Method for detecting sensitization of cosmetics containing hexylcinnamyl aldehyde

Abstract

The invention relates to a method for detecting sensitization of cosmetics containing hexylcinnamyl aldehyde, which comprises the following steps: reacting a to-be-detected cosmetic sample solution with an amino acid derivative stock solution, testing response values of amino acid derivatives before and after the reaction through liquid chromatography, and calculating the consumption percentage of the amino acid derivatives so as to judge the sensitization. The detection method provided by the invention is not only suitable for evaluating the potential sensitization of the raw material hexylcinnamyl aldehyde for cosmetics, but also suitable for evaluating the potential sensitization of finished cosmetics (containing hexylcinnamyl aldehyde perfumes with different contents), and has the characteristics of high sensitivity, good accuracy, wide application range, low price, high sensitivity and the like. And the requirements on quick screening and quick detection in enterprise production and practical application are met.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetic toxicology detection, and relates to a method for detecting the allergenicity of cosmetics containing hexyl cinnamaldehyde, and in particular to a method for detecting skin allergic reactions caused by cosmetics containing hexyl cinnamaldehyde. Background Art

[0002] For cosmetic fragrances, 26 fragrances labelled under current EU legislation (26 EU fragrances) are used to a greater or lesser extent: amyl cinnamaldehyde, benzyl benzoate, benzyl salicylate, butylphenyl methylpropional, citral, citronellol, coumarin, eugenol, geraniol, hexyl cinnamaldehyde, hydroxyisohexyl 3-cyclohexenecarboxaldehyde (HICC), α-isomethylionone and linalool in quantities exceeding 175 tonnes, and amyl cinnamyl alcohol, anise alcohol, benzyl alcohol, benzyl cinnamate, cinnamaldehyde, cinnamyl alcohol, farnesol, hydroxycitronellal, isoeugenol, d-limonene, methyl 2-octynoate, oakmoss (Evernia prunastri) and treemoss (Evernia furfuracea) in quantities less than 175 tonnes.

[0003] The regulations on cosmetics point out 26 aromatic compound allergens. Hexylcinnamaldehyde is a commonly used fragrance in cosmetic ingredients and is also an aromatic compound allergen. However, the current methods for detecting allergens are only used to evaluate the potential allergenicity of single-ingredient cosmetic raw materials or chemical raw materials for cosmetics with known and simple compositions, and cannot be used to detect complex mixtures of unknown compositions or finished cosmetic products. This limits the direct prediction of allergic reactions in finished cosmetic products and widens the distance for the clinical detection and application of allergic reactions caused by cosmetics.

[0004] Therefore, it is very necessary to provide a method for detecting the sensitization of hexyl cinnamaldehyde in mixtures of complex unknown compositions or finished cosmetic products. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting the allergenicity of cosmetics containing hexyl cinnamaldehyde. The detection method is not only applicable to the potential allergenicity evaluation of hexyl cinnamaldehyde, a raw material for cosmetics, but also applicable to the potential allergenicity evaluation of finished cosmetics (containing different contents of hexyl cinnamaldehyde). The detection method has high sensitivity, good accuracy, wide application range, and low price, and meets the needs of enterprises for rapid screening and rapid testing in production and practical applications.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, the detection method comprising: reacting a cosmetic sample solution to be tested with an amino acid derivative stock solution, testing the response value of the amino acid derivative before and after the reaction by liquid chromatography, calculating the consumption percentage of the amino acid derivative, and thereby determining the allergenicity.

[0008] The detection method provided by the present invention is not only applicable to the potential allergenicity evaluation of hexyl cinnamaldehyde, a raw material for cosmetics, but also applicable to the potential allergenicity evaluation of finished cosmetics (containing different contents of hexyl cinnamaldehyde), and does not require the preparation of a series of concentration standard solutions. The detection method has the advantages of high sensitivity, good accuracy, a wide range of applications, low price, short analysis cycle, low operating requirements for practitioners, etc., and meets the needs of enterprises for rapid screening and rapid testing in production and practical applications.

[0009] In the process of detecting the content of the amino acid derivative fragments of the present invention, the matrix of the finished product will not interfere with the characteristic peaks of the amino acid derivative fragments in liquid chromatography.

[0010] The present invention utilizes amino acid derivative fragments and allergenic test objects to simulate skin proteins for co-incubation, resulting in a decrease in the amount of the amino acid derivative fragments. The amount of the amino acid derivative fragments is measured and the consumption percentage of the amino acid derivative fragments is calculated, thereby determining the skin allergenicity of the test object.

[0011] Preferably, the amino acid derivative stock solution comprises N-(2-(1-naphthyl)acetyl)-L-cysteine ​​(NAC) stock solution and N-(2-(1-naphthyl)acetyl)-L-lysine (NAL) stock solution.

[0012] Preferably, the concentration of the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution is 0.667 μmol / L, and the volume ratio of the cosmetic sample solution to be tested to the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution is 1:3.

[0013] Preferably, the preparation method of the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution comprises: mixing and dissolving N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and a phosphate buffer having a pH of 8.0±0.1 (e.g., 7.9, 8, 8.1, etc.) to obtain the stock solution.

[0014] In the present invention, the preparation method of the phosphate buffer with a pH of 8.0±0.1 includes: taking 0.18g of anhydrous disodium hydrogen phosphate and 4.02g of anhydrous sodium dihydrogen phosphate and dissolving them in 300mL of distilled water, adding 1mL of 0.1mmol / L EDTA solution per 300mL of buffer, mixing evenly, measuring the final solution pH to be 8.0±0.1, and the preparation is completed.

[0015] Preferably, the concentration of the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution is 0.667 μmol / L, and the volume ratio of the cosmetic sample solution to be tested to the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution is 1:3.

[0016] Preferably, the preparation method of the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution comprises: mixing and dissolving N-(2-(1-naphthyl)acetyl)-L-lysine and a phosphate buffer solution having a pH of 10.2±0.1 to obtain the stock solution.

[0017] In the present invention, the preparation method of the phosphate buffer with a pH of 10.2±0.1 includes: taking 4.26g of anhydrous sodium dihydrogen phosphate and dissolving it in 286mL of distilled water, adding 14mL of 0.1mol / L NaOH solution, and adding 1mL of 0.1mmol / L EDTA solution per 300mL of buffer. Stirring evenly, measuring the final solution pH of 10.2±0.1, and the preparation is completed.

[0018] Preferably, the cosmetic sample solution includes caprylic / caprylic triglyceride (molecular weight 464.635), hyaluronic acid (molecular weight thousands to millions of Daltons), tocopherol acetate (molecular weight 472.75), xanthan gum (molecular weight 2×10 6 to 20×10 6 or carbomer (molecular weight is theoretically estimated to be between 7×10 5 to 4×10 6 Any one or a combination of at least two of the above.

[0019] The detection method provided by the present invention can be applied to the allergenicity detection of cosmetic stock solutions containing multiple complex ingredients, especially containing the above-mentioned high molecular weight ingredients. The detection method avoids the interference of ingredients in the cosmetic matrix and has the advantages of high sensitivity, good accuracy and wide application range.

[0020] Preferably, the detection method comprises:

[0021] (1) reacting a cosmetic sample solution to be tested with a stock solution of N-(2-(1-naphthyl)acetyl)-L-cysteine, testing the response value of N-(2-(1-naphthyl)acetyl)-L-cysteine ​​before and after the reaction by liquid chromatography, and calculating the consumption percentage of N-(2-(1-naphthyl)acetyl)-L-cysteine; reacting a cosmetic sample solution to be tested with a stock solution of N-(2-(1-naphthyl)acetyl)-L-lysine, testing the response value of N-(2-(1-naphthyl)acetyl)-L-lysine before and after the reaction by liquid chromatography, and calculating the consumption percentage of N-(2-(1-naphthyl)acetyl)-L-lysine;

[0022] (2) taking an average value M of the consumption percentages of N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and N-(2-(1-naphthyl)acetyl)-L-lysine obtained in step (1), and judging the allergenicity of the cosmetics containing cinnamaldehyde or hexylcinnamaldehyde based on the average value M.

[0023] Preferably, the cosmetic sample does not co-elute with N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and N-(2-(1-naphthyl)acetyl)-L-lysine, and the judgment standard in step (2) is: 0% ≤ average value M < 4.9%, the reaction level is level 1, and the sensitization is negative; 4.9% ≤ average value M ≤ 100%, the reaction level is level 2, and the sensitization is positive.

[0024] Preferably, the reaction is a light-protected reaction.

[0025] Preferably, the reaction temperature is 20-30°C (for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc.), and the reaction time is 23-25h (for example, 23h, 23.5h, 24h, 24.5h, 25h, etc.).

[0026] Preferably, after the reaction, the reaction liquid is subjected to solid-liquid separation, and the supernatant is taken for liquid chromatography testing.

[0027] Preferably, the solid-liquid separation method comprises filtration.

[0028] Preferably, the mobile phase of the liquid chromatography comprises phase A, phase B and phase C; the phase A is an aqueous solution containing 0.1% trifluoroacetic acid, the phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid, and the phase C is tetrahydrofuran.

[0029] Preferably, the elution method adopted by the liquid chromatography is gradient elution.

[0030] Preferably, the elution procedure of the liquid chromatography is as follows: taking the sum of the volumes of phase A, phase B and phase C as 100%, at minute 0, the volume percentage of phase A is 82-72% (for example, 82%, 80%, 78%, 76%, 74%, 72%, etc.), the volume percentage of phase B is 17-27% (for example, 17%, 18%, 20%, 22%, 24%, 26%, 27%, etc.), and the volume percentage of phase C is 1%; then the volume percentage changes uniformly to minute 9, the volume percentage of phase A is 45-55% (for example, 45%, 46%, 48%, 50%, 52%, 54%, 55%, etc.), the volume percentage of phase B is 44.5-54.5% (for example, 4 4.5%, 46%, 48%, 50%, 52%, 54.5%, etc.), the volume percentage of phase C is 0.5%; then change at a uniform speed to the 10th minute, the volume percentage of phase A is 0%, the volume percentage of phase B is 100%, and the percentage of phase C is 0%; maintain this ratio for 13 minutes and then change at a uniform speed to the 14th minute, the volume percentage of phase A is 82-72% (for example, it can be 82%, 80%, 78%, 76%, 74%, 72%, etc.), the volume percentage of phase B is 17-27% (for example, it can be 17%, 18%, 20%, 22%, 24%, 26%, 27%, etc.), and the volume percentage of phase C is 1%; 14-20min, the ratio remains constant.

[0031] Preferably, the stationary phase of the liquid chromatography column and the guard column is C18.

[0032] In the present invention, the chromatographic column of the liquid chromatography can be HORIZON-C18, with an inner diameter of 3.0 mm, a length of 150 mm, and a particle size of 2.7 μm; the protective column can be Phenomenon C-18, 4×2.0 mm.

[0033] The present invention confirms that the test conditions are effective by an external standard method, and compared with a series of standard solution methods, the steps are simple, the analysis cycle is shortened, and the requirements for practitioners are low. A specific chromatographic column is selected and used in conjunction with a specific elution method, so that the peaks in the liquid chromatogram of the mixture can be effectively separated, and other peaks and the matrix will not interfere with the characteristic peaks of the amino acids; if other chromatographic columns or other elution conditions are used, other interfering peaks in the liquid chromatogram cannot be effectively separated from the characteristic peaks of the amino acids, and the matrix will also interfere with the characteristic peaks of the amino acids, thereby affecting the accuracy and sensitivity of the test.

[0034] It is worth noting that a small amount of tetrahydrofuran is added during gradient elution in the present invention. Tetrahydrofuran is a strong eluent with a greater elution capacity than isopropanol. It is often used to regenerate columns and elute strongly retained components. It can also improve separation and peak shape. In the present invention, it can be seen that the NAC chromatographic peaks without tetrahydrofuran are not completely separated and the two peaks overlap seriously. After adding tetrahydrofuran, different substances are completely separated.

[0035] Preferably, the response value is the peak area of ​​a characteristic peak in liquid chromatography.

[0036] Preferably, the detector of the liquid chromatography is a DAD detector.

[0037] Preferably, the ultraviolet absorption wavelength of the liquid chromatography is 281 nm.

[0038] Preferably, the flow rate of the liquid chromatography is 0.1-0.5 mL / min, for example, it can be 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, etc., preferably 0.3 mL / min.

[0039] Preferably, the column temperature of the liquid chromatography is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc., preferably 25°C.

[0040] Preferably, the injection volume of the liquid chromatography is 3-25 μL, for example, 3 μL, 5 μL, 10 μL, 12 μL, 15 μL, 17 μL, 20 μL, etc., preferably 20 μL.

[0041] Preferably, the detection method further includes a blank control, a matrix control and a positive control.

[0042] In the present invention, the control test includes control test A, control test B and control test C. Control test A is to illustrate the stability of the detection instrument, control test B is to illustrate the stability of the amino acid derivative fragment during the detection process, and control test C is to illustrate that the matrix of the cosmetics other than the allergen has no interference with the reaction to determine whether the peak in the test substance overlaps with NAC or NAL. Positive control D is to select the known positive substance phenylacetaldehyde acetonitrile to confirm whether the experiment is established.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] The present invention provides a method for detecting the allergenicity of cosmetics containing hexyl cinnamaldehyde fragrance. The method is not only applicable to the potential allergenicity evaluation of hexyl cinnamaldehyde, a raw material for cosmetics, but also applicable to the potential allergenicity evaluation of finished cosmetics (containing different amounts of hexyl cinnamaldehyde). The method has the advantages of high sensitivity, good accuracy, wide application range, low price, short analysis cycle, low operating requirements for practitioners, etc., and meets the needs of enterprises for rapid screening and rapid detection in production and practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the liquid chromatogram of the NAC blank control sample in the control group A of Example 1.

[0046] Figure 2 This is the liquid chromatogram of the NAL blank control sample in the control group A of Example 1.

[0047] Figure 3 This is the co-elution liquid chromatogram of hexylcinnamaldehyde NAC in Example 1.

[0048] Figure 4 This is the co-elution liquid chromatogram of hexylcinnamaldehyde NAL in Example 1.

[0049] Figure 5 This is the NAC liquid chromatogram of the sample matrix control (control C) in Example 1.

[0050] Figure 6 This is the NAL liquid chromatogram of the sample matrix control (control C) in Example 1.

[0051] Figure 7 This is the liquid chromatogram of the NAC positive control sample in the control group D of Example 1.

[0052] Figure 8 This is the liquid chromatogram of the NAL positive control sample in the control group D of Example 1.

[0053] Fig. 9 This is the NAC liquid chromatogram of the sample hexyl cinnamaldehyde (content 0.01%) of Example 1.

[0054] Fig.10 This is the NAL liquid chromatogram of the sample hexyl cinnamaldehyde (content 0.01%) of Example 1.

[0055] Fig.11 This is the NAC liquid chromatogram of the sample matrix control (control C) in Example 4.

[0056] Fig.12 This is the NAC liquid chromatogram of the sample matrix control (control C) in Example 5. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0058] Example 1

[0059] This embodiment provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, comprising the following steps:

[0060] (1) Preparation of amino acid derivative fragment standard solution

[0061] 1-1. Configuration of NAC standard solution:

[0062] Step 1: Dissolve 0.18g of anhydrous disodium hydrogen phosphate and 4.02g of anhydrous sodium dihydrogen phosphate in 300mL of distilled water. Add 1mL of 0.1mmol / L EDTA solution to every 300mL of buffer and mix well. Measure the final solution pH between 7.9-8.1. After the preparation is complete, check the pH before each use.

[0063] Step 2: Weigh an appropriate amount of NAC and prepare a 6.667 μmol / L solution with pH 8.0 phosphate buffer for later use;

[0064] Step 3: Take 15 mL of pH 8.0 phosphate buffer in a 50 mL centrifuge tube, add 1 mL of water, 100 μL of trifluoroacetic acid, and 3.9 mL of acetonitrile, and oscillate to mix to prepare NAC dilution solution.

[0065] Step 4: dilute the NAC stock solution with a concentration of 0.667 μmol / L with the NAC diluent to obtain a NAC standard solution with a concentration of 0.156 μmol / L.

[0066] 1-2. Preparation of NAL standard solution:

[0067] Step 1: Dissolve 4.26g of anhydrous sodium dihydrogen phosphate in 286mL of distilled water, add 14mL of 0.1mol / LNaOH solution, add 1mL of 0.1mmol / L EDTA solution per 300mL of buffer, stir evenly, and measure the final solution pH between 10.1-10.3. After the preparation is completed, check the pH before each use;

[0068] Step 2: Weigh an appropriate amount of NAL and prepare a 6.667 μmol / L solution with pH 10.2 phosphate buffer for later use;

[0069] Step 3: Take 15 mL of pH 10.2 phosphate buffer in a 50 mL centrifuge tube, add 1 mL of water, 100 μL of trifluoroacetic acid, and 3.9 mL of acetonitrile, and shake to mix to prepare NAL dilution solution.

[0070] Step 4: dilute the NAL stock solution with a concentration of 0.667 μmol / L with NAL diluent to obtain a NAL standard solution with a concentration of 0.156 μmol / L.

[0071] In this embodiment, in order to ensure the accuracy of the test results, the NAC standard solution and the NAL standard solution are both prepared and used immediately, and the shelf life of the NAC stock solution and the NAL stock solution is 24 hours.

[0072] In this embodiment, the external standard method is used to confirm that the test conditions are accurate and effective. Compared with the series standard solution method, it has simple steps, shortened analysis cycle, and lower requirements for practitioners.

[0073] (2) Pretreatment of cosmetic samples:

[0074] Step 1: Take 150 μL of the tested cosmetic samples with different cinnamaldehyde contents respectively. The cosmetics used in this example are moisturizing creams (the contents of cinnamaldehyde fragrances in the tested cosmetic samples are shown in Table 1);

[0075] Table 1

[0076]

[0077] Step 2: Prepare liquid test solution group M and group N (the preparation methods of group M and group N are shown in Table 2), react at 25°C in the dark for 24 hours, and obtain the tested solution group M and group N after the reaction;

[0078] Table 2

[0079]

[0080]

[0081] Step 3: Add the reaction termination fixative to the test solutions of group M and group N obtained in step 2, and filter them with a filter membrane of 0.45 μm to obtain pretreated samples.

[0082] (3) Preparation of the control group (see Table 3):

[0083] Table 3

[0084]

[0085] Among them, the control group A can be prepared on the day of injection, and does not need to be placed for 24 hours. Its function is to verify the stability of high-performance liquid chromatography; the control group B needs the same treatment method as the sample, that is, it needs to react for 24 hours and filter. It needs to be measured at the beginning and end of the injection sequence in the subsequent liquid chromatography test to ensure the stability of the amino acid fragment during the test; the control groups C and D need the same treatment method as the sample, that is, they need to react for 24 hours and filter. When the average polypeptide consumption percentage of the positive control is: NAC 6%-30%, SD <10%. NAL 75%-100%, SD <10%, it meets one of the conditions for the establishment of the test.

[0086] (4) High performance liquid chromatography detection:

[0087] Test objects: amino acid fragment standard solution, pretreated samples, control group (control group AD);

[0088] High performance liquid chromatograph: model LC-20AT, manufacturer: Shimadzu;

[0089] Chromatographic column: HORIZON-C18, 3.0 mm × 150 mm, 2.7 μm;

[0090] Chromatographic column guard column: Phenomenon C-18, 4×2.0 mm;

[0091] Detector: DAD detector;

[0092] UV absorption wavelength: 281nm; flow rate: 0.3mL / min;

[0093] Column temperature: 25°C;

[0094] Injection volume: 20 μL;

[0095] Mobile phase: Phase A is an aqueous solution containing 0.1% trifluoroacetic acid, Phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid (acetonitrile is chromatographically pure, purchased from Merck), and Phase C is tetrahydrofuran (chromatographically pure, purchased from Thermo Fisher);

[0096] Elution mode: gradient elution (see Table 4), the process of NAC or NAL gradient elution is: based on the sum of the volumes of phase A, phase B and phase C as 100%, in the 0th minute, the volume percentage of phase A is 75%, the volume percentage of phase B is 24%, and the volume percentage of phase C is 1%; then change at a constant speed to the 9th minute, the volume percentage of phase A is 50%, the volume percentage of phase B is 49.5%, and the volume percentage of phase C is 0.5%; then change at a constant speed to the 10th minute, the volume percentage of phase A is 0%, the volume percentage of phase B is 100%, and the volume percentage of phase C is 0%; keep this ratio for 13 minutes and then change at a constant speed to the 14th minute, the volume percentage of phase A is 75%, the volume percentage of phase B is 24%, and the volume percentage of phase C is 1%; 14-20 minutes, keep this ratio unchanged.

[0097] Table 4

[0098]

[0099]

[0100] During the detection process, the liquid phase injection sequence includes: standard solution, control A, control C, control B, positive control (D), and test substance. The liquid phase injection sequence of NAC is shown in Table 5, and the liquid phase injection sequence of NAL is shown in Table 6.

[0101] Table 5

[0102]

[0103] Table 6

[0104]

[0105]

[0106] Figure 1 This is the liquid chromatogram of the NAC blank control sample in the control group A, verifying the system stability of the HPLC;

[0107] Figure 2 This is the liquid chromatogram of the NAL blank control sample in the control group A, verifying the system stability of the HPLC;

[0108] Figure 3 This is the liquid chromatogram of the co-elution of hexylcinnamaldehyde NAC, which is used to examine whether the sample and buffer have interference with the amino acid fragments;

[0109] Figure 4 This is the co-elution liquid chromatogram of hexylcinnamaldehyde NAL, which is used to examine whether the sample and buffer have interference with the amino acid fragments;

[0110] Figure 5 is the NAC liquid chromatogram of the sample matrix control (control C), which shows that there is no peak in the sample that overlaps with the NAC fragment peak;

[0111] Figure 6 is the NAL liquid chromatogram of the sample matrix control (control C), which shows that there is no peak in the sample that overlaps with the NAL fragment peak;

[0112] Figure 7 This is the liquid chromatogram of the NAC positive control sample in the control group D. When the average amino acid fragment consumption percentage of the positive control is: cysteine ​​fragment 6%-30%, SD <10%, it meets one of the test establishment conditions;

[0113] Figure 8 This is the liquid chromatogram of the NAL positive control sample in the control group D. When the average amino acid fragment consumption percentage of the positive control is: lysine fragment 6%-30%, SD <10%, it meets one of the test establishment conditions;

[0114] Fig. 9 This is the NAC liquid chromatogram of the sample hexylcinnamaldehyde (content 0.01%);

[0115] Fig.10 This is the NAL liquid chromatogram of the sample hexylcinnamaldehyde (content 0.01%).

[0116] (5) Analysis of test results:

[0117] Step 1: Take NAC / NAL standard solution and inject it three times continuously to get RSD NAC =1.68%, RSD NAL =1.98% (as shown in Table 7 below), meeting the system suitability requirements. According to the external standard method, the average amino acid derivative consumption percentage of the positive control, the amino acid derivative consumption value SD of the test substance, the average concentration of reference solution A (μM), the average concentration of reference solution C (μM), the peak area RSD% of reference solutions B and C (n=9), the peak area RSD% of solvent control C (n=3), and the stability were determined to confirm that the test conditions were valid (as shown in Table 8 below);

[0118] Table 7

[0119] title Injection volume (μL) area NAC-1 20.0 2475 NAC-2 20.0 2431 NAC-3 20.0 2514 NAL-1 20.0 3307 NAL-2 20.0 3210 NAL-3 20.0 3186

[0120] Table 8

[0121]

[0122]

[0123] Step 2: Based on the data obtained by the external standard method in the first step, the spectrum of the pretreated sample was analyzed. It can be seen that NAC and NAL in the pretreated sample showed sharp peaks, and there was basically no matrix interference near the peak, so the concentration consumption percentage of NAC and NAL in the pretreated sample was obtained (the results are shown in Table 9), and the calculation formula is as follows:

[0124] Consumption value of amino acid fragments = (1-mean value of peptide peak area in pretreated samples / mean value of peptide peak area in sample control) × 100%

[0125] Table 9

[0126]

[0127] Fig. 9 The liquid chromatogram of the cosmetic sample test solution (hexyl cinnamaldehyde) for NAC consumption is shown in Figure 2. Fig.10 The chromatographic peak at 574 min is the peak of residual NAC after the sample solution and NAC were mixed and reacted for 24 hours;

[0128] Fig.10 The liquid chromatogram of NAL consumption by the cosmetic sample test solution (hexyl cinnamaldehyde) is shown in Figure 2. Figure 5 The chromatographic peak at 451 min is the peak of residual NAL after the sample solution and NAL were mixed and reacted for 24 hours;

[0129] (6) Determination of sensitization:

[0130] Step 1: The cosmetic sample did not co-elute with NAC and NAL, and the 1:50 cysteine ​​fragment and 1:50 lysine fragment models were used for determination (see Table 10).

[0131] Table 10

[0132] Mean percentage of cysteine ​​and lysine consumption Reaction level DPRA prediction results 0% ≤ Mean of consumption percentage < 4.9% Level 1 Negative 4.9%≤mean of consumption percentage≤100% Level 2 Positive

[0133] The results show that the detection method provided in the present application has high sensitivity and stability, and the detection result is still positive when the content of hexylcinnamaldehyde in the cosmetic sample is as low as 0.01%.

[0134] Example 2

[0135] This embodiment provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, which differs from Embodiment 1 only in that in step (4) high performance liquid chromatography detection:

[0136] Flow rate: 0.2 mL / min;

[0137] Column temperature: 25°C;

[0138] Injection volume: 25 μL;

[0139] Elution method: Gradient elution as shown in Table 11:

[0140] Table 11

[0141]

[0142]

[0143] For other details, refer to Example 1.

[0144] The test results are shown in Table 12:

[0145] Table 12

[0146]

[0147] The results showed that the cosmetic allergenicity tests for the above-mentioned hexyl cinnamaldehyde content were all positive.

[0148] Example 3

[0149] This embodiment provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, which differs from Embodiment 1 only in that in step (4) high performance liquid chromatography detection:

[0150] Flow rate: 0.5 mL / min;

[0151] Column temperature: 20°C;

[0152] Injection volume: 15 μL;

[0153] Elution method: Gradient elution as shown in Table 13:

[0154] Table 13

[0155]

[0156]

[0157] For other details, refer to Example 1.

[0158] The test results are shown in Table 14:

[0159] Table 14

[0160]

[0161] The results showed that the cosmetic allergenicity tests for the above-mentioned hexyl cinnamaldehyde content were all positive.

[0162] Example 4

[0163] This example provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, which differs from Example 1 only in that the gradient elution procedure in step (4) is different (the percentage content of mobile phase C is distributed to mobile phase A, as shown in Table 15), and the rest is referred to Example 1;

[0164] Table 15

[0165]

[0166] Test results such as Fig.11 As shown, the results showed that the elution effect became worse when the mobile phase elution program was changed, and there were overlapping peaks for the NAC peak area, making it impossible to detect the sensitization.

[0167] Example 5

[0168] This example provides a method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, which differs from Example 1 only in that the gradient elution procedure in step (4) is different (the percentage content of mobile phase C is distributed to mobile phase B, as shown in Table 16), and the rest is referred to Example 1;

[0169] Table 16

[0170]

[0171]

[0172] Test results such as Fig.12 As shown, the results showed that the elution effect became worse when the mobile phase elution program was changed, and there were overlapping peaks for the NAC peak area, making it impossible to detect the sensitization.

[0173] Comparative Example 1

[0174] The difference from Example 1 is that the DPRA method is used, the chromatographic column is replaced with Agilent ZORBAX SB-C18, 4.6 mm×150 mm, 3.5 μm, the flow rate is 1.0 mL / min, the injection volume is 10 μL, the elution method is different, and the sample is centrifuged and then filtered for testing after reaction. The elution procedure is as shown in Table 17, specifically: taking the sum of the volumes of water and acetonitrile as 100%, within 0-10 min, 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%; within 10-11 min, the volume percentage of water is continuously reduced from 64% to 10%, and the volume percentage of acetonitrile is continuously increased from 36% to 90%; within 11-14 min, the volume percentage of water is constant at 10%, and the volume percentage of acetonitrile is constant at 90%; within 14-14.5 min, the volume percentage of water is continuously increased from 10% to 87%, and the volume percentage of acetonitrile is continuously reduced from 90% to 13%; within 14.5-22 min, the volume percentage of water is constant at 87%, and the volume percentage of acetonitrile is constant at 13%.

[0175] The result showed that the content was below 0.5%, and the test result was negative for sensitization, with reduced sensitivity.

[0176] Table 17

[0177]

[0178]

[0179] Comparative Example 2

[0180] Take 0.2g of the same cosmetic sample as in Example 1 to conduct a mouse local lymph node test (LLNA:DA) sensitization test.

[0181] The results showed that when the concentration of cinnamaldehyde in the cosmetic sample was 3% and 5%, SI>1.8, and the sensitization test showed positive sensitization. When the concentration was 0.25%, 0.5%, 0.7%, 1.0%, and 1.5%, it showed negative sensitization.

[0182] By comparing Example 1 and Comparative Example 2, it can be seen that the method provided by this embodiment has higher sensitivity and lower detection limit, and cinnamaldehyde can be detected when the concentration is 0.01%, while the method provided by Comparative Example 2 can only detect cinnamaldehyde when the concentration is not less than 3%.

[0183] Comparative Example 3

[0184] 0.2 g of the same cosmetic sample as in Example 1 was taken to conduct a mouse local lymph node test (LLNA: BrdU-ELISA) sensitization test.

[0185] The results showed that when the concentration of cinnamaldehyde in the cosmetic sample was 3%, SI = 1.6, when the concentration of cinnamaldehyde was 5%, SI>1.6, the sensitization test showed sensitization positive, and when the concentration was 0.25%, 0.5%, 0.7%, 1.0%, and 1.5%, it showed sensitization negative.

[0186] By comparing Example 1 and Comparative Example 3, it can be seen that the method provided by this embodiment has higher sensitivity and lower detection limit, and cinnamaldehyde can be detected when the concentration is 0.01%, while the method provided by Comparative Example 3 can only detect cinnamaldehyde when the concentration is not less than 3%.

[0187] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the allergenicity of cosmetics containing hexylcinnamaldehyde, characterized in that: The detection method comprises: reacting a cosmetic sample solution to be tested with an amino acid derivative stock solution, testing the response value of the amino acid derivative before and after the reaction by liquid chromatography, calculating the consumption percentage of the amino acid derivative, and thus judging the allergenicity.

2. The detection method according to claim 1, characterized in that: The amino acid derivative stock solution includes N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution and N-(2-(1-naphthyl)acetyl)-L-lysine stock solution; Preferably, the concentration of the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution is 0.667 μmol / L, and the volume ratio of the cosmetic sample solution to be tested to the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution is 1:3; Preferably, the preparation method of the N-(2-(1-naphthyl)acetyl)-L-cysteine ​​stock solution comprises: mixing and dissolving N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and a phosphate buffer solution having a pH of 8.0±0.1 to obtain the stock solution; Preferably, the concentration of the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution is 0.667 μmol / L, and the volume ratio of the cosmetic sample solution to be tested to the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution is 1:3; Preferably, the preparation method of the N-(2-(1-naphthyl)acetyl)-L-lysine stock solution comprises: mixing and dissolving N-(2-(1-naphthyl)acetyl)-L-lysine and a phosphate buffer solution having a pH of 10.2±0.1 to obtain the stock solution.

3. The detection method according to claim 1 or 2, characterized in that: The cosmetic sample solution comprises any one of caprylic / capric triglyceride, hyaluronic acid, tocopherol acetate, xanthan gum or carbomer, or a combination of at least two of them.

4. The detection method according to any one of claims 1 to 3, characterized in that: The detection method comprises: (1) reacting a cosmetic sample solution to be tested with a stock solution of N-(2-(1-naphthyl)acetyl)-L-cysteine, testing the response value of N-(2-(1-naphthyl)acetyl)-L-cysteine ​​before and after the reaction by liquid chromatography, and calculating the consumption percentage of N-(2-(1-naphthyl)acetyl)-L-cysteine; reacting a cosmetic sample solution to be tested with a stock solution of N-(2-(1-naphthyl)acetyl)-L-lysine, testing the response value of N-(2-(1-naphthyl)acetyl)-L-lysine before and after the reaction by liquid chromatography, and calculating the consumption percentage of N-(2-(1-naphthyl)acetyl)-L-lysine; (2) taking an average value M of the consumption percentages of N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and N-(2-(1-naphthyl)acetyl)-L-lysine obtained in step (1), and judging the allergenicity of the cosmetics containing cinnamaldehyde or hexylcinnamaldehyde based on the average value M.

5. The detection method according to claim 4, characterized in that: The cosmetic sample does not co-elute with N-(2-(1-naphthyl)acetyl)-L-cysteine ​​and N-(2-(1-naphthyl)acetyl)-L-lysine, and the judgment standard in step (2) is: 0% ≤ average value M < 4.9%, the reaction level is level 1, and the sensitization is negative; 4.9% ≤ average value M ≤ 100%, the reaction level is level 2, and the sensitization is positive.

6. The detection method according to any one of claims 1 to 5, characterized in that: The reaction is a light-proof reaction; Preferably, the reaction temperature is 20-30°C and the reaction time is 23-25h; Preferably, after the reaction, the reaction liquid is subjected to solid-liquid separation, and the supernatant is subjected to liquid chromatography testing; Preferably, the solid-liquid separation method comprises filtration.

7. The detection method according to any one of claims 1 to 6, characterized in that: The mobile phase of the liquid chromatography comprises phase A, phase B and phase C; the phase A is an aqueous solution containing 0.1% trifluoroacetic acid, the phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid, and the phase C is tetrahydrofuran; Preferably, the elution method adopted by the liquid chromatography is gradient elution; Preferably, the elution procedure of the liquid chromatography is: based on the sum of the volumes of phase A, phase B and phase C as 100%, at the 0th minute, the volume percentage of phase A is 82-72%, the volume percentage of phase B is 17-27%, and the volume percentage of phase C is 1%; then it changes at a uniform speed to the 9th minute, the volume percentage of phase A is 45-55%, the volume percentage of phase B is 44.5-54.5%, and the volume percentage of phase C is 0.5%; then it changes at a uniform speed to the 10th minute, the volume percentage of phase A is 0%, the volume percentage of phase B is 100%, and the percentage of phase C is 0%; maintain this ratio for 13 minutes and then change at a uniform speed to the 14th minute, the volume percentage of phase A is 82-72%, the volume percentage of phase B is 17-27%, and the volume percentage of phase C is 1%; from 14 to 20 minutes, the ratio remains constant.

8. The detection method according to any one of claims 1 to 7, characterized in that: The stationary phase of the liquid chromatography column and the guard column is C18; Preferably, the response value is the peak area of ​​a characteristic peak of liquid chromatography; Preferably, the detector of the liquid chromatography is a DAD detector; Preferably, the ultraviolet absorption wavelength of the liquid chromatography is 281 nm.

9. The detection method according to any one of claims 1 to 8, characterized in that: The flow rate of the liquid chromatography is 0.1-0.5 mL / min; Preferably, the column temperature of the liquid chromatography is 20-30°C; Preferably, the injection volume of the liquid chromatography is 3-25 μL.

10. The detection method according to any one of claims 1 to 9, characterized in that: The detection method also includes a blank control, a matrix control and a positive control.

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