A method for determining six natural pigments in cosmetics by high performance liquid chromatography

Abstract

The application provides a method for determining six natural pigments in cosmetics by high performance liquid chromatography. The steps are as follows: preparing a standard stock solution: accurately weighing curcumin, shikonin, lutein, cochineal, astaxanthin and cyanidin standard, and preparing a standard stock solution; preparing a mixed standard working solution: mixing the standard according to a certain mass ratio, and preparing a mixed standard working solution; preparing a sample solution to be determined: the natural pigments in the sample are fully extracted by vortex, ultrasonic and centrifugation; sample on machine: the treated solution is filtered through a filter membrane and is determined by high performance liquid chromatography; and result analysis: the six natural pigments are qualitatively determined by retention time, and the natural pigments are quantitatively determined by a standard curve. The application can simultaneously detect a plurality of natural pigments, is simple to operate, has high sensitivity, is suitable for detecting multiple batches of cosmetics, and provides technical support for product quality control in the cosmetic industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetic analysis, in particular to a high performance liquid chromatography determination method for six natural pigments in cosmetics. BACKGROUND

[0002] With the continuous improvement of living standards, as one of the beauty ways, cosmetics have become a world fashion. Adding pigments to cosmetics is to increase the aesthetic, attractiveness and recognition of the product. Pigments can provide a series of different colors for cosmetics, making the product more attractive and meeting the specific needs of consumers. In some cases, pigments can also be used to cover up or reduce skin blemishes such as dullness, red blood vessels, etc. According to statistics, pigments are one of the "three evils" of cosmetic raw materials that can cause skin dysfunction, with a damage rate of 43.9%. The other two evils are fragrances and preservatives. Pigments in cosmetics can cause people to be allergic to cosmetics, and along with the phenomenon of cosmetic allergy, symptoms such as burning, itching, epidermal exfoliation, and mild pain may occur. The safety of cosmetics has become the focus of attention. However, some improperly used or selected pigments can have a negative impact on human health, so safety assessment and regulation are needed. Detecting pigments in cosmetics provides a favorable basis for establishing national standard test methods in the future, and has great significance for the health supervision of cosmetics and the development of the cosmetics industry.

[0003] The detection method of pigments in cosmetics generally uses gas chromatography and liquid chromatography tandem mass spectrometry, but the application of gas chromatography is limited by the polarity and boiling point of the compound, liquid chromatography tandem mass spectrometry instrument is expensive, the detection cost is high, the popularization rate is low, and the matrix effect of oil sample is large, which is not easy to quantify, limiting the application of the method, and high performance liquid chromatography has high sensitivity on antioxidants. The purpose of the present application is to construct a rapid, accurate and simple operation method for determining six natural pigments in cosmetics by high performance liquid chromatography, and to apply it to the detection of multiple batches of cosmetics to provide technical support for product quality control in the cosmetics industry. SUMMARY

[0004] The technical problem to be solved is to construct a rapid, accurate and simple operation method for determining six natural pigments in cosmetics by high performance liquid chromatography, and to apply it to the detection of multiple batches of cosmetics to provide technical support for product quality control in the cosmetics industry.

[0005] Technical scheme: A high performance liquid chromatography determination method for six natural pigments in cosmetics, the six natural pigments include: curcumin, shikonin, lutein, cochineal, astaxanthin, and cyanidin; the method specifically includes the following steps:

[0006] (1) Preparation of standard stock solution: accurately weigh 10 mg of each of the six natural pigment standard samples into a 10 mL volumetric flask, and dilute to volume with 70% chromatographically pure methanol to prepare a standard stock solution with a concentration of 1 mg / mL; (2) Preparation of mixed standard working solution: accurately weigh 10 mg of each of the six natural pigment standard samples into a 10 mL volumetric flask, and dilute to volume with 70% chromatographically pure methanol to prepare a mixed standard stock solution with a concentration of 1 mg / mL. Prepare the mixed standard stock solution with a concentration gradient of 0.10 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL using chromatographically pure methanol to prepare a mixed standard working solution;

[0007] (3) Preparation of sample solution to be tested: if the sample to be tested is a cream, powder, water, or shampoo sample, add 70% chromatographically pure methanol and ultrasonically extract for 30 min. Transfer to a centrifuge tube and centrifuge at 6000-8000 r / min for 10-15 min. Filter the supernatant through a membrane, and the filtrate is used for high-performance liquid chromatography. If the sample to be tested is a lipstick sample, add chromatographically pure tetrahydrofuran and vortex for 2-3 min. Then add chromatographically pure acetonitrile, vortex for 2-3 min, and ultrasonically extract for 30 min. Transfer to a centrifuge tube and centrifuge at 6000-8000 r / min for 10-15 min. Filter the supernatant through a membrane, and the filtrate is used for high-performance liquid chromatography.

[0008] (4) Sample analysis: use a high-performance liquid chromatograph with a diode array detector, a C18 chromatographic column, a column temperature of 35°C, a mobile phase A of 1% formic acid, a mobile phase B of acetonitrile, and an elution gradient program of 0-5 min, 5%-15% A and 95%-85% B; 5-10 min, 15%-30% A and 85%-70% B; 10-15 min, 30%-50% A and 70%-50% B; 15-22 min, 50%-5% A and 50%-95%; 22-25 min, 5% A and 95% B; a flow rate of 1.0 mL / min; a sample injection volume of 10 μL; and detection wavelengths of 430 nm for curcumin and lutein, 475 nm for astaxanthin, 510 nm for shikonin and cochineal, and 530 nm for cyanidin.

[0009] (5) Result analysis: inject the standard stock solution prepared in step (1), the mixed standard working solution prepared in step (2), and the filtrate in step (3) into the high-performance liquid chromatograph, and perform gradient elution and detection under the conditions of step (4). Determine the retention time of each natural pigment based on the peak time of the standard stock solution, and prepare a standard curve based on the mass concentration corresponding to the peak area of each natural pigment in the mixed standard working solution to obtain a linear regression equation for determining the content of different natural pigments in the sample.

[0010] Preferably, the step (3) specifically comprises the following steps:

[0011] (a) If the sample is a cream, powder, water agent and shampoo sample, accurately weigh 1.0 g of the sample (accurate to 0.001 g) into a 10 mL colorimetric tube, add 70% chromatographically pure methanol to the calibration mark, ultrasonic extraction for 30 min, transfer to a 10 mL centrifuge tube, centrifuge at 6000-8000 r / min for 10-15 min, filter the supernatant on a membrane, and the filtrate is used for high performance liquid chromatography determination;

[0012] (b) If the sample is a lipstick sample, accurately weigh 1.0 g of the sample (accurate to 0.001 g) into a 50 mL conical flask, add 1.5 mL of chromatographically pure tetrahydrofuran, and vortex for 2-3 min, then add chromatographically pure acetonitrile to the calibration mark, vortex for 2-3 min, ultrasonic extraction for 30 min, take 10 mL of the solution and transfer it to a 10 mL centrifuge tube, centrifuge at 6000-8000 r / min for 10-15 min, filter the supernatant on a membrane, and the filtrate is used for high performance liquid chromatography determination. Preferably, the filter membrane specification in the step (3) is 13 mm x 0.22 μm.

[0013] Preferably, the high performance liquid chromatograph in the step (4) is a Thermo Ultimate3000.

[0014] Preferably, the chromatographic column in the step (4) is a Thermo Hypersil BDS C18 chromatographic column. Preferably, the chromatographic column in the step (4) has a specification of 4.6 mm x 250 mm, and the filling particle size is 5 μm.

[0015] Beneficial effects:

[0016] 1. The present application can simultaneously detect a plurality of natural pigments, and the pretreatment operation is simple, which greatly shortens the detection period. The detection method of the present application is rapid and accurate, and has high sensitivity.

[0017] 2. The present application can detect natural pigments in multiple batches of cosmetics, rapidly evaluate the safety performance of the cosmetics, and can be well applied to the actual detection of cosmetics, thereby providing technical support for product quality control in the cosmetic industry. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a high performance liquid chromatogram of a mixed standard solution of six natural pigments;

[0019] Figure 2 is a high performance liquid chromatogram of natural pigment components in a water agent cosmetic;

[0020] Figure 3A high performance liquid chromatogram of a natural pigment ingredient in a lipstick type cosmetic. DETAILED DESCRIPTION

[0021] The application will be further described in conjunction with the accompanying drawings and examples. The following examples are illustrative of specific embodiments of the application, but the application is not to be limited to the embodiments.

[0022] Example 1

[0023] 1. Reagents and materials

[0024] 1.1 Methanol, tetrahydrofuran, acetonitrile: chromatographically pure.

[0025] 1.2 Formic acid: analytically pure.

[0026] 1.3 Ultra-pure water.

[0027] 1.4 Natural pigment standard: curcumin, lutein, astaxanthin, shikonin, cochineal, cyanidin (purity≥98%).

[0028] 1.5 Preparation of natural pigment standard solution: accurately weigh 10 mg of each of the six natural pigment standards into a 10 mL brown volumetric flask, and make up to the mark with 70% chromatographically pure methanol to prepare a standard stock solution with a concentration of 1 mg / mL.

[0029] 1.6 Preparation of mixed standard working solution: accurately weigh 10 mg of each of the six natural pigment standards into a 10 mL brown volumetric flask, and make up to the mark with 70% chromatographically pure methanol to prepare a mixed standard stock solution with a concentration of 1 mg / mL. Prepare mixed standard working solutions with concentration gradients of 0.10 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL from the mixed standard stock solution using chromatographically pure methanol.

[0030] 2. Instruments and equipment

[0031] High performance liquid chromatograph with a diode array detector, analytical balance, high-speed refrigerated centrifuge, high-frequency digital ultrasonic cleaner, and multi-tube vortex shaker.

[0032] 3. High performance liquid chromatography conditions

[0033] 3.1 Chromatographic column: Thermo Hypersil BDS C18 column, 5 μm, 4.6 mm x 250 mm;

[0034] 3.2 Mobile phase: mobile phase A was 1% formic acid solution, mobile phase B was acetonitrile, elution gradient program: 0-5 min, 5%-15% A and 95%-85% B; 5-10 min, 15%-30% A and 85%-70% B; 10-15 min, 30%-50% A and 70%-50% B; 15-22 min, 50%-5% A and 50%-95%; 22-25 min, 5% A and 95% B;

[0035] 3.3 Flow rate: 1.0 μL / min;

[0036] 3.4 Column temperature: 35 °C;

[0037] 3.5 Injection volume: 10 μL;

[0038] 3.6 Detection wavelength: 430 nm for curcumin and lutein; 475 nm for astaxanthin; 510 nm for shikonin and carmine; 530 nm for cyanidin.

[0039] 4. Detection of standard solution

[0040] Under the above chromatographic conditions, the prepared six natural pigment standard stock solutions were gradient eluted and detected to determine the peak time of each natural pigment, and the retention time was used for qualitative analysis. Under the same conditions, the prepared mixed standard working solution was gradient eluted and detected to obtain the high performance liquid chromatography separation diagram of the mixed standard solution of the six different natural pigments, and the results are shown in Figure 1 The standard curve was drawn by taking the peak area (Y) of each natural pigment in the mixed standard working solution corresponding to the mass concentration (X, μg / mL), and the linear regression equation was obtained to determine the content of different natural pigments in the sample, and the detection limit (S / N=3) and the quantitative limit (S / N=10) were calculated, and the results are shown in Table 1.

[0041] Table 1 Linear equation, correlation coefficient, detection limit and quantitative limit of six natural pigments in cosmetics

[0042]

[0043] 5. Precision and recovery experiment

[0044] The recovery experiment was carried out in three levels in the blank matrix of water-based cosmetics, and each addition level was repeated for 6 times to determine the recovery rate, and the results are shown in Table 2. The results showed that the recovery rate of the six natural pigments in the blank matrix sample at three addition levels was 89.6%-102.5%, and the relative standard deviation was 0.73%-3.56%. It showed that the method met the analysis and detection of six natural pigments in cosmetics.

[0045] Table 2 Recovery and relative standard deviation of six natural pigments in water-like liquid cosmetics

[0046]

[0047]

[0048] Example 2

[0049] Determination of natural pigments in water-based cosmetics

[0050] 1. Reagents and materials

[0051] Methanol (chromatographic pure), acetonitrile (chromatographic pure), formic acid (analytical pure), filter membrane (13 mm x 0.22 μm), ultrapure water.

[0052] 2. Instruments and equipment

[0053] High performance liquid chromatograph with diode array detector, analytical balance, high-speed refrigerated centrifuge, high-frequency digital ultrasonic cleaner, multi-tube vortex shaker.

[0054] 3. High performance liquid chromatography conditions

[0055] 3.1 Column: Thermo Hypersil BDS C18 column, 5 μm, 4.6 mm x 250 mm;

[0056] 3.2 Mobile phase: mobile phase A is 1% formic acid solution, mobile phase B is acetonitrile, elution gradient program: 0-5 min, 5%-15% A and 95%-85% B; 5-10 min, 15%-30% A and 85%-70% B; 10-15 min, 30%-50% A and 70%-50% B; 15-22 min, 50%-5% A and 50%-95%; 22-25 min, 5% A and 95% B;

[0057] 3.3 Flow rate: 1.0 μL / min;

[0058] 3.4 Column temperature: 35°C;

[0059] 3.5 Injection volume: 10 μL;

[0060] 3.6 Detection wavelength: 0-5 min is 475 nm; 5-10 min is 510 nm; 10-15 min is 430 nm; 15-25 min is 530 nm.

[0061] 4. Sample determination

[0062] A kind of water agent cosmetics, accurately weigh 1.0 g sample (precise to 0.001 g) is placed in 10 mL cuvette, 70% chromatographically pure methanol is added to the scale, ultrasonic extraction is 30 min, is transferred to 10 mL centrifuge tube, is centrifuged at 7000 r / min for 10 min, the supernatant is filtered membrane, is determined under the above chromatographic conditions, and the results are shown in Figure 2 According to the retention time of six natural pigments, it can be known that the sample contains natural pigments astaxanthin and curcumin.

[0063] Example 3

[0064] Determination of natural pigments in lip gloss cosmetics

[0065] 1. Reagents and materials

[0066] Methanol (chromatographically pure), tetrahydrofuran (chromatographically pure), acetonitrile (chromatographically pure), formic acid (analytically pure), filter membrane (13 mm x 0.22 μm), ultrapure water.

[0067] 2. Instruments and equipment

[0068] High performance liquid chromatograph, detector is diode array detector, analytical balance, high-speed refrigerated centrifuge, high-frequency numerical control ultrasonic cleaner, multi-tube vortex shaker.

[0069] 3. High performance liquid chromatography conditions

[0070] 3.1 Chromatographic column: Thermo Hypersil BDS C18 column, 5 μm, 4.6 mm x 250 mm;

[0071] 3.2 Mobile phase: mobile phase A is 1% formic acid solution, mobile phase B is acetonitrile, elution gradient program: 0-5 min, 5%-15% A and 95%-85% B; 5-10 min, 15%-30% A and 85%-70% B; 10-15 min, 30%-50% A and 70%-50% B; 15-22 min, 50%-5% A and 50%-95%; 22-25 min, 5% A and 95% B;

[0072] 3.3 Flow rate: 1.0 μL / min;

[0073] 3.4 Column temperature: 35℃;

[0074] 3.5 Injection volume: 10 μL;

[0075] 3.6 Detection wavelength: 0-5 min is 475 nm; 5-10 min is 510 nm; 10-15 min is 430 nm; 15-25 min is 530 nm.

[0076] 4. Sample determination

[0077] A lipstick sample, 1.0 g of sample (accurate to 0.001 g) was accurately weighed into a 50 mL conical flask, 2 mL of chromatographically pure tetrahydrofuran was added, followed by chromatographically pure acetonitrile to the calibration line, 10 mL of the solution was transferred into a 10 mL centrifuge tube, centrifuged at 6500 r / min for 15 min, the supernatant was filtered through a membrane, and determination was carried out under the above chromatographic conditions. The results are shown in Table 1. Figure 3 As can be seen from the retention time of the six natural pigments, the sample contains natural pigment cyanidin.

[0078] Obviously, the above examples are only examples for the purpose of clear illustration, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. It is not necessary and impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A high-performance liquid chromatography method for the determination of six natural pigments in cosmetics, characterized in that: The six natural pigments include: curcumin, shikonin, lutein, carmine, astaxanthin, and cyanidin; the method specifically includes the following steps: (1) Preparation of standard stock solution: Accurately weigh 10 mg of each of the six natural pigment standards, place them in a 10 mL volumetric flask, and dilute to volume with 70% chromatographic grade methanol solution to prepare a standard stock solution with a concentration of 1 mg / mL. (2) Preparation of mixed standard working solutions: Accurately weigh 10 mg of each of the six natural pigment standards and place them in a 10 mL volumetric flask. Dilute to volume with 70% chromatographic grade methanol solution to prepare a mixed standard stock solution with a concentration of 1 mg / mL. Prepare mixed standard working solutions with concentration gradients of 0.10 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 12.5 μg / mL, 25 μg / mL and 50 μg / mL using chromatographic grade methanol. (3) Preparation of the sample solution: If the sample to be tested is a cream, loose powder, aqueous solution or shampoo, add 70% chromatographic grade methanol, extract by ultrasonication for 30 min, transfer to a centrifuge tube, centrifuge at 6000-8000 r / min for 10-15 min, filter the supernatant through a membrane, and use the filtrate for high performance liquid chromatography (HPLC) analysis; if the sample to be tested is a lipstick, add chromatographic grade tetrahydrofuran and vortex for 2-3 min, then add chromatographic grade acetonitrile, vortex for 2-3 min to mix, extract by ultrasonication for 30 min, transfer to a centrifuge tube, centrifuge at 6000-8000 r / min for 10-15 min, filter the supernatant through a membrane, and use the filtrate for HPLC analysis; (4) Sample loading: The instrument used was a high-performance liquid chromatograph (HPLC), the detector was a diode array detector, the chromatographic column was a C18 column, the column temperature was 35℃, the mobile phase A was 1% formic acid solution, the mobile phase B was acetonitrile, and the elution gradient program was as follows: 0-5 min, 5%-15% A and 95%-85% B; 5-10 min, 15%-30% A and 85%-70% B; 10-15 min, 30%-50% A and 70%-50% B; 15-22 min, 50%-5% A and 50%-95%; 22-25 min, 5% A and 95% B; the flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelengths were: curcumin and lutein 430 nm; astaxanthin 475 nm; shikonin and carmine 510 nm; cyanidin 530 nm. (5) Results analysis: The standard stock solution prepared in step (1), the mixed standard working solution prepared in step (2), and the filtrate in step (3) were injected into the high performance liquid chromatograph and gradient elution and detection were performed under the conditions in step (4). The retention time of each natural pigment was determined by the peak time of the standard stock solution. A standard curve was plotted based on the mass concentration corresponding to the peak area of ​​each natural pigment in the mixed standard working solution to obtain a linear regression equation, so as to determine the content of different natural pigments in the sample.

2. The high-performance liquid chromatography method for determining six natural pigments in cosmetics according to claim 1, characterized in that: Step (3) specifically includes the following steps: (a) If the sample is a cream, loose powder, aqueous solution or shampoo, accurately weigh 1.0 g of the sample and place it in a 10 mL colorimetric tube, add 70% chromatographic grade methanol to the mark, extract by sonication for 30 min, transfer to a 10 mL centrifuge tube, centrifuge at 6000-8000 r / min for 10-15 min, filter the supernatant through a membrane, and use the filtrate for high performance liquid chromatography determination; (b) If the sample is a lipstick, accurately weigh 1.0 g of the sample and place it in a 50 mL Erlenmeyer flask. Add 1.5 mL of chromatographic grade tetrahydrofuran and vortex for 2-3 min. Then add chromatographic grade acetonitrile to the mark, vortex for 2-3 min to mix, and then sonicate for 30 min. Take 10 mL of the solution and transfer it to a 10 mL centrifuge tube. Centrifuge at 6000-8000 r / min for 10-15 min. Filter the supernatant through a membrane and use the filtrate for high performance liquid chromatography analysis.

3. The high-performance liquid chromatography method for determining six natural pigments in cosmetics according to claim 1, characterized in that: The filter membrane in step (3) has a size of 13mm × 0.22μm.

4. The high-performance liquid chromatography method for determining six natural pigments in cosmetics according to claim 1, characterized in that: The high-performance liquid chromatograph used in step (4) is a Thermo Ultimate 3000.

5. The high-performance liquid chromatography method for determining six natural pigments in cosmetics according to claim 1, characterized in that: The chromatographic column used in step (4) is a Thermo Hypersil BDS C18 column.

6. The high-performance liquid chromatography method for determining six natural pigments in cosmetics according to claim 1, characterized in that: In step (4), the chromatographic column has a size of 4.6 mm × 250 mm and the particle size of the packed particles is 5 μm.

Images