating and changing the appearance, or correcting the odor of the human body and keeping a good state. The hyaluronic acid in the cosmetic has effects of keeping moisture, lubricating, preventing sunburn, thickening, stabilizing emulsification, resisting aging, and repairing after sunburn.
In a particular embodiment, the method of the invention is used for the determination of the hyaluronic acid content in a hair care product. The hair care products can be further divided into pet hair care products and human hair care products according to different applicable objects. According to different effects of the product, the hair care product can also comprise an anti-hair loss product, a hair follicle regeneration promoting product, a hair improving product, a hair perming product, a hair dyeing product and a hair styling product, and has the effects of moisturizing, inhibiting bacteria, repairing, preventing hair loss, promoting hair follicle regeneration and the like.
In a specific embodiment, the method of the present invention is used for the determination of the amount of hyaluronic acid in an oral care product. The oral care product comprises a composition for treating oral ulcer, a tooth-planting product, a tooth-washing product, a product for relieving xerostomia, an oral cleaning product, a saliva substitute and the like, and has the effects of cleaning oral cavity, inhibiting bacteria, resisting inflammation, repairing, moisturizing, thickening, inducing bone regeneration and the like.
By utilizing the method for determining the content of the hyaluronic acid, disclosed by the invention, the hyaluronic acid disaccharide can be eluted by using a salt-free buffer solution by using the ion exchange column for analyzing the organic acid, so that the damage to a liquid phase pipeline system can be reduced, and the hyaluronic acid can be effectively detected for a longer time. By using the method of the invention, the used chromatographic column can run for a long time, the situations of blockage and the like do not occur, the chromatographic column and the like do not need to be cleaned frequently, and the running time lasts more than 3600 minutes.
Examples
The present invention will be described in detail with reference to examples. It should be understood, however, that the present invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The numerical ranges recited in the present invention each include data for both endpoints of the numerical range, and also include each of the specific values in the numerical range, and the numerical values can be combined with the endpoints at will to form a new subrange.
Example 1
1 reagents and materials
Sodium dihydrogen phosphate (national group chemical Co., Ltd.), disodium hydrogen phosphate (national group chemical Co., Ltd.), phosphoric acid (national group chemical Co., Ltd.)
Hyaluronic Acid (HA) control (Huaxi Rui Da biomedicine Co., Ltd.), hyaluronidase (HAase) (Huaxi Rui Da biomedicine Co., Ltd.)
2 chromatographic conditions
A chromatographic column: MCI GEL CK08EH chromatography column (Mitsubishi chemical, 8X 300mm, 5 μm);
mobile phase: 1% phosphoric acid solution;
flow rate: 0.6 ml/min;
sample introduction amount: 20 mu L of the solution; column temperature: 40 ℃;
detection wavelength: 232 nm.
Description of chromatographic conditions:
wherein, the mobile phase, the flow rate and the column temperature are recommended parameters of the chromatographic column, and the sample amount is a common value. The detection wavelength is the maximum ultraviolet absorption wavelength of HA disaccharide.
3 preparation of solution
And (3) enzymolysis buffer solution: weighing sodium dihydrogen phosphate (NaH)2PO4·2H2O)27.4g, disodium hydrogen phosphate (Na)2HPO4·12H2O)8.8g is put into a 1000mL volumetric flask, diluted to the scale by adding water and shaken up to obtain 0.2mol/L Na2HPO4-NaH2PO4And (4) a buffer solution. The buffer was diluted 40-fold to obtain an enzymatic buffer (5mM/L Na)2HPO4-NaH2PO4Buffer, ph 6.0).
Control solution: precisely weighing about 50mg of sodium hyaluronate reference substance in a 50mL volumetric flask, dissolving the sodium hyaluronate reference substance in an enzymolysis buffer solution, fixing the volume to a scale, and uniformly mixing. Placing 0.2mL of the above solution in a 10mL volumetric flask, adding 0.5mL of hyaluronidase, mixing, sealing, performing enzymolysis at 42 deg.C for 2h, boiling for 2min to inactivate enzyme, fixing the volume of mobile phase to the scale, and filtering with 0.22 μm filter membrane to obtain reference solution.
Test article (may also be referred to as a test sample) solution: precisely measuring 0.5mL of test solution, adding 1mL of hyaluronidase, mixing, sealing, performing enzymolysis at 42 deg.C for 2h, boiling for 2min to inactivate enzyme, metering volume to 10mL of mobile phase, and filtering with 0.22 μm filter membrane to obtain test solution.
4 determination of
And respectively taking a reference substance and 20 mu L of test solution, injecting a sample, detecting according to the chromatographic conditions, and calculating the content of the sodium hyaluronate in the test solution by the external standard method peak area.
5 calculation of
Calculating the HA content of the test solution according to the following formula:
wherein, X represents HA content in sample solution in mg/mL
Peak area of As-sample solution
Peak area of Ar-control solution
Wr-weight of control, mg
Z-reference content
h-loss on drying of control
In addition, the theoretical plate number (N) reflects column efficiency parameters of the chromatographic column, and the calculation formula is as follows:
n ═ 5.54 × (retention time/half peak width)2
The theoretical plate number is generally given by automatic calculation of chromatographic workstation data processing software.
6 results
The results of measurement of HA content in the test samples are shown in Table 1. The chromatogram is shown in FIG. 1.
TABLE 1 chromatographic analysis results of the test article in example 1
And (4) continuously detecting different samples to be detected under the conditions, wherein the total running time of the chromatographic column exceeds 3600 minutes, and the chromatographic column still runs normally.
Example 2
The content of sodium hyaluronate in the test solution was measured under the same conditions as in example 1 except that the mobile phase of the chromatography in example 1 was changed to a 0.5 wt% phosphoric acid solution and the flow rate was changed to 0.4 ml/min. The results of the measurement of HA content in the test sample are shown in Table 2
TABLE 2 chromatographic analysis results of the test article in example 2
Example 3
The content of sodium hyaluronate in the sample solution was measured by changing the mobile phase of the chromatography in example 1 to a 0.01% acetic acid solution and the column temperature to 80 ℃ under the same conditions as in example 1. The results of measurement of HA content in the test samples are shown in Table 3.
TABLE 3 chromatographic analysis results of the test article in example 3
Example 4
The flow rate was changed to 0.5ml/min, the column temperature was changed to 30 ℃, and the sodium hyaluronate content in the test solution was measured under the same conditions as in example 1. The results of measurement of HA content in the test samples are shown in Table 4.
Table 4 chromatographic analysis results of the test article in example 4
Example 5
The content of sodium hyaluronate in the test solution was measured by changing the flow rate to 0.3ml/min and the column temperature to 45 ℃ under the same conditions as in example 1. The results of measurement of HA content in the test article are shown in Table 5
TABLE 5 chromatographic analysis results of the test article in example 5
Example 6
The flow rate was changed to 1.0ml/min, the detection wavelength of the chromatogram in example 1 was changed to 235nm, and the content of sodium hyaluronate in the test solution was measured under the same conditions as in example 1. The results of measurement of HA content in the test articles are shown in Table 6.
TABLE 6 chromatographic analysis results of the test article in example 6
Example 7
The sample solution in example 1 was changed to 10 tablets containing HA, after grinding and grinding, 1g to 10mL volumetric flasks were precisely weighed, an appropriate amount of enzymatic hydrolysis buffer was added to dissolve the tablets sufficiently, filtration was carried out, 0.5mL filtrate was subjected to enzymatic hydrolysis, the flow rate was changed to 0.8mL/min, the amount of sample was changed to 100. mu.L, the column temperature was changed to 60 ℃, and the other conditions were the same as in example 1, and the sodium hyaluronate content in the sample solution was measured. The results of measurement of HA content in the test articles are shown in Table 7.
TABLE 7 chromatographic analysis results of the test article in example 7
Example 8
The column of example 1 was changed to a SilGreen GH0830078H column, and the content of sodium hyaluronate in the test solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test samples are shown in Table 8.
TABLE 8 chromatographic analysis results of the test article in example 8
Example 9
The content of sodium hyaluronate in the sample solution was measured by changing the amount of the sample to be chromatographed in example 1 to 50. mu.l and the detection wavelength of the chromatograph in example 1 to 220nm under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 9.
TABLE 9 chromatographic analysis results of the test articles in example 9
Example 10
The column temperature of the column in example 1 was changed to 95 ℃ and the sodium hyaluronate content of the sample solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 10.
TABLE 10 chromatographic analysis results of the test article in example 10
Example 11
The content of sodium hyaluronate in the sample solution was measured by changing the mobile phase of the chromatography in example 1 to a 1.2wt% phosphoric acid solution and the column temperature of the column to 25 ℃ under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 11.
TABLE 11 chromatographic analysis results of the test articles in example 11
Example 12
The content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1 except that the mobile phase of the chromatography in example 1 was changed to a 1wt% acetic acid solution, the flow rate was changed to 0.7ml/min, the amount of the sample was changed to 5. mu.l, the column temperature of the column was changed to 50 ℃ and the detection wavelength was set to 235 nm.
The results of measurement of HA content in the test articles are shown in Table 12.
TABLE 12 chromatographic analysis results of the test article in example 12
Comparative example 1
The chromatographic column in example 1 was replaced by an amino column, and the detection conditions were as follows:
a chromatographic column: luna NH2(4.6×250mm，5μm)；
Mobile phase: 0.4mol/L NaH2PO4A solution; flow rate: 0.6 ml/min; sample introduction amount: 20 mu L of the solution;
column temperature: 35 ℃; detection wavelength: 232 nm.
The results of measurement of HA content in the test samples are shown in Table 8.
TABLE 8 chromatographic analysis results of the test article in comparative example 1
Comparative example 2
Sodium dihydrogenphosphate 0.15 wt% was added to the mobile phase of the chromatogram in example 1, and the content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 13.
TABLE 13 chromatographic analysis results of the test article in example 13
Comparative example 3
The content of sodium hyaluronate in the test sample solution was measured by changing the mobile phase of the chromatography in example 1 to a 1.6 wt% phosphoric acid solution under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 14.
TABLE 14 chromatographic analysis results of the test article in example 14
Comparative example 4
The content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1 except that the flow rate of the mobile phase of the chromatography in example 1 was changed to 1.5ml/min and the amount of the sample was changed to 10. mu.l, instead of water.
The results of measurement of the HA content in the test samples are shown in Table 15, and HA disaccharide HAs no chromatographic peak under the chromatographic conditions.
TABLE 15 chromatographic analysis results of the test articles in example 15
Comparative example 5
The column temperature of the chromatogram in example 1 was changed to 100 ℃, and the content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 16. Under the condition, the mobile phase is close to a boiling state, so that a large amount of gas is generated in a chromatographic system, and the detection cannot be carried out.
TABLE 16 chromatographic analysis results of the test article in example 16
Comparative example 6
The detection wavelength of the chromatogram in example 1 was changed to 240nm, and the content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 17. At the wavelength, HA disaccharide absorption is weak, the chromatographic peak area is small, and the detection result error is large.
TABLE 17 chromatographic analysis results of the test articles in example 17
Comparative example 7
The column temperature of the chromatogram in example 1 was changed to 20 ℃, and the content of sodium hyaluronate in the sample solution was measured under the same conditions as in example 1.
The results of measurement of HA content in the test articles are shown in Table 18. The column effect of the chromatographic column is obviously reduced at the temperature, and the error of the detection result is larger.
TABLE 18 chromatographic analysis results of the test articles in example 18
Reference example 1
The sample solution in example 1 was changed to a blank sample solution containing no HA, and the chromatogram of the sample solution was measured under the same conditions as in example 1. The results are shown in FIG. 2.
Reference example 2
The sample solution in example 1 was changed to a control sample solution of HA disaccharide, and the chromatogram of the sample solution was measured under the same conditions as in example 1. The results are shown in FIG. 3.
The blank sample is a sample without HA added in the formula, and the blank sample HAs no chromatographic peak consistent with the retention time (10.3min) of HA disaccharide, which indicates that other components in the formula do not interfere with the determination of HA.
As is clear from table 9, the method for measuring the hyaluronic acid content of the present invention has a high theoretical plate number, and can efficiently detect hyaluronic acid, and unlike comparative example 1 shown in table 10, it requires a high concentration of buffer salt in the mobile phase (aqueous phase) to elute hyaluronic acid disaccharide, and can reduce damage to the liquid phase piping system, and efficiently detect hyaluronic acid over a longer period of time.
As shown in table 10, in comparative example 2, the total operating time of the column was less than 2000min although the theoretical plate number was higher because salt was added to the mobile phase than in example 1, and the hyaluronic acid could not be efficiently detected for a long period of time because the liquid phase line system was damaged by salt.
In comparative example 3, the concentration of the phosphoric acid solution in the mobile phase was increased to 1.6 wt%, and the total column running time was less than 1000min although the theoretical plate number was high, because the concentration of the phosphoric acid solution was too high, which not only affected the baseline stability, but also failed to detect hyaluronic acid efficiently for a long period of time.
In comparative example 4, the mobile phase was changed to water, the flow rate was changed to 1.5ml/min, and HA disaccharide had no chromatographic peak under the chromatographic conditions. In comparative example 5, the column temperature of the chromatogram was changed to 100 ℃ and the column temperature was too high to be detected.
In comparative example 6, the detection wavelength was changed to 240nm, at which the HA disaccharide absorption was weak, the chromatographic peak area was small, and the error in the detection result was large. In comparative example 7, the column temperature of the chromatogram was changed to 20 ℃, and the column efficiency of the chromatogram was significantly reduced at this temperature, resulting in a large error in the detection result.
In contrast to these comparative examples, hyaluronic acid was effectively detected under the liquid chromatography conditions of the examples.
While embodiments and specific examples of the invention have been described, the invention is not limited to the specific embodiments and applications described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Claims (18)
1. A method of determining the amount of hyaluronic acid, comprising:
carrying out enzymolysis on a sample to be detected containing hyaluronic acid by utilizing hyaluronidase;
detecting hyaluronic acid after enzymatic hydrolysis by hyaluronidase using an ion exchange column for analyzing organic acids using a mobile phase not containing salt; and
calculating the content of hyaluronic acid contained in the sample to be detected based on the detection result;
the ion exchange column for analyzing the organic acid is an MCI GEL CK08EH chromatographic column or a SilGreen GH0830078H chromatographic column;
the mobile phase of the high performance liquid chromatography is a phosphoric acid solution or an acetic acid solution with the concentration of 0.01-1.2 wt%;
the flow rate of the mobile phase is 0.3-1.0 ml/min;
the column temperature of a chromatographic column used in the high performance liquid chromatography is 25-95 ℃.
2. The method according to claim 1, wherein the flow rate of the mobile phase is 0.4 to 0.8 ml/min.
3. The method of any one of claims 1 to 2, wherein the column temperature of the chromatographic column used in the high performance liquid chromatography is from 30 ℃ to 80 ℃.
4. The method of any one of claims 1 to 2, wherein the column temperature of the column used in the high performance liquid chromatography is 40 ℃ to 60 ℃.
5. The method according to any one of claims 1 to 2, wherein the detection wavelength in high performance liquid chromatography is 220 to 235 nm.
6. The method according to claim 3, wherein the detection wavelength is 220 to 235nm in high performance liquid chromatography.
7. The method according to any one of claims 1 to 2, wherein the amount of the sample to be sampled is 5 to 100. mu.L when the sample is analyzed by high performance liquid chromatography.
8. The method according to claim 3, wherein the amount of the sample to be sampled is 5 to 100. mu.L when the sample is analyzed by high performance liquid chromatography.
9. The method according to claim 5, wherein the amount of the sample to be sampled is 5 to 100. mu.L when the sample is analyzed by high performance liquid chromatography.
10. The method according to any one of claims 1-2, wherein the detected content of citric acid in the solution of hyaluronic acid is less than 0.1% o.
11. The method of claim 3, wherein the detected content of citric acid in the solution of hyaluronic acid is less than 0.1% o.
12. The method of claim 5, wherein the detected content of citric acid in the solution of hyaluronic acid is less than 0.1% o.
13. The method of claim 9, wherein the detected content of citric acid in the solution of hyaluronic acid is less than 0.1% o.
14. The method according to any one of claims 1 to 2, which is used for determination of the content of hyaluronic acid in health food, drugs, medical instruments, cosmetics, hair care products, oral care products, and paper products.
15. The method according to claim 3, which is used for determination of the hyaluronic acid content in health foods, pharmaceuticals, medical devices, cosmetics, hair care products, oral care products, and paper products.
16. The method according to claim 5, which is used for determination of the hyaluronic acid content in health foods, pharmaceuticals, medical devices, cosmetics, hair care products, oral care products, and paper products.
17. The method according to claim 9, which is used for determination of the hyaluronic acid content in health foods, pharmaceuticals, medical devices, cosmetics, hair care products, oral care products, and paper products.
18. The method according to claim 12, which is used for determination of the hyaluronic acid content in health foods, pharmaceuticals, medical devices, cosmetics, hair care products, oral care products, and paper products.
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| CN113287708A (en) * | 2021-07-12 | 2021-08-24 | 千世泰生物科技(青岛)有限公司 | Solid beverage containing sodium hyaluronate and method for detecting content of sodium hyaluronate in solid beverage |
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