CN110045045B - A kind of detection method of hyaluronic acid content in facial mask - Google Patents
A kind of detection method of hyaluronic acid content in facial mask Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 65
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 65
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 65
- 230000001815 facial effect Effects 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 title abstract description 25
- 239000012488 sample solution Substances 0.000 claims abstract description 39
- 239000000243 solution Substances 0.000 claims abstract description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000003085 diluting agent Substances 0.000 claims abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 14
- -1 amine formate Chemical class 0.000 claims abstract description 8
- 238000005259 measurement Methods 0.000 claims description 7
- 238000010812 external standard method Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 abstract description 12
- 238000012360 testing method Methods 0.000 abstract description 8
- 239000007864 aqueous solution Substances 0.000 abstract description 6
- 239000012085 test solution Substances 0.000 description 6
- 239000013068 control sample Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
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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
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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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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明公开了一种面膜中透明质酸含量的检测方法，包括分别在透明质酸‑TRC和面膜样品中添加稀释剂溶解成对照溶液和试样溶液，其中稀释剂为20%的0.05mol/L甲酸胺水溶液与80%的甲醇混合的流动相，然后向HPLC仪分别注入15‑25μL的对照溶液和试样溶液，通过示差折光检测器检测，得到透明质酸的峰面积后计算试样溶液中透明质酸的含量，本发明通过设置最佳比例的流动相溶液，使用三氟乙酸调整流动相最佳pH为4.00，设置流动相最佳流速为1.0ml/min，使用普通示差折光检测器，使得本发明的透明质酸的保留时间缩短为10‑15min内，在对面膜中的透明质酸含量进行大批量检测时，可以缩短总的检测时长，减少流动相溶液的用量，提高检测效率，减少检测成本。The invention discloses a method for detecting the content of hyaluronic acid in a facial mask, comprising adding a diluent to the hyaluronic acid-TRC and the facial mask sample to dissolve into a control solution and a sample solution, wherein the diluent is 20% of 0.05mol/ L amine formate aqueous solution mixed with 80% methanol as mobile phase, then inject 15-25 μL of control solution and sample solution into HPLC instrument respectively, detect by differential refraction detector, and calculate the sample solution after obtaining the peak area of hyaluronic acid The content of hyaluronic acid in the present invention is set by setting the mobile phase solution of the optimal ratio, using trifluoroacetic acid to adjust the optimal pH of the mobile phase to 4.00, setting the optimal flow rate of the mobile phase to 1.0ml/min, and using a common refractive index detector. , so that the retention time of the hyaluronic acid of the present invention is shortened to within 10-15min, and when the hyaluronic acid content in the facial mask is detected in large quantities, the total detection time can be shortened, the consumption of the mobile phase solution can be reduced, and the detection efficiency can be improved. , reduce testing costs.
Description
Technical Field
The invention relates to the field of detection of hyaluronic acid, in particular to a method for detecting the content of hyaluronic acid in a mask.
Background
The conventional detection method for hyaluronic acid is an HPLC method, wherein a mobile phase is usually KCl-phosphate buffer solution, the retention time of hyaluronic acid in the method is usually about 20min, and if the detection time for large-batch hyaluronic acid is long, the consumption of the mobile phase solution is increased due to the long detection time, and the detection cost is also increased; in addition, the conventional detection method usually uses an ELSD or CAD detector, which is troublesome and leads to the increase of the detection cost.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for detecting the content of hyaluronic acid in a mask, which is rapid and accurate and has low detection cost.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for detecting the content of hyaluronic acid in a facial mask comprises the following steps:
(1) preparation of control solution: adding a diluent into hyaluronic acid-TRC to dissolve into a control solution with the concentration of 0.4-0.6mg/ml, wherein the diluent is a mobile phase, and the mobile phase is a mixed solution of 20% of 0.05mol/L of amine formate water solution and 80% of methanol;
(2) preparation of sample solution: adding the diluent into the mask sample to be dissolved into a sample solution with the concentration of 450-550 mg/ml;
(3) measurement of the content of hyaluronic acid in the sample solution: and a chromatographic column and a sample injector are arranged in the HPLC instrument, 15-25 mu L of the control solution and the sample solution are respectively injected into the HPLC instrument by adopting the sample injector, the detection is carried out by a differential refraction detector, the peak areas of the hyaluronic acid in the control solution and the sample solution are respectively recorded, and the content of the hyaluronic acid in the sample solution is obtained by calculating the peak areas according to an external standard method.
The mobile phase is combined with 0.05mol/L of amine formate aqueous solution with the proportion of 20% and 80% of methanol, and the optimal pH value of the mobile phase is 4.00, so that the retention time of hyaluronic acid is controlled within 10-12min, the detection time is greatly shortened, the using amount of the mobile phase is reduced, and the use cost is reduced. The invention uses the differential refraction detector, which is simpler and reduces the detection cost.
The mobile phase is adjusted to pH 3.50-4.50 by trifluoroacetic acid. Trifluoroacetic acid in the mobile phase can improve the peak shape, overcome the problems of peak broadening and tailing, combine with positive charge and polar group of hyaluronic acid to reduce polarity retention and shield the residual polar surface on the hyaluronic acid, and has small interference on the detection of hyaluronic acid at low wavelength because the ultraviolet maximum absorption peak of trifluoroacetic acid is lower than 200nm, and in addition, trifluoroacetic acid is volatile and can be conveniently removed from the sample.
In addition, the trifluoroacetic acid has strong acidity and can be used for adjusting the pH of the mobile phase, and in the invention, the stability of hyaluronic acid is best when the pH of the mobile phase is 4.00.
The flow rate of the mobile phase is 0.5-1.5ml/min, and when the flow rate of the mobile phase is set to be 1.0ml/min, the peak shape of hyaluronic acid is good, the retention time is short, the detection time is further reduced, and the detection efficiency is improved.
The retention time of the hyaluronic acid is 10-16min, the retention time of the original hyaluronic acid is shortened, the total detection time can be shortened when the content of the hyaluronic acid in the mask is detected in a large batch, the detection efficiency is improved, and the detection cost is greatly reduced.
The chromatographic column is NH2The chromatographic column has a column temperature of 30-50 ℃.
The content of hyaluronic acid in the sample solution is not less than 0.1%.
The invention has the beneficial effects that: according to the invention, by setting a mobile phase solution of 0.05mol/L of amine formate aqueous solution with an optimal proportion of 20% and 80% of methanol, using trifluoroacetic acid to adjust the optimal pH of the mobile phase to 4.00, setting the optimal flow rate of the mobile phase to 1.0ml/min, and using a common differential refraction detector, the retention time of the invention is shortened to 10-15min, and when the hyaluronic acid content in the facial mask is detected in a large batch, the total detection time can be shortened, the using amount of the mobile phase solution is reduced, the detection efficiency is improved, and the detection cost is reduced.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1:
a method for detecting the content of hyaluronic acid in a facial mask comprises the following steps:
(1) preparation of control solution: adding a diluent into hyaluronic acid-TRC to dissolve into a control solution with the concentration of 0.4mg/ml, wherein the diluent is a mobile phase, the mobile phase is a mixed solution of 20% of 0.05mol/L amine formate aqueous solution and 80% of methanol, the pH of the solution is adjusted to be 3.50 by trifluoroacetic acid, and the flow rate of the mobile phase is 0.5 ml/min;
(2) preparation of sample solution: adding the diluent into the mask sample to dissolve the mask sample into a sample solution with the concentration of 450 mg/ml;
(3) measurement of the content of hyaluronic acid in the sample solution: the HPLC instrument is provided with a chromatographic column and a sample injector, wherein the chromatographic column is NH2And the column temperature of the chromatographic column is 30 ℃, 25 mu L of the control solution and the sample solution are respectively injected into an HPLC (high performance liquid chromatography) instrument by adopting the sample injector, the detection is carried out by a differential refraction detector, the peak areas of the hyaluronic acid in the control solution and the sample solution are respectively recorded, and the content of the hyaluronic acid in the sample solution is obtained by calculating the peak areas according to an external standard method.
The retention time of hyaluronic acid of example 1 was 10 min.
The content of hyaluronic acid in the sample solution is not less than 0.1%.
Example 2:
a method for detecting the content of hyaluronic acid in a facial mask comprises the following steps:
(1) preparation of control solution: adding a diluent into hyaluronic acid-TRC to dissolve into a control solution with the concentration of 0.5mg/ml, wherein the diluent is a mobile phase, the mobile phase is a mixed solution of 20% of 0.05mol/L amine formate aqueous solution and 80% of methanol, the pH of the solution is adjusted to be 4.00 by trifluoroacetic acid, and the flow rate of the mobile phase is 1.0 ml/min;
(2) preparation of sample solution: adding the diluent into the mask sample to dissolve the mask sample into a sample solution with the concentration of 500 mg/ml;
(3) measurement of the content of hyaluronic acid in the sample solution: the HPLC instrument is provided with a chromatographic column and a sample injector, wherein the chromatographic column is NH2And the temperature of the chromatographic column is 40 ℃, 20 mu L of the control solution and the sample solution are respectively injected into an HPLC (high performance liquid chromatography) instrument by adopting the sample injector, the detection is carried out by a differential refraction detector, the peak areas of the hyaluronic acid in the control solution and the sample solution are respectively recorded, and the content of the hyaluronic acid in the sample solution is obtained by calculating the peak areas according to an external standard method.
The retention time of hyaluronic acid of example 2 was 13 min.
The content of hyaluronic acid in the sample solution is not less than 0.1%.
Example 3:
a method for detecting the content of hyaluronic acid in a facial mask comprises the following steps:
(1) preparation of control solution: adding a diluent into hyaluronic acid-TRC to dissolve into a control solution with the concentration of 0.6mg/ml, wherein the diluent is a mobile phase, the mobile phase is a mixed solution of 20% of 0.05mol/L amine formate aqueous solution and 80% of methanol, the pH of the solution is adjusted to be 4.50 by trifluoroacetic acid, and the flow rate of the mobile phase is 1.5 ml/min;
(2) preparation of sample solution: adding the diluent into the mask sample to dissolve the mask sample into a sample solution with the concentration of 550 mg/ml;
(3) measurement of the content of hyaluronic acid in the sample solution: the HPLC instrument is provided with a chromatographic column and a sample injector, wherein the chromatographic column is NH2And the temperature of the chromatographic column is 50 ℃, 15 mu L of the control solution and the sample solution are respectively injected into an HPLC (high performance liquid chromatography) instrument by adopting the sample injector, the peak areas of the hyaluronic acid in the control solution and the sample solution are respectively recorded by detecting through a differential refraction detector, and the content of the hyaluronic acid in the sample solution is obtained by calculating the peak areas according to an external standard method.
The retention time of hyaluronic acid of example 3 was 16 min.
The content of hyaluronic acid in the sample solution is not less than 0.1%.
The accuracy of the invention was tested as follows:
hyaluronic acid linearity test
Hyaluronic acid-TRC was prepared as a test solution of 0.1mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml and 1.0mg/ml using the diluent, and 20. mu.L of the test solution was injected into the HPLC apparatus using the sample injector, and the peak area was recorded for each concentration, and the table is shown in Table 1.
| Concentration (mg/ml) | Peak area | Concentration (mg/ml) | Peak area |
| 0.100 | 201.850 | 0.500 | 100.9250 |
| 0.098 | 201.798 | 0.502 | 100.9450 |
| 0.300 | 605.550 | 0.600 | 122.1115 |
| 0.298 | 605.480 | 0.601 | 122.1205 |
| 0.400 | 807.400 | 1.002 | 201.8512 |
| 0.402 | 807.411 | 1.000 | 2018500 |
And (3) making a linear regression equation by using the concentration to the peak area to obtain a correlation coefficient r2 of the linear equation, which is 0.9999. This gave the hyaluronic acid of the present example which was linear well within the concentration range of 0.1mg/ml to 1.0 mg/ml.
Hyaluronic acid accuracy test
(1) Preparation of control solution: precisely weighing about 25mg of hyaluronic acid control sample, placing the hyaluronic acid control sample in a 50ml volumetric flask, adding a proper amount of diluent, dissolving, and fixing the volume by using the diluent to obtain a control solution.
(2) Preparation of a test solution: precisely weighing hyaluronic acid control samples and 99% test sample facial masks of 20mg, 25mg and 30mg respectively, placing in six 100ml volumetric flasks respectively, adding appropriate amount of diluent, dissolving, and diluting to constant volume with diluent to obtain samples of 80%, 100% and 120%.
The measured recovery rates are shown in Table 2.
This makes it possible to obtain the present example with good measurement accuracy.
Third, hyaluronic acid precision investigation
The sample solution having a concentration of 100% in the hyaluronic acid accuracy test was continuously injected into the HPLC apparatus 6 times using a sample injector, and the peak area and the accuracy were measured as shown in table 3.
This makes it possible to obtain the present embodiment with high precision.
Fourthly, hyaluronic acid solution stability test
The test solution with a concentration of 100% in the hyaluronic acid accuracy test was divided into 7 portions, which were placed at room temperature for 0, 2, 4, 8, 12, 24, and 48 hours, respectively, and injected into the HPLC apparatus using a sample injector, and the changes in peak area were recorded, and the stability of the test solution was measured as shown in table 4.
This resulted in good solution stability within 48 hours at room temperature.
Method durability test
Preparing a test solution with the concentration of 100% in a hyaluronic acid accuracy test, changing conditions such as the pH value of a mobile phase, the temperature of a chromatographic column and the like in a chromatographic condition in a small range, and inspecting the influence on the retention time of hyaluronic acid and the influence on a content measurement result. The results obtained when the mobile phase pH was 4.00 are shown in table 5.
The above embodiments do not limit the scope of the present invention, and those skilled in the art can make equivalent modifications and variations without departing from the overall concept of the present invention.
Claims (3)
1. A method for detecting the content of hyaluronic acid in a facial mask is characterized by comprising the following steps:
(1) preparation of control solution: adding a diluent into hyaluronic acid-TRC to dissolve into a control solution with the concentration of 0.4-0.6mg/ml, wherein the diluent is a mobile phase, and the mobile phase is a mixed solution of 20% of 0.05mol/L of amine formate water solution and 80% of methanol; adjusting the pH of the mobile phase to 3.50-4.50 by trifluoroacetic acid;
(2) preparation of sample solution: adding the diluent into the mask sample to be dissolved into a sample solution with the concentration of 450-550 mg/ml;
(3) measurement of the content of hyaluronic acid in the sample solution: the HPLC instrument is provided with a chromatographic column and a sample injector, and the sample injector is adopted to feed the HPLC instrumentRespectively injecting 15-25 μ L of the control solution and sample solution, detecting with a differential refraction detector, respectively recording the peak areas of hyaluronic acid in the control solution and sample solution, and calculating the content of hyaluronic acid in the sample solution according to the peak area by an external standard method, wherein the chromatographic column is NH2The chromatographic column has a column temperature of 30-50 ℃.
2. The method for detecting the content of hyaluronic acid in a facial mask according to claim 1, wherein the flow rate of the mobile phase is 0.5-1.5 ml/min.
3. The method for detecting the content of hyaluronic acid in a facial mask according to claim 1, wherein the content of hyaluronic acid in the sample solution is not less than 0.1%.
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