A method for detecting intermediate benzenediol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide in cosmetics by high performance liquid chromatography

Abstract

The application provides a high-performance liquid chromatography detection method for cosmetic intermediates resorcinol, ferulic acid, phenethyl resorcinol and benzoyl peroxide, which comprises the following steps: accurately weighing resorcinol, phenethyl resorcinol, benzoyl peroxide and ferulic acid standard products respectively to prepare a mixed standard solution, and precisely pipetting the mixed standard solution to obtain a series of standard working solutions by gradient concentration dilution; mixing and stirring the sample to be detected and methanol, vortexing, centrifuging, taking the supernatant, filtering, and reserving the filtrate; and injecting the standard working solution and the treated sample solution into a liquid chromatograph respectively to perform quantitative analysis and testing. The detection method is simple in operation, rapid in analysis, strong in specificity, high in separation degree, and can be used as a detection method for the four whitening components in cosmetics, thereby providing technical support for the efficacy component detection of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic efficacy ingredient analysis, and particularly relates to a high-performance liquid chromatography detection method for resorcinol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide in cosmetics. BACKGROUND

[0002] Resorcinol is often added to spot-removing and whitening cosmetics in cosmetics due to its whitening and preservative effects. Since resorcinol can effectively prevent the generation of melanin, it has a whitening effect. However, long-term and excessive use of resorcinol can cause irreversible damage to the human body, such as skin irritation. Ferulic acid is a phenolic plant widely existing in the plant kingdom and a derivative of cinnamic acid. Ferulic acid has a strong antioxidant effect and thus has a protective effect on irradiated cells. Ferulic acid can reduce pigment deposition through competition and thus has a spot-removing and whitening effect. Therefore, ferulic acid is often added to spot-removing and whitening cosmetics to achieve the spot-removing and whitening effect. Phenylethyl resorcinol, also known as SymWhite 377, can effectively inhibit the synthesis of melanin and thus improve skin color, and thus has a whitening effect. Meanwhile, phenylethyl resorcinol can also be added to cosmetics as an antioxidant and has an anti-wrinkle effect. Due to the good stability, whitening, spot-removing, and anti-wrinkle effects, phenylethyl resorcinol is widely used in various spot-removing and whitening cosmetics. Benzoyl peroxide, also known as BPO, is a banned cosmetic ingredient. However, due to its ability to quickly kill bacteria and remove acne, benzoyl peroxide is often illegally added to cosmetics together with other antibiotics.

[0003] At present, the detection methods for the four acids are mostly used in food, medicine and cosmetics. For example, there is a document using gas chromatography-mass spectrometry / mass spectrometry to detect resorcinol and phenylethyl resorcinol in cosmetics [1] . There is also a high-performance liquid chromatography method for detecting ferulic acid in cosmetics [2] . However, there is no document indicating the simultaneous detection of resorcinol, benzoyl peroxide, ferulic acid and phenylethyl resorcinol in cosmetics. SUMMARY

[0004] In order to overcome the defects in the prior art, the present application provides a high-performance liquid chromatography detection method for resorcinol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide in cosmetics. The detection method is simple to operate, fast in analysis, strong in specificity and high in separation degree. The detection method can be used as a detection method for the four whitening ingredients in cosmetics and provides technical support for the efficacy ingredient detection of cosmetics.

[0005] To achieve the above object, the present application adopts the following technical solutions:

[0006] The present application provides a high-performance liquid chromatography detection method for resorcinol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide in cosmetics, which comprises the following steps:

[0007] Preparation of standard working solution: accurately weigh the standard samples of resorcinol, phenylethyl resorcinol, benzoyl peroxide and ferulic acid respectively to prepare a mixed standard solution, and then dilute the mixed standard solution to obtain a series of standard working solutions with gradient concentrations;

[0008] Pre-treatment of sample: mix the sample to be tested with methanol, vortex, centrifuge, take the supernatant, filter, and reserve the filtrate;

[0009] Detection: inject the standard working solution and the treated sample solution into the liquid chromatograph respectively, and perform quantitative analysis test.

[0010] Further, the standard working solution is prepared by using methanol, preferably analytical pure methanol.

[0011] Further, the above-mentioned standard working solution is a solution prepared and used immediately, and is stored in low temperature and away from light.

[0012] Further, the methanol used in the pre-treatment of the sample is analytical pure methanol.

[0013] Further, the vortexing time is 30 s-90 s, preferably 60 s.

[0014] Further, the ultrasonic time is 10-20 min, preferably 15 min.

[0015] Further, the centrifugation condition is 8000-12 000 r / min for 3-10 min, preferably 10 000 r / min for 5 min.

[0016] Further, the above-mentioned filtration uses a microporous filter membrane with an average pore size of 0.45 μm.

[0017] Further, the test conditions of the above-mentioned liquid chromatograph are as follows:

[0018] Chromatographic column: SVEA C4 chromatographic column (250 mm x 4.6 mm, 5 μm); column temperature: 25 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0019] The mobile phase and elution conditions are as follows: the mobile phase A is 0.1% phosphoric acid solution, and the mobile phase B is acetonitrile; gradient elution, gradient elution program: 0-8 min, 85% A; 8-8.1 min, 85%-80% A; 8.1-20 min, 80%-60% A; 20-40 min, 60%-40% A; 40-40.10 min, 40%-85% A; 50 min, 85% A.

[0020] Compared with the prior art, the present application has the following technical effects:

[0021] In the selection of the constant volume solvent of the standard, a large number of tests show that the use of methanol has a good target peak shape, and the separation degree of each compound meets the requirements in the chromatography.

[0022] In the selection of the extraction solution of the cosmetic sample, a large number of tests show that the use of methanol as the extraction solvent has a good extraction effect and a high recovery rate.

[0023] In the selection of the extraction time, a large number of tests show that the extraction efficiency of each compound is the highest when the vortex is 1 min and the ultrasonic time is 15 min, especially for the thermally unstable benzoyl peroxide, and the ultrasonic time does not affect the stability.

[0024] (4) In the selection of the mobile phase, a large number of tests show that when 0.1% phosphoric acid and acetonitrile are used as the mobile phase, the four acid peak shapes obtained are symmetrical, and the baseline is stable. The total ion chromatogram of the four compounds can be completely separated.

[0025] The detection method provided by the present application is simple to operate, fast in analysis, strong in specificity, high in separation degree, and can be used as a detection method for the four whitening components in the cosmetic, thereby providing technical support for the efficacy component detection of the cosmetic. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the high performance liquid chromatogram of the resorcinol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide standard solution in an embodiment of the present application (concentration: 100 μg·mL −1 ); wherein, 1 represents resorcinol, 2 represents ferulic acid, 3 represents phenylethyl resorcinol, and 4 represents benzoyl peroxide;

[0027] Figure 2 shows the recovery rate under different ultrasonic times for sample pretreatment;

[0028] Figure 3 shows the high performance liquid chromatogram of the standard solution when the water phase of the mobile phase is water (20 μg / mL) (the chromatographic peak of ferulic acid is not shown); wherein, 1 represents resorcinol, 3 represents phenylethyl resorcinol, and 4 represents benzoyl peroxide;

[0029] Figure 4 shows the high performance liquid chromatogram of the standard solution under the C18 chromatographic column (20 μg / mL) (the peak of benzoyl peroxide is not shown); wherein, 1 represents resorcinol, 2 represents ferulic acid, and 3 represents phenylethyl resorcinol;

[0030] Figure 5The high performance liquid chromatogram of the reference solution under the C8 chromatographic column (20 μg / mL) is shown (the peak of the benzoyl peroxide is not shown); wherein, 1 represents resorcinol, 2 represents ferulic acid, and 3 represents phenylethyl resorcinol. DETAILED DESCRIPTION

[0031] The present application is described in detail and specifically below through specific examples and drawings, so that the present application can be better understood. However, the following examples do not limit the scope of the present application.

[0032] In the examples, the methods are used unless otherwise specified, and the reagents used are commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.

[0033] The instruments, reagents and materials involved in the following examples are as follows:

[0034] Sartorius CP224S and 225D1CN electronic balance (Sartorius, Germany); MS3 vortex mixer (IKA, Germany); 5810R desktop centrifuge (Eppendof, Germany); MilliQ Reference A+ ultrapure water instrument (Millipore, USA); Agilent 1260 high performance liquid chromatograph (Agilent, USA); 5800 ultrasonic instrument (Branson, USA)

[0035] Resorcinol standard, purity 99.25 % (Beijing Bailingwei Technology Co., Ltd.); benzoyl peroxide standard, purity 99.2 % (Shanghai Ampcie Standard Technology Service Co., Ltd.); ferulic acid, purity 99.4 % (China Institute for Drug Control); phenylethyl resorcinol, purity 98.11 % (Shanghai Aladdin Biochemical Technology Co., Ltd.); methanol (analytical pure); acetonitrile, methanol (chromatographic pure, Merk, Germany); phosphoric acid (analytical pure, Shanghai Lingfeng Chemical Reagent Co., Ltd.). Water is ultrapure water. EXAMPLE

[0036] The present example provides a high performance liquid chromatography detection method for resorcinol, ferulic acid, phenylethyl resorcinol and benzoyl peroxide in cosmetics, comprising the following steps:

[0037] Prepare the standard: accurately weigh 10 mg of resorcinol, phenylethyl resorcinol and benzoyl peroxide standard, respectively, and place them in different 10 mL brown volumetric flasks. Dissolve and dilute to the mark with methanol, and shake well.

[0038] The concentration of each is 1 mg·mL −1Resorcinol, phenethyl resorcinol and benzoyl peroxide standard stock solution. Accurately weigh 10 mg of ferulic acid standard, place it in a 10 mL brown volumetric flask, dissolve with methanol and dilute to the mark, shake well, accurately pipette 1 mL into a 25 mL brown volumetric flask, shake well, and prepare a ferulic acid standard stock solution with a concentration of 0.04 mg·mL −1 .

[0039] Preparation of mixed standard solution: accurately pipette 2.0 mL of resorcinol standard stock solution, 4.0 mL of ferulic acid standard stock solution, 1.0 mL of phenethyl resorcinol standard stock solution and 0.4 mL of benzoyl peroxide standard stock solution into the same 20 mL brown volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed standard solution with a mass concentration of 100, 8, 50, 20 μg·mL −1 of resorcinol, ferulic acid, phenethyl resorcinol and benzoyl peroxide, respectively.

[0040] Standard working solution: accurately pipette the mixed standard solution, dilute with methanol to prepare a standard working solution, with a resorcinol concentration of 1.00 μg / mL, 5.00 μg / mL, 10.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, 100 μg / mL; a ferulic acid concentration of 0.08 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL; a phenethyl resorcinol concentration of 0.50 μg / mL, 2.50 μg / mL, 5.00 μg / mL, 12.5 μg / mL, 25.0 μg / mL, 50.0 μg / mL; and a benzoyl peroxide concentration of 0.20 μg / mL, 1.00 μg / mL, 2.00 μg / mL, 5.00 μg / mL, 10.0 μg / mL, 20.0 μg / mL.

[0041] Sample pretreatment: accurately weigh about 0.2 g of sample (accurate to 0.0001 g), place it in a 25 mL plastic centrifuge tube, dilute to the mark with methanol, mix well, vortex for 1 min, ultrasonic for 15 min, centrifuge at 10 000 r / min for 5 min, take the supernatant, pass through a 0.45 μm filter membrane, and the filtrate is ready for use.

[0042] Detection: inject the series of standard solutions in (4) and the filtrate in (5) into the liquid chromatograph for quantitative analysis test.

[0043] The molecular formula, molecular weight and structure of each component are as follows:

[0044] Resorcinol, molecular formula and molecular weight is C6H6O2 / 110.11, the specific structural formula as shown in formula (1).

[0045] Ferulic acid, molecular formula and molecular weight is C 10 H 10 O4 / 194.18, the specific structural formula as shown in formula (2).

[0046] Phenylethyl resorcinol, molecular formula and molecular weight is C 14 H 14 O2 / 214.27, the specific structural formula as shown in formula (3).

[0047] Benzoyl peroxide, molecular formula and molecular weight is C 14 H 10 O4 / 242.23, the specific structural formula as shown in formula (4).

[0048]

[0049] Liquid chromatography conditions

[0050] Chromatographic column: SVEA C4 chromatographic column (250 mm x 4.6 mm, 5 μm); column temperature: 25 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL.

[0051] The mobile phase and elution conditions are as follows: mobile phase A is 0.1% phosphoric acid solution, mobile phase B is acetonitrile; gradient elution, gradient elution program: 0~8 min, 85% A; 8~8.1 min, 85%~80% A; 8.1~20 min, 80%~60% A; 20~40 min, 60%~40% A; 40~40.10 min, 40%~85% A; 50 min, 85% A.

[0052] The high performance liquid chromatogram of the standard solution is shown in Figure 1 Table 1. Linear equation, correlation coefficient, linear range and detection limit of four kinds of compounds.

[0053] Table 1. Linear equation, correlation coefficient, linear range and detection limit of four kinds of compounds.

[0054]

[0055] Examples

[0056] This example optimizes and verifies the method provided in Example 1, and the specific steps and results are as follows:

[0057] 1) Optimization of extraction time and extraction method

[0058] During the experiment, the vortex was observed for 1 min, and the ultrasonic treatment was observed for 0, 15, 30, 60, and 90 min, respectively. It was found that after ultrasonic treatment for 15 min, the recovery rate of each compound could reach more than 85%. With the increase of ultrasonic time, the recovery rate of benzoyl peroxide decreased significantly with the increase of time due to the instability of benzoyl peroxide under heat. The recovery rate of ferulic acid did not decrease significantly. The recovery rate of resorcinol and phenethyl resorcinol did not change significantly between 15 min and 90 min of ultrasonic treatment (see Figure 2 ). Therefore, the pretreatment method of vortexing for 1 min and ultrasonic treatment for 15 min was finally selected as the preparation method of the test solution.

[0059] 2) Optimization of mobile phase

[0060] In the experiment, the effect of water and 0.1% phosphoric acid as the water phase of the mobile phase on the chromatographic peaks of the four compounds was also investigated. It was found that when the water phase of the mobile phase was water, ferulic acid was retained on the chromatographic column and was not easy to be eluted. Under this chromatographic condition, no peak was obtained (see Figure 3 ). When a small amount of weak acid was added, ferulic acid could form a peak under this condition. Therefore, 0.1% phosphoric acid was finally selected as the water phase of the mobile phase.

[0061] 3) Optimization of chromatographic column

[0062] In the experiment, according to the characteristics of resorcinol, ferulic acid, phenethyl resorcinol, and benzoyl peroxide, three types of chromatographic columns, C18 column, C8 column, and C4 column, were selected for experimental study. It was found that on C8 and C18 chromatographic columns, benzoyl peroxide had a long peak time and was not easy to form a peak (see Figure 4 and Figure 5 ). The use of C4 column could obtain a suitable retention time of benzoyl peroxide and a satisfactory peak shape. Therefore, C4 column was finally selected as the chromatographic column.

[0063] 4) Stability investigation

[0064] Benzoyl peroxide is unstable under heat and generates benzene free radicals, which destroy its structure. Ferulic acid is a light-unstable compound and is easily decomposed under light. In order to further investigate the stability of the solution, the standard working solution was taken and placed in brown and transparent liquid phase vials, respectively, and stored in a refrigerator (4°C) and at room temperature (30°C). The changes of the four compounds with time were compared: the samples were analyzed at 0, 4, 8, 12, and 24 h, and the peak areas of the two components were recorded. The peak area at 0 h was taken as 100%, and the recovery rate of each point was investigated.

[0065] The results show that the standard solution is placed in a brown liquid phase vial and refrigerated at 4°C, which is more conducive to the stability of ferulic acid and benzoyl peroxide, and the peak area recovery rate is more than 95% at 24h. When placed in a transparent liquid phase vial and at room temperature, the content of ferulic acid and benzoyl peroxide is less than 85% at 24h. The peak area of resorcinol and phenethyl resorcinol does not change significantly within 24 hours under the above conditions. Therefore, the control solution and the test solution should be prepared immediately before use, stored at low temperature and away from light, and injected in time.

[0066] Example

[0067] This example uses the method of Example 1 to detect cosmetics, and the specific experimental steps and results are as follows:

[0068] (1) A commercially available cosmetic matrix was used as the test object (resorcinol, ferulic acid, phenethyl resorcinol and benzoyl peroxide were not detected), and the standard was added according to the following Table 2, and parallel tests were performed (n=6), and the recovery rate and RSD are shown in Table 2.

[0069] The results show that under high, medium and low standard addition concentration conditions, the recovery rate of the four substances is in the range of 93.26%~107.66%, and the relative deviation RSD (n=6) is in the range of 0.9%~2.41%. It is shown that the recovery rate of the method of the present application is good.

[0070] Table 2 Recovery rate results n =6)

[0071]

[0072] (2) Using the method provided in Example 1, 30 batches of commercially available cream and emulsion, liquid water-based (facial mask, toner, essence, cream, gel, etc.) were determined according to the law. One batch of ferulic acid was detected, with a content of 1.34%. Six batches of phenethyl resorcinol were detected, ranging from 0.0057% to 0.47%; three batches of samples had inconsistent detection results with the sample label, so they could be used for positive sample verification and actual sample detection.

[0073] The specific embodiments of the present application are described in detail above, but they are only examples, and the present application is not limited to the specific embodiments described above. Any equivalent modification and substitution of the present application by those skilled in the art is also within the scope of the present application. Therefore, any equivalent transformation and modification made without departing from the spirit and scope of the present application should be covered within the scope of the present application.

[0074] References

[0075] [1] Xiao Z J, Tong L Y, Li G R, et al. Determination of five phenolic substances in whitening cosmetics by gas chromatography-mass spectrometry / mass spectrometry[J]. China Surfactants, 2022, 52(1): 84-90.

[0076] [2] Zhang B, Qian Y F, Min C Y, et al. Simultaneous determination of seven whitening ingredients in cosmetics by ultra performance liquid chromatography[J]. China Surfactants, 2016, 46(2): 118-122.

Claims

1. A method for detecting hydroquinone, ferulic acid, phenylethyl hydroquinone, and benzoyl peroxide in a cosmetic product by high performance liquid chromatography, characterized by, It comprises the following steps: The standard working solution is prepared by accurately weighing the resorcinol, phenylethyl resorcinol, benzoyl peroxide and ferulic acid standard solution, respectively, mixing the standard solution, and then diluting the mixed standard solution to obtain a series of standard working solutions; The sample is pretreated by mixing the sample to be tested with methanol, vortexing, ultrasonicating, centrifuging, taking the supernatant, filtering, and reserving the filtrate; Detection: The standard working solution and the treated sample solution are injected into the liquid chromatograph, respectively, for quantitative analysis test; The test conditions of the liquid chromatograph are as follows: The chromatographic column is SVEA C4 chromatographic column with a specification of 250mm*4.6mm*5μm, the column temperature is 25℃, the flow rate is 1.0mL / min, and the injection volume is 10μL; The mobile phase and elution conditions are as follows: the mobile phase A is 0.1% phosphoric acid solution, and the mobile phase B is acetonitrile; gradient elution, gradient elution program: 0~8 min, 85% A; 8~8.1 min, 85%~80% A; 8.1~20 min, 80%~60% A; 20~40 min, 60%~40% A; 40~40.10 min, 40%~85% A; 50 min, 85% A. The standard working solution is prepared by using methanol. The standard working solution is prepared on site and stored in low temperature and dark. The methanol used in the sample pretreatment is analytical pure methanol.

2. The high performance liquid chromatography detection method according to claim 1, characterized in that, The vortexing time is 30s-90s.

3. The method of high performance liquid chromatography detection according to claim 1, wherein, The ultrasonicating time is 10-20min.

4. The method of high performance liquid chromatography detection of claim 1, wherein, The centrifuging condition is 8000-12 000r / min for 3-10min.

5. The high performance liquid chromatography detection method according to claim 1, characterized in that, The filter used is a microporous filter membrane with an average pore size of 0.45μm.

6. The high performance liquid chromatography detection method of claim 1, wherein, ​ 7. The high performance liquid chromatography detection method of claim 1, wherein, ​ 8. The method of high performance liquid chromatography detection of claim 1, wherein, ​

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