Detection method for simultaneously determining seven active components in cosmetics

Abstract

The invention provides a detection method for simultaneously determining seven active components in cosmetics, constructs a high performance liquid chromatography determination method which is efficient, accurate and simple to operate, and can simultaneously detect the contents of Ectoin, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin and madecassoside in the cosmetics, so that the contents of Ectoin, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin and madecassoside in the cosmetics can be detected at the same time. And the method is applied to detection of large-batch cosmetics and provides technical support for product quality control in the cosmetic industry.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics analysis, and in particular relates to a detection method for simultaneously determining ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin and madecassoside in cosmetics. Background Art

[0002] Active ingredients are chemical components in cosmetics that contribute to moisturizing, whitening, anti-aging, and anti-wrinkle effects. Natural active ingredients are biologically active chemicals extracted from nature. These substances typically originate from plants, animals, and microorganisms, such as olive extract, gentian extract, Centella asiatica extract, lactobacillus fermentation products, and amino acid derivatives. They possess unique structures and bioactivities, and have biological functions such as defending against predators and competing for resources, thus possessing significant application value in the cosmetics field.

[0003] With the development of society and the improvement of living standards, people's demand for cosmetics has gradually shifted from basic cosmetics with simple chemical ingredients to functional cosmetics containing natural active ingredients that can address deeper skin problems. Common natural active ingredients include phenolic acids, flavonoids, flavanols, glycosides, and sesquiterpenoids. These active ingredients have excellent soothing, anti-wrinkle, anti-aging, antioxidant, moisturizing, and oil-control effects, and are widely used in cosmetics.

[0004] The main methods for detecting active ingredients in cosmetics are gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, and liquid chromatography-tandem mass spectrometry. Gas chromatography and gas chromatography-mass spectrometry have limited applicability due to their boiling point requirements for the target compound. Liquid chromatography-tandem mass spectrometry is expensive, has high detection costs, and is not widely used. Some samples may experience matrix interference and require special pretreatment such as column cleanup, resulting in cumbersome operation steps. Liquid chromatography, on the other hand, is widely used due to its wide range of applications, accuracy, and sensitivity. However, current methods for detecting active ingredients in cosmetics are limited by their single detection component and have thus failed to achieve widespread application. Therefore, in order to protect the rights and interests of consumers and ensure the quality and safety of such cosmetics, it is urgently necessary to develop a high-performance liquid chromatography method for the determination of multiple natural active ingredients in cosmetics to provide technical support for quality control in the cosmetics industry. Summary of the Invention

[0005] Technical problem to be solved: In response to the above technical problems, the purpose of the present invention is to provide a detection method for the simultaneous determination of seven active ingredients in cosmetics, to construct an efficient, accurate and easy-to-operate high-performance liquid chromatography determination method, which can simultaneously detect the content of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin and madecassoside in cosmetics, and to apply it to the detection of large quantities of cosmetics, providing technical support for product quality control in the cosmetics industry.

[0006] Technical solution: A method for simultaneously determining seven active ingredients in cosmetics, the seven active ingredients being ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside; The detection method includes the following steps: S1. Preparation of standard stock solutions: Weigh 10 mg of each of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside into a 10 mL volumetric flask, dissolve in methanol, and dilute to the mark to prepare a 1 mg / mL standard stock solution. S2. Preparation of mixed standard working solutions: Pipette 5.00µL, 25.0µL, 50.0µL, 100.0µL, 250.0µL, and 500.0µL of the standard stock solution into the same 10mL volumetric flask, and dilute to the mark with methanol to prepare mixed standard working solutions of 0.50µg / mL, 2.50µg / mL, 5.00µg / mL, 10.0µg / mL, 25.0µg / mL, and 50.0µg / mL; S3. Sample solution preparation: Accurately weigh 1.0 g of sample into a 10 mL stoppered colorimetric tube. Add methanol to the mark. Vortex for 10-120 seconds to mix thoroughly. Ultrasonicate for 10-30 minutes. Allow to cool to room temperature. Centrifuge. Filter the supernatant through a microporous membrane. Submit the filtrate for HPLC analysis. S4. Qualitative and quantitative detection: HPLC was used to perform gradient elution and detection of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside in the samples. Qualitative analysis was performed based on the peak elution time of each component in the mixed standard solution and the corresponding spectra. A standard curve was drawn based on the mass concentration (X, mg / L) corresponding to the peak area (Y) of each active ingredient in the mixed standard working solution. A linear regression equation was obtained to determine the content of different active ingredients in the sample.

[0007] Furthermore, in step S3, the centrifugal speed is 8000-14000 r / min, and the centrifugal time is 5-10 min.

[0008] Furthermore, the microporous filter membrane in step S3 is a 0.22µm microporous filter membrane.

[0009] Furthermore, the high performance liquid chromatography detection conditions in step S4 are as follows: Chromatographic column: Reverse-phase C18 column, 5 μm, 4.6 mm × 250 mm; Detector: diode array detector; Column temperature: 30°C; Flow rate: 1.0 mL / min; Injection volume: 10-20 μL; Wavelength: Bisabolol, Madecassoside 205nm; Ectoin, Hydroxytyrosol, Salicin, Oleuropein 220nm; Gentipicroside 270nm; Mobile phase: A-methanol; B: 0.05% phosphoric acid solution; Gradient elution program: 0~3 min, 90%B; 3~6 min, 90%→60%B; 6~9.5 min, 60%B; 9.5~20 min, 60%→15%B; 20~30 min, 15%B; 30~33 min, 15%→90%B; 33~38 min, 90%B. Beneficial effects

[0010] The present invention constructs a high-performance liquid chromatography determination method for natural active ingredients in cosmetics, which can simultaneously determine seven active ingredients: ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside. The method is characterized by high efficiency, accuracy, and ease of operation, and is applied to the detection of large quantities of cosmetics, greatly shortening the detection cycle, saving time and labor costs, and providing technical support for product quality control in the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 1 is a HPLC chromatogram of a mixed standard solution of seven active ingredient standards in Example 1 at a wavelength of 205 nm; Figure 2 The HPLC chromatogram of the mixed standard solution of the seven active ingredient standards in Example 1 at a wavelength of 220 nm is shown; Figure 3 1 is a HPLC chromatogram of a mixed standard solution of seven active ingredient standards in Example 1 at a wavelength of 270 nm; Figure 4 This is the HPLC chromatogram of the active ingredients of the emulsion cosmetics in Example 2 at a wavelength of 205 nm; Figure 5 This is the HPLC chromatogram of the active ingredients of the emulsion cosmetics in Example 2 at a wavelength of 220 nm; Figure 6 This is a high performance liquid chromatogram of the active ingredients of the emulsion cosmetics in Example 2 at a wavelength of 270 nm; Figure 7 This is a high performance liquid chromatogram of the active ingredients of the cream-like cosmetics in Example 3 at a wavelength of 205 nm; Figure 8 This is a high performance liquid chromatogram of the active ingredients of the cream-like cosmetics in Example 3 at a wavelength of 220 nm; Figure 9 This is the HPLC chromatogram of the active ingredients of the cream-like cosmetics in Example 3 at a wavelength of 270 nm. DETAILED DESCRIPTION

[0012] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are intended to explain the present invention, but the present invention is not limited to the following embodiments: 1. Reagents and Materials Methanol: chromatographic grade; phosphoric acid: premium grade; first-grade water; microporous filter membrane (13 mm × 0.22 μm); seven active ingredient standard substances: ectoine (CAS: 96702-03-3, purity: 99.9%, batch number: 2422725), hydroxytyrosol (CAS: 10597-60-1, purity: 98.0%, batch number: 2418807), oleuropein (CAS: 5534-13-4, purity: 98.0%, batch number: 2418683), bisabolol (CAS: 515-6 8-5, purity: 95.0%, batch number: 2418994,), gentiopicroside (CAS: 20831-76-9, purity: 99.8%, batch number: 2358782,), salicin (CAS: 138-52-3, purity: 99.1%, batch number: 2424700,), and madecassoside (CAS: 34540-22-2, purity: 98.0%, batch number: 2413106,) were purchased from Anpuyun Laboratory Supplies (Shanghai) Co., Ltd. 2. Instruments and Equipment The high-performance liquid chromatograph was Thermo UltiMate 3000 (with a diode array detector), an analytical balance (Mettler ME55 / 02), a high-speed refrigerated centrifuge (TGL-16M, 16,000 r / min), a CNC ultrasonic cleaner (YT-500DE), and a vortex oscillator (IKA, MS 3). Example 1

[0013] Preparation of standard stock solutions: Weigh 10 mg of each of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside into a 10 mL volumetric flask, dissolve in methanol, and dilute to the mark to prepare a 1 mg / mL standard stock solution. (2) Preparation of mixed standard working solutions: Pipette 5.00µL, 25.0µL, 50.0µL, 100.0µL, 250.0µL, and 500.0µL of the standard stock solution into the same 10mL volumetric flask, dilute to the mark with methanol, and prepare mixed standard working solutions of 0.50µg / mL, 2.50µg / mL, 5.00µg / mL, 10.0µg / mL, 25.0µg / mL, and 50.0µg / mL; (3) High performance liquid chromatography detection: Chromatographic column: Aglient HC-C18 column, 5 μm, 4.6 mm × 250 mm; Detector: Diode array detector (DAD) Column temperature: 30°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Wavelength: Bisabolol, Madecassoside 205nm; Ectoin, Hydroxytyrosol, Salicin, Oleuropein 220nm; Gentipicroside 270nm; Mobile phase: A-methanol; B: 0.05% phosphoric acid solution; Gradient elution program: 0~3 min, 90%B; 3~6 min, 90%→60%B; 6~9.5 min, 60%B; 9.5~20 min, 60%→15%B; 20~30 min, 15%B; 30~33 min, 15%→90%B; 33~38 min, 90%B.

[0014] The prepared mixed standard solution was subjected to gradient elution and detection under the above-mentioned HPLC conditions to obtain a HPLC separation diagram of the active ingredient mixed standard solution. The chromatogram is shown in FIG. Figure 1-3 As shown, the seven active ingredients all showed good separation, the separation between adjacent peaks was greater than 1.5, the peak shape was well symmetrical, and there was no tailing or front extension.

[0015] (4) Linear relationship, detection limit and quantification limit Different volumes of standard stock solution were diluted with methanol to prepare mixed standard working solutions with a concentration gradient of 0.50 µg / mL, 2.50 µg / mL, 5.00 µg / mL, 10.0 µg / mL, 25.0 µg / mL, and 50.0 µg / mL. Following the above-mentioned HPLC detection conditions, samples were injected sequentially, gradient elution was performed, and detection was performed. HPLC chromatograms of the active ingredient mixed standard solutions were obtained. A standard curve was plotted with the peak area of ​​each active ingredient in the mixed standard working solution as the ordinate (Y) and the corresponding mass concentration as the abscissa (X, μg / mL). The linear regression equation and correlation coefficient (R) were obtained. Separate mixed standard solutions were prepared and serially diluted. The limits of detection and quantification were calculated using a 3-fold noise ratio (S / N = 3) and a 10-fold noise ratio (S / N = 10), respectively. The results are shown in Table 1.

[0016] Table 1 Linear equations, correlation coefficients, detection limits, and quantification limits of seven active ingredients Component name Linear equations Correlation coefficient R <![CDATA[Detection limit / mg·kg -1 > <![CDATA[Quantitation limit / mg·kg -1 > Ectoin Y=0.4030X-0.0948 0.9992 0.6 1.5 Hydroxytyrosol Y=0.7471X-0.2267 0.9992 0.6 1.5 salicin Y=0.4006X-0.0222 0.9990 1.0 2.5 Oleuropein Y=0.5638X-0.1848 0.9991 1.0 2.5 Madecassoside Y=0.1852X-0.0871 0.9991 2.5 5.0 Bisabolol Y=1.2302X-0.0111 0.9994 2.5 5.0 Gentiopicroside Y=0.6833X-0.2435 0.9991 0.8 2.5 As shown in Table 1, the seven active ingredients showed good linear relationships, with R values ​​greater than 0.999. The detection limits and quantification limits were 0.6-2.5 mg / kg and 1.5-5.0 mg / kg, respectively. The method has high sensitivity and can fully meet the detection requirements of the seven compounds.

[0017] (5) Spike recovery rate Following the sample preparation method, spike recovery experiments were conducted in cosmetic blank matrices at low, medium, and high concentration levels. Six replicates were performed for each spike level, and the average recoveries and relative standard deviations (RSDs) were calculated. The results are shown in Table 2. The results showed that the recoveries of the seven active ingredients at the three spike levels ranged from 89.9% to 103.8%, with relative standard deviations (RSDs) ranging from 0.4% to 4.8%. This method demonstrates good accuracy and is suitable for the simultaneous determination of seven active ingredients in cosmetics.

[0018] Table 2 Spiked recoveries and relative standard deviations of seven active ingredients

[0019] (6) Precision A mixed solution of seven active ingredients with a concentration of 10 μg / mL was taken and injected continuously for 6 times according to the HPLC conditions for separation and determination. The peak area of ​​each active component was calculated. The relative standard deviations (RSDs) (n=6) of the peak areas of ectoine, hydroxytyrosol, salicin, oleuropein, madecassoside, bisabolol, and gentiopicroside were 2.72%, 1.96%, 1.80%, 2.47%, 2.88%, 1.82%, and 1.67%, respectively. The results showed that the system had good precision.

[0020] (7) Repeatability The same batch of positive aqueous samples were taken and 6 replicates were prepared in parallel according to the sample solution preparation method. The samples were separated and determined according to the HPLC conditions. The content of each active ingredient in the samples was determined according to the standard curve method. The average contents of ectoine, hydroxytyrosol, salicin, oleuropein, madecassoside, bisabolol, and gentiopicroside were calculated to be 67.4, 50.8, 47.9, 45.2, 48.6, 50.2, and 51.8 mg / kg, respectively. The relative standard deviations (RSDs) (n=6) were 1.60%, 2.36%, 2.14%, 2.28%, 1.29%, 1.61%, and 2.06%, respectively, indicating that the method had good repeatability.

[0021] (8) Stability A mixed solution of seven active ingredients with a concentration of 10 μg / mL was taken and placed in an automatic sampler. According to the HPLC conditions, the samples were injected, separated and determined at 0, 2, 6, 12 and 24 hours, respectively. The peak area of ​​each active ingredient at different time was calculated. The relative standard deviations (RSDs) of the peak areas of the seven components, including ectoine, hydroxytyrosol, oleuropein, salicin, gentiopicroside, madecassoside and bisabolol, were 2.37%, 3.00%, 2.45%, 2.66%, 3.63%, 0.65% and 2.67%, respectively. The results showed good stability. Example 2

[0022] A method for simultaneously determining seven active ingredients in emulsion cosmetics comprises the following steps: S1. Sample solution preparation: Accurately weigh 1.0 g of sample into a 10 mL stoppered colorimetric tube. Add methanol to the mark. Vortex for 30 seconds to mix thoroughly. Ultrasonic extraction for 20 minutes. Allow to cool to room temperature. Centrifuge at 14,000 rpm for 5 minutes. Filter the supernatant through a microporous filter. The filtrate is then used for HPLC analysis. S2. Quantitative detection: According to the HPLC detection conditions and methods in Example 1, the ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside in the sample were detected and the contents were calculated. Example 3

[0023] A method for simultaneously determining seven active ingredients in cream-like cosmetics comprises the following steps: S1. Sample solution preparation: Accurately weigh 1.0 g of sample into a 10 mL stoppered colorimetric tube. Add methanol to the mark. Vortex for 30 seconds to mix thoroughly. Ultrasonic extraction for 20 minutes. Allow to cool to room temperature. Centrifuge at 14,000 rpm for 5 minutes. Filter the supernatant through a microporous filter. The filtrate is then used for HPLC analysis. S2. Quantitative detection: According to the HPLC detection conditions and methods in Example 1, the ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside in the sample were detected and the contents were calculated.

[0024] The chromatogram of the emulsion cosmetics in Example 2 is as follows: Figure 4-6 As shown in FIG, hydroxytyrosol and oleuropein were detected, and their contents were 263.0 mg / kg and 274.2 mg / kg respectively; the chromatogram of the cream-like cosmetics in Example 3 is shown in FIG. Figure 7-9 As shown, ectoine and bisabolol were detected, with contents of 229.1 mg / kg and 371.8 mg / kg respectively.

[0025] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for simultaneously determining seven active ingredients in cosmetics, characterized by: The seven active ingredients are ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin and madecassoside; The detection method includes the following steps: S1. Preparation of standard stock solutions: Weigh 10 mg of each of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside into a 10 mL volumetric flask, dissolve in methanol, and dilute to the mark to prepare a 1 mg / mL standard stock solution. S2. Preparation of mixed standard working solutions: Pipette 5.00µL, 25.0µL, 50.0µL, 100.0µL, 250.0µL, and 500.0µL of the standard stock solution into the same 10mL volumetric flask, and dilute to the mark with methanol to prepare mixed standard working solutions of 0.50µg / mL, 2.50µg / mL, 5.00µg / mL, 10.0µg / mL, 25.0µg / mL, and 50.0µg / mL; S3. Sample solution preparation: Accurately weigh 1.0 g of sample into a 10 mL stoppered colorimetric tube. Add methanol to the mark. Vortex for 10-120 seconds to mix thoroughly. Ultrasonicate for 10-30 minutes. Allow to cool to room temperature. Centrifuge. Filter the supernatant through a microporous membrane. Submit the filtrate for HPLC analysis. S4. Qualitative and quantitative detection: HPLC was used to perform gradient elution and detection of ectoine, hydroxytyrosol, oleuropein, bisabolol, gentiopicroside, salicin, and madecassoside in the samples. Qualitative analysis was performed based on the peak elution time of each component in the mixed standard solution and the corresponding spectra. A standard curve was drawn based on the mass concentration (X, mg / L) corresponding to the peak area (Y) of each active ingredient in the mixed standard working solution. A linear regression equation was obtained to determine the content of different active ingredients in the sample.

2. The method for simultaneously determining seven active ingredients in cosmetics according to claim 1, characterized in that: In step S3, the centrifugal speed is 8000-14000 r / min, and the centrifugal time is 5-10 min.

3. The method for simultaneously determining seven active ingredients in cosmetics according to claim 1, characterized in that: The microporous filter membrane in step S3 is a 0.22µm microporous filter membrane.

4. The method for simultaneously determining seven active ingredients in cosmetics according to claim 1, characterized in that: The high performance liquid chromatography detection conditions in step S4 are as follows: Chromatographic column: Reverse-phase C18 column, 5 μm, 4.6 mm × 250 mm; Detector: diode array detector; Column temperature: 30°C; Flow rate: 1.0 mL / min; Injection volume: 10-20 μL; Wavelength: Bisabolol, Madecassoside 205nm; Ectoin, Hydroxytyrosol, Salicin, Oleuropein 220nm; Gentipicroside 270nm; Mobile phase: A-methanol; B : 0.05% phosphoric acid solution; Gradient elution program: 0-3 min, 90% B; 3-6 min, 90%→60% B; 6-9.5 min, 60% B; 9.5~20min, 60%→15%B; 20~30min, 15%B; 30~33min, 15%→90%B; 33~38min, 90%B.

Images