阅读说明：本技术 一种含肉桂醛的化妆品致敏性的检测方法 (Method for detecting sensitization of cosmetics containing cinnamaldehyde ) 是由 吴熙 王平 郭煜堂 刁璇 王晓炜 李军 秦美蓉 黄泽愉 陈宁 冼静雯 王钢力 于 2019-10-14 设计创作，主要内容包括：本发明提供了一种含肉桂醛的化妆品致敏性的检测方法,所述检测方法包括：将待测化妆品样品溶液和多肽贮备液反应,通过液相色谱测试多肽反应前后的响应值,计算多肽的消耗百分比,从而判断致敏性；该检测方法不仅适用于化妆品用原料肉桂醛潜在的致敏性评价,还适用于成品化妆品(含有不同含量的肉桂醛)潜在致敏性评价,该检测方法具有灵敏度高、准确性好、使用范围广、价格低廉,满足企业生产和实际应用中对快筛快检的需求。(The invention provides a method for detecting sensitization of cosmetics containing cinnamaldehyde, which comprises the following steps: reacting a cosmetic sample solution to be detected with a polypeptide stock solution, testing response values before and after the polypeptide reaction through liquid chromatography, and calculating the consumption percentage of the polypeptide so as to judge the allergenicity; the detection method is suitable for potential sensitization evaluation of the cosmetic raw material cinnamaldehyde, is also suitable for potential sensitization evaluation of finished products (containing cinnamaldehyde with different contents), has the advantages of high sensitivity, good accuracy, wide application range and low price, and meets the requirements of enterprise production and practical application on quick screening and quick detection.)

1. A method for detecting sensitization of cosmetics containing cinnamaldehyde, which is characterized by comprising the following steps: and (3) reacting the cosmetic sample solution to be detected with the polypeptide stock solution, testing response values before and after the polypeptide reaction through liquid chromatography, and calculating the consumption percentage of the polypeptide so as to judge the sensitization.

2. The method for detecting sensitization of cinnamaldehyde-containing cosmetics according to claim 1, wherein the polypeptide stock solution comprises a cysteine polypeptide stock solution and a lysine polypeptide stock solution;

preferably, the preparation method of the cysteine polypeptide stock solution comprises the following steps: preparing cysteine polypeptide stock solution with the concentration of 0.667mmol/L by using phosphate buffer solution with the pH of 7.5 +/-0.5;

preferably, the cysteine polypeptide is 90-99.9% pure;

preferably, the preparation method of the phosphate buffer solution with the pH of 7.5 +/-0.5 comprises the following steps: mixing a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L and a disodium hydrogen phosphate solution with the concentration of 0.1mol/L to obtain a phosphate buffer solution with the pH value of 7.5 +/-0.5;

preferably, the volume of the added sodium phosphate dibasic solution is 82mL, calculated as the volume of the added sodium phosphate monobasic solution is 18 mL;

preferably, the preparation method of the lysine polypeptide stock solution comprises: preparing lysine polypeptide stock solution with the concentration of 0.667mmol/L by using ammonium acetate buffer solution with the pH value of 10.2;

preferably, the lysine polypeptide is 90-99.9% pure;

preferably, the preparation method of the ammonium acetate buffer solution with the pH of 10.2 comprises the following steps: dissolving ammonium acetate in water, and adjusting pH value with ammonia water to obtain ammonium acetate buffer solution with pH of 10.2;

preferably, the volume of water added is 200mL, based on the amount of ammonium acetate added as 1.542 g.

3. The method for detecting sensitization of cosmetics containing cinnamaldehyde according to claim 1 or 2, wherein the method comprises:

(1) reacting a cosmetic sample solution to be tested with the cysteine polypeptide stock solution, testing the response values of the cysteine polypeptide before and after the reaction by using liquid chromatography, and calculating the consumption percentage of the cysteine polypeptide;

(2) reacting a cosmetic sample solution to be tested with a lysine polypeptide stock solution, testing response values of lysine polypeptide before and after the reaction by using liquid chromatography, and calculating the consumption percentage of the lysine polypeptide;

(3) taking the average value M of the consumption percentage of the cysteine polypeptide obtained in the step (1) and the consumption percentage of the lysine polypeptide obtained in the step (2), and judging the sensitization of the cosmetics containing the cinnamaldehyde according to the average value M.

4. The method for detecting sensitization of cosmetics containing cinnamaldehyde according to claim 3, wherein the reaction in the step (1) comprises: taking 750 mu L of cysteine polypeptide stock solution, 200 mu L of acetonitrile and 50 mu L of cosmetic sample solution to be detected for mixing reaction;

preferably, the reaction in step (2) comprises: and mixing 750 mu L of lysine polypeptide stock solution and 250 mu L of cosmetic sample solution to be detected for reaction.

5. The method for detecting sensitization of cosmetics containing cinnamaldehyde according to claim 3 or 4, wherein the reaction is a light-shielding reaction;

preferably, the reaction time is 15-24 h;

preferably, the detection method further comprises separating a reactant obtained after the reaction;

preferably, the separation method comprises the steps of firstly centrifuging, and then filtering the lower clear liquid obtained by centrifuging;

preferably, the speed of the centrifugation is 2000-6000 r/min;

preferably, the time of centrifugation is 5-15 min.

6. The method for detecting sensitization of cosmetics containing cinnamaldehyde according to any one of claims 3 to 5, wherein the solution of a cosmetic sample to be detected is an acetonitrile solution of a cosmetic sample to be detected;

preferably, the addition amount of the acetonitrile is 0.75-2.25mL calculated by the addition amount of the cosmetic sample to be detected being 0.5-1.5 g.

7. The method for detecting sensitization of cinnamaldehyde-containing cosmetics according to any one of claims 3 to 6, wherein the response value is a peak area of a characteristic peak of liquid chromatography;

preferably, the detector of the liquid chromatograph is a DAD detector;

preferably, the ultraviolet absorption wavelength of the liquid chromatogram is 220 nm;

preferably, the flow rate of the liquid chromatography is 0.5-2mL/min, preferably 1 mL/min;

preferably, the column temperature of the liquid chromatography is 20-30 ℃, preferably 25 ℃;

preferably, the sample size of the liquid chromatography is 3-20 μ L, preferably 10 μ L;

preferably, the inner diameter of the chromatographic column of the liquid chromatogram is 4.6mm, the length is 150mm, and the particle size is 3.5 μm;

preferably, the chromatographic column of the liquid chromatography is Agilent ZORBAX SB-C18;

preferably, the chromatographic column protective column of the liquid chromatography is Phenomenon C-18.

8. The method for detecting sensitization of cinnamaldehyde-containing cosmetics according to any one of claims 3 to 7, wherein the mobile phase of the liquid chromatography comprises water and acetonitrile;

preferably, the water is an aqueous solution containing 0.1% trifluoroacetic acid, and the acetonitrile is an acetonitrile solution containing 0.085% trifluoroacetic acid;

preferably, the liquid chromatography adopts gradient elution;

preferably, the flow of gradient elution is as follows: the volume percentage of water is continuously reduced from 87% to 64% and the volume percentage of acetonitrile is continuously increased from 13% to 36% in 0-10min, wherein the sum of the volumes of the water and the acetonitrile is 100%; within 10-11min, the volume percentage of water is continuously reduced from 64 percent to 10 percent, and the volume percentage of acetonitrile is continuously increased from 36 percent to 90 percent; in 11-14min, the volume percentage of water is constant at 10 percent, and the volume percentage of acetonitrile is constant at 90 percent; within 14-14.5min, the volume percentage of water is continuously increased to 87% from 10%, and the volume percentage of acetonitrile is continuously decreased to 13% from 90%; within 14.5-22min, the volume percentage of water is constant at 87%, and the volume percentage of acetonitrile is constant at 13%.

9. The method for detecting sensitization of a cinnamaldehyde-containing cosmetic according to any one of claims 3 to 8, wherein the method further comprises a blank control, a solvent control, a sample control, and a positive control.

10. The method for detecting sensitization of cosmetics containing cinnamaldehyde according to any one of claims 3 to 9, wherein the cosmetic sample is not co-eluted with the cysteine polypeptide and the lysine polypeptide, and the judgment criterion in the step (3) is: the average value M is more than or equal to 0 percent and less than or equal to 6.38 percent, the reaction grade is grade 1, and the allergenicity is negative; 6.38% < average M is less than or equal to 22.62%, the reaction grade is grade 2, and the allergenicity is positive; 22.62% < average M is less than or equal to 42.47%, the reaction grade is grade 3, and the sensitization is positive; 42.47% < average M is less than or equal to 100%, the reaction grade is grade 4, and the sensitization is positive.

Technical Field

The invention belongs to the field of cosmetic toxicology detection, relates to a method for detecting sensitization of cosmetics containing cinnamaldehyde, and particularly relates to a method for detecting skin allergy caused by cosmetics containing cinnamaldehyde.

Background

Cinnamaldehyde is a perfume which is commonly used in cosmetic ingredients, and 26 kinds of aromatic compound allergens are specified in the regulations on cosmetics, and cinnamaldehyde also belongs to one kind of the aromatic compound allergens. Meanwhile, because the cinnamaldehyde has the functions of promoting blood circulation and preventing corrosion and sterilization, people apply the effect to cosmetic products such as massage liquid and the like. However, this method is currently used only for evaluation of potential sensitization of cosmetic raw materials of a single component or cosmetic chemical raw materials of a known composition and simple components, and cannot be used for detection of a mixture of a complex unknown composition or a finished cosmetic product. This limits the direct prediction of allergy in finished cosmetic products, and widens the distance of clinical applications of allergy-causing cosmetic products.

Therefore, it is necessary to provide a method for detecting cinnamaldehyde sensitization in a complex mixture or a finished cosmetic product of unknown composition.

Disclosure of Invention

The invention aims to provide a method for detecting the sensitization of cosmetics containing cinnamaldehyde, which is not only suitable for evaluating the potential sensitization of the cinnamaldehyde serving as a raw material for cosmetics, but also suitable for evaluating the potential sensitization of finished cosmetics (containing cinnamaldehyde with different contents), has the advantages of high sensitivity, good accuracy, wide application range and low price, and meets the requirements of enterprise production and practical application on quick screening and quick detection.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a method for detecting the sensitization of cosmetics containing cinnamaldehyde, which comprises the following steps: and (3) reacting the cosmetic sample solution to be detected with the polypeptide stock solution, testing response values before and after the polypeptide reaction through liquid chromatography, and calculating the consumption percentage of the polypeptide so as to judge the sensitization.

The detection method provided by the invention is not only suitable for evaluating the potential sensitization of the cinnamaldehyde serving as the raw material for cosmetics, but also suitable for evaluating the potential sensitization of finished cosmetics (containing cinnamaldehyde with different contents), and does not need to prepare a series of concentration standard solutions.

In the process of detecting the content of the polypeptide, the matrix of the finished product does not interfere with the characteristic peak of the polypeptide in liquid chromatogram.

The invention utilizes the co-incubation of the polypeptide and the skin-simulating protein of the test object with sensitization to reduce the amount of the polypeptide, and calculates the consumption percentage of the polypeptide by measuring the amount of the polypeptide so as to judge the skin sensitization of the test object.

In the present invention, the polypeptide stock solution includes a cysteine polypeptide stock solution and a lysine polypeptide stock solution.

In the present invention, the preparation method of the cysteine polypeptide stock solution comprises: cysteine polypeptide is prepared into cysteine polypeptide stock solution with the concentration of 0.667mmol/L by phosphate buffer solution with the pH of 7.5 +/-0.5.

In the present invention, the cysteine polypeptide is 90-99.9% pure, e.g., 90%, 92%, 95%, 97%, 99.9%, etc.

In the present invention, the preparation method of the phosphate buffer solution with pH of 7.5 ± 0.5 comprises: mixing 0.1mol/L sodium dihydrogen phosphate solution and 0.1mol/L disodium hydrogen phosphate solution to obtain phosphate buffer solution with pH of 7.5 + -0.5.

In the present invention, the volume of disodium hydrogen phosphate solution added was 82mL, based on the volume of sodium dihydrogen phosphate solution added being 18 mL.

In the present invention, the method for preparing the lysine polypeptide stock solution comprises: lysine polypeptide is prepared into a lysine polypeptide stock solution with the concentration of 0.667mmol/L by using an ammonium acetate buffer solution with the pH value of 10.2.

In the present invention, the lysine polypeptide has a purity of 90-99.9%, e.g., 90%, 92%, 95%, 97%, 99.9%, etc.

In the invention, the preparation method of the ammonium acetate buffer solution with the pH of 10.2 comprises the following steps: dissolving ammonium acetate in water, and adjusting pH value with ammonia water to obtain ammonium acetate buffer solution with pH of 10.2.

In the present invention, the volume of water added was 200mL, based on the amount of ammonium acetate added of 1.542 g.

In the present invention, the detection method includes:

(1) and (3) reacting the cosmetic sample solution to be detected with the cysteine polypeptide stock solution, testing the response values of the cysteine polypeptide before and after the reaction by using liquid chromatography, and calculating the consumption percentage of the cysteine polypeptide.

(2) And (3) reacting the cosmetic sample solution to be detected with the lysine polypeptide stock solution, testing response values of the lysine polypeptide before and after the reaction by using liquid chromatography, and calculating the consumption percentage of the lysine polypeptide.

(3) Taking the average value M of the consumption percentage of the cysteine polypeptide obtained in the step (1) and the consumption percentage of the lysine polypeptide obtained in the step (2), and judging the sensitization of the cosmetics containing the cinnamaldehyde according to the average value M.

In the present invention, the reaction in step (1) comprises: and mixing 750 mu L of cysteine polypeptide stock solution, 200 mu L of acetonitrile and 50 mu L of cosmetic sample solution to be detected for reaction.

In the present invention, the reaction in step (2) comprises: and mixing 750 mu L of lysine polypeptide stock solution and 250 mu L of cosmetic sample solution to be detected for reaction.

In the present invention, the reaction is a reaction protected from light.

In the present invention, the reaction time is 15-24h, such as 15h, 18h, 20h, 22h, 24h, etc.

In the invention, the detection method further comprises the step of separating a reactant obtained after the reaction.

In the invention, the separation method comprises the steps of firstly centrifuging, and then filtering the lower clear liquid obtained by centrifuging.

In the present invention, the centrifugation rate is 2000-6000r/min, such as 2000r/min, 2500r/min, 3000r/min, 3500r/min, 4000r/min, 4500r/min, 5000r/min, 5500r/min, 6000r/min, etc.

In the invention, the centrifugation time is 5-15 min.

In the invention, the cosmetic sample solution to be detected is acetonitrile solution of the cosmetic sample to be detected.

In the present invention, the amount of acetonitrile to be added is 0.75 to 2.25mL (e.g., 0.75mL, 1mL, 1.25mL, 1.5mL, 1.75mL, 2mL, 2.25mL, etc.) based on the amount of the cosmetic sample to be tested which is 0.5 to 1.5g (e.g., 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, etc.).

In the present invention, the response value is a peak area of a characteristic peak of liquid chromatography.

In the present invention, the detector of the liquid chromatography is a DAD detector.

In the present invention, the ultraviolet absorption wavelength of the liquid chromatography is 220 nm.

In the present invention, the flow rate of the liquid chromatography is 0.5 to 2mL/min, for example, 0.5mL/min, 0.75mL/min, 1mL/min, 1.25mL/min, 1.5mL/min, 1.75mL/min, 2mL/min, etc., preferably 1 mL/min.

In the present invention, the column temperature of the liquid chromatography is 20 to 30 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ and the like, preferably 25 ℃.

In the present invention, the amount of the sample to be applied to the liquid chromatography is 5 to 20. mu.L, for example, 5. mu.L, 8. mu.L, 10. mu.L, 12. mu.L, 15. mu.L, 17. mu.L, 20. mu.L, etc., preferably 10. mu.L.

In the present invention, the inner diameter of the column for liquid chromatography is 4.6mm, the length is 150mm, and the particle size is 3.5. mu.m.

In the invention, the chromatographic column of the liquid chromatogram is Agilent ZORBAX SB-C18.

In the invention, the chromatographic column protective column of the liquid chromatogram is Phenomenon C-18.

In the present invention, the mobile phase of the liquid chromatography comprises water and acetonitrile.

The water adopted by the mobile phase in the invention is an aqueous solution containing 0.1 percent of trifluoroacetic acid; acetonitrile is a solution of 0.085% trifluoroacetic acid in acetonitrile.

In the invention, the elution mode adopted by the liquid chromatography is gradient elution.

In the present invention, the flow of gradient elution is as follows: the volume percentage of water is continuously reduced from 87% to 64% and the volume percentage of acetonitrile is continuously increased from 13% to 36% in 0-10min, wherein the sum of the volumes of the water and the acetonitrile is 100%. Within 10-11min, the volume percentage of water is continuously reduced from 64 percent to 10 percent, and the volume percentage of acetonitrile is continuously increased from 36 percent to 90 percent. Within 11-14min, the volume percentage of water is constant at 10%, and the volume percentage of acetonitrile is constant at 90%. Within 14-14.5min, the volume percentage of water is continuously increased from 10% to 87%, and the volume percentage of acetonitrile is continuously decreased from 90% to 13%. Within 14.5-22min, the volume percentage of water is constant at 87%, and the volume percentage of acetonitrile is constant at 13%.

The method confirms that the test conditions are effective through an external standard method, and compared with a series of standard solution methods, the method has the advantages of simple steps, short analysis period and low requirement on practitioners. A specific chromatographic column is selected and matched with a specific elution mode, so that peaks in a liquid chromatogram of the mixture can be effectively separated, and other peaks and matrixes cannot interfere with characteristic peaks of the polypeptide; if other chromatographic columns or other elution conditions are used, other interference peaks in the liquid chromatogram cannot be effectively separated from the characteristic peaks of the polypeptide, and the matrix can also interfere with the characteristic peaks of the polypeptide, so that the accuracy and the sensitivity of the test can be influenced.

In the present invention, the detection method further comprises a blank control, a solvent control, a sample control and a positive control.

In the present invention, the control test includes a control test a for explaining the stability of the detecting instrument, a control test B for explaining the stability of the polypeptide during the detecting process, a control test C for explaining that the solvent does not interfere with the reaction, and a sample control for determining whether the peak in the test substance overlaps with the peak of the cysteine polypeptide or the lysine polypeptide.

In the invention, the cosmetic sample and the cysteine polypeptide and the lysine polypeptide are not co-eluted, and the judgment standard in the step (3) is as follows: the average value M is more than or equal to 0 percent and less than or equal to 6.38 percent, the reaction grade is grade 1, and the sensitization is negative. 6.38% < average M < 22.62%, reaction grade 2, positive sensitization. 22.62% < average M < 42.47%, reaction grade 3, positive sensitization. 42.47% < average M is less than or equal to 100%, the reaction grade is grade 4, and the sensitization is positive.

Compared with the prior art, the invention has the following beneficial effects:

the method for detecting the sensitization of the cosmetics containing the cinnamaldehyde is not only suitable for evaluating the potential sensitization of the cinnamaldehyde serving as the raw material for cosmetics, but also suitable for evaluating the potential sensitization of finished cosmetics (containing different contents of cinnamaldehyde), has the advantages of high sensitivity, good accuracy, wide application range, low price, short analysis period, low operation requirement on workers and the like, and meets the requirement on quick screening and quick detection in enterprise production and practical application.

Drawings

FIG. 1 is a liquid chromatogram of a blank control of cysteine polypeptide in control group A;

FIG. 2 is a liquid chromatogram of a blank lysine polypeptide control in control group A;

FIG. 3 is a liquid chromatogram of a blank control of cysteine polypeptide in control group C;

FIG. 4 is a liquid chromatogram of a blank lysine polypeptide control in control group C;

FIG. 5 is a cysteine polypeptide liquid chromatogram of a sample control (control D);

FIG. 6 is a liquid chromatogram of lysine polypeptide from a sample control (control D);

FIG. 7 is a liquid chromatogram of a positive control for cysteine polypeptide in control E;

FIG. 8 is a liquid chromatogram of a lysine polypeptide positive control in control E;

FIG. 9 is a liquid chromatogram of a cosmetic sample test solution for cysteine polypeptide consumption;

FIG. 10 is a liquid chromatogram of a cosmetic sample test solution for lysine polypeptide consumption;

FIG. 11 is a liquid chromatogram of a standard solution (concentration of 0.58mM) of cysteine polypeptide in comparative example 1;

FIG. 12 is a liquid chromatogram of a standard solution (concentration of 0.58mM) of lysine polypeptide in comparative example 1.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.