Method for detecting various whitening functional components in cosmetics

Abstract

The invention relates to the technical field of analysis and detection, and discloses a method for detecting various whitening functional components in cosmetics, which comprises the following steps: sequentially adding dichloromethane and water into a to-be-detected sample for extraction, and taking the uppermost layer and the lowermost layer of liquid to obtain an extracting solution; carrying out liquid chromatography separation on the extracting solution according to a specific elution procedure, and collecting eluent; and carrying out diode array detection, series electric fog type detection or evaporative light scattering detection on the eluent. By adopting the method disclosed by the invention, synchronous retention and effective separation of various whitening functional components with remarkable polarity difference in the cosmetics on the chromatographic column can be realized, and detection of components covered with ultraviolet absorption and components without ultraviolet absorption can be realized, so that the method has relatively high detection sensitivity and accuracy, and interference of other components in the cosmetics on detection can be avoided.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method for detecting multiple whitening ingredients in cosmetics. Background Technology

[0002] Whitening cosmetics are one of the core categories in the current cosmetics market. Determining the types and contents of whitening ingredients in whitening cosmetics through chemical analysis provides a scientific basis for evaluating the whitening efficacy and potential allergic risks of the products, which is of great practical significance.

[0003] Existing detection methods for various whitening ingredients in cosmetics can simultaneously retain and effectively separate ingredients with similar polarity (such as arbutin and kojic acid). However, for strongly polar whitening ingredients (such as acetylglucosamine, tranexamic acid, and niacinamide) and weakly polar whitening ingredients (such as phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate), differentiated detection methods are required for separate analysis.

[0004] For example, patent CN112684050A uses high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to simultaneously detect multiple highly polar whitening ingredients, including niacinamide, but it cannot detect weakly polar whitening ingredients. When the polarity of whitening ingredients varies greatly, existing methods cannot achieve simultaneous retention and effective separation of highly polar and weakly polar components on the chromatographic column. Different extraction conditions and chromatographic analysis methods are required for different polarity components, making the operation process cumbersome. In addition, HPLC-MS / MS requires expensive mass spectrometry detectors, making it difficult to widely apply in routine detection. Summary of the Invention

[0005] To address the aforementioned technical problem—namely, the difficulty of achieving simultaneous retention and effective separation of multiple components with significant polarity differences in cosmetics on a chromatographic column using existing technologies—this invention provides a method for detecting multiple whitening functional ingredients in cosmetics. Using this method, simultaneous retention and effective separation of multiple whitening functional ingredients with significant polarity differences in cosmetics can be achieved on a chromatographic column, covering both UV-absorbing and non-UV-absorbing components. It exhibits high detection sensitivity and accuracy and avoids interference from other components in the cosmetics.

[0006] The specific technical solution of this invention is as follows: A method for detecting multiple whitening ingredients in a cosmetic product, comprising the following steps: S1: Add dichloromethane and water to the sample to be tested in sequence for extraction, and take the uppermost and lowermost liquids to obtain the extract; S2: Perform liquid chromatography separation of the extract according to the following elution procedure and collect the eluent: The eluent composition from 0 min to 2-3 min is 94-100% v / v buffer + balance acetonitrile; The eluent composition gradually changes until min 11-13, becoming buffer solution 50-60% v / v + acetonitrile balance; The eluent composition gradually changes until min 15-17, becoming acetonitrile 35-45% v / v + isopropanol balance; The eluent composition gradually changed from 17 to 19 min to 10-20% v / v acetonitrile + balance isopropanol, and remained unchanged until 27 to 29 min; S3: Perform diode array detection, series electro-fogging detection, or evaporative light scattering detection on the eluent.

[0007] In the pretreatment process of the test sample, the present invention uses dichloromethane and water as extractants, which can achieve one-time extraction of hydrophilic (strongly polar) and lipophilic (weakly polar) whitening ingredients (without the need for step extraction and separation of upper and lower liquid layers after each extraction). This simplifies the pretreatment process of the test sample and helps to reduce operational errors. In addition, the extractants in the present invention can also avoid the solvent effect that causes abnormal peak shapes in the chromatogram.

[0008] In the process of liquid chromatography separation of the extract, this invention uses three mobile phases: buffer, acetonitrile, and isopropanol, and performs gradient elution according to a specific elution program. This method can achieve good elution results for whitening ingredients with significant polarity differences (including hydrophilic ingredients such as acetylglucosamine, tranexamic acid, and nicotinamide, as well as lipophilic ingredients such as phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate). Furthermore, each whitening ingredient exhibits high separation degree and specificity, avoiding interference from other components in cosmetics in the detection.

[0009] This invention employs either diode array detection in series with electro-fogging or evaporative light scattering (ELS) for the detection of eluents. Diode array detection provides good detection of skin-whitening ingredients with UV absorption (such as niacinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate), while electro-fogging or ELS can effectively detect skin-whitening ingredients without UV absorption (such as acetylglucosamine and tranexamic acid). Therefore, this invention covers the detection of both UV-absorbing and non-UV-absorbing ingredients, solving the detection blind spot for non-UV-absorbing ingredients, avoiding the high cost of mass spectrometry, and ensuring detection accuracy.

[0010] Through the above methods, this invention employs specific extractants, eluents, and elution procedures, as well as specific eluent detection methods, to achieve efficient detection of various whitening ingredients with significant polarity differences in cosmetics (such as acetyl glucosamine, tranexamic acid, nicotinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate). It has high detection sensitivity and accuracy, and can avoid interference from other components in cosmetics.

[0011] Preferably, the whitening active ingredient includes a hydrophilic component and a lipophilic component; the hydrophilic component includes at least one of acetylglucosamine, tranexamic acid, and niacinamide; the lipophilic component includes at least one of phenylethyl resorcinol, glycyrrhizin, and tetraisopalmitate ascorbate.

[0012] Preferably, in step S2, the conditions for liquid chromatography separation are: HSS T3 column, column temperature 25~45℃, injection volume 1~5μL, and flow rate 0.1~0.5mL / min.

[0013] Using an HSS T3 chromatographic column can achieve good retention and separation of components with significant polarity differences in cosmetics, and the obtained chromatographic peaks have good peak shapes, which helps to improve the sensitivity and accuracy of detection.

[0014] As a preferred option, in step S3: During the diode array detection process, the scanning range is 200~400nm; The conditions for the electro-fogging detection are: atomization temperature 30~50℃, acquisition frequency 2~20Hz, and filtration constant 3.6~5.0; or, the conditions for the evaporative light scattering detection are: gas pressure 35~50psi, drift tube temperature 40~60℃, gain 100~1000, and sprayer mode is cooling.

[0015] Preferably, in the elution procedure of step S2, after 27 to 29 minutes, the eluent composition gradually changes to 94 to 100% v / v buffer + acetonitrile balance.

[0016] Preferably, in the elution process of step S2, the buffer solution is an aqueous solution of ammonium formate or ammonium acetate with a concentration of 0.1~0.2wt%, and the pH value is adjusted to 3.5~4.5 using formic acid or acetic acid.

[0017] As a preferred method, qualitative and / or quantitative analysis of the whitening efficacy components in the sample to be tested is performed based on the detection results obtained in step S3, as follows: Qualitative analysis: The standard working solution was tested according to steps S2 and S3 to determine the retention time of each whitening active ingredient to be tested; based on the retention time of the chromatographic peaks in the test results obtained in step S3, it was determined whether the sample to be tested contained each whitening active ingredient to be tested. Quantitative analysis: The standard working solution was tested according to steps S2 and S3 to obtain the concentration-peak area standard curve of each whitening efficacy ingredient to be tested; based on the peak area of ​​the corresponding chromatographic peak in the test results obtained in step S3, combined with the standard curve, the content of each whitening efficacy ingredient to be tested in the sample was calculated.

[0018] Preferably, in step S3, the eluent is detected using a diode array tandem electro-fogging method; the retention times for each whitening active ingredient to be tested are as follows: Acetyl glucosamine: 1.7~1.9 min; Tranexamic acid: 2.5~2.7 min; Nicotinamide: 3.6~3.9 min; Phenethyl resorcinol: 16.3~17.25 min; Glycyrrhizin: 17.3~18.2 min; Ascorbate tetraisopalmitate: 23.0~24.3 min.

[0019] Preferably, step S1 includes the following steps: adding dichloromethane to the sample to be tested, vortexing and mixing, then adding water, vortexing and mixing again, centrifuging, and filtering the top and bottom layers of liquid to obtain the extract; the filter membrane used in the filtration process has a pore size of 0.1~0.45μm; the vortex mixing time is 30~90s; the centrifugation speed is 5000~10000r / min, and the time is 4~10min.

[0020] Preferably, the cosmetic is a lotion, cream, ointment, or oil.

[0021] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses dichloromethane and water as extractants, which can achieve one-time extraction of hydrophilic whitening ingredients and lipophilic whitening ingredients, while avoiding the abnormal peak shape in the chromatogram caused by solvent effect.

[0022] (2) The present invention uses three mobile phases: buffer, acetonitrile and isopropanol, combined with a specific elution program and chromatographic column design, which can achieve a good separation effect on whitening ingredients with significant polarity differences in cosmetics. It can separate these ingredients from the extract and achieve good separation between these ingredients during the elution process. In addition, it can avoid interference from other ingredients in cosmetics on the detection.

[0023] (3) The present invention uses diode array detection series electro-fogging detection or evaporative light scattering detection to detect the eluent, which can cover the detection of components with and without ultraviolet absorption. This solves the detection blind zone of components without ultraviolet absorption, avoids the high cost of mass spectrometry, and ensures the accuracy of detection. Attached Figure Description

[0024] Figure 1 This is a typical chromatogram of the standard working solution obtained using the diode array detection-electro-fogging detection tandem detection system in Example 1.

[0025] Figure 2 This is a typical chromatogram of acetyl glucosamine and tranexamic acid in the standard working solution obtained using the diode array detection-evaporative light scattering detection tandem detection system in Example 1.

[0026] Figure 3 Chromatograms of acetyl glucosamine and tranexamic acid obtained using a Waters BEH C18 column.

[0027] Figure 4 Chromatograms of acetyl glucosamine, tranexamic acid, phenylethyl resorcinol, and glycyrrhizin obtained using a Waters HILIC column.

[0028] Figure 5 The chromatograms of acetyl glucosamine and tranexamic acid obtained using 0.1 wt% formic acid aqueous solution as mobile phase A are shown.

[0029] Figure 6 The chromatograms of acetyl glucosamine and tranexamic acid obtained using the elution procedure in Table 8 are shown.

[0030] Figure 7 The chromatograms of phenylethyl resorcinol and glycyrrhizin obtained using the elution procedure in Table 9 are shown.

[0031] Figure 8 The recovery rates of six whitening active ingredients were calculated using different extraction agents. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments.

[0033] A method for detecting multiple whitening ingredients in a cosmetic product, comprising the following steps: S1: Add dichloromethane and water to the sample to be tested in sequence for extraction, and take the uppermost and lowermost liquids to obtain the extract; S2: Perform liquid chromatography separation of the extract according to the following elution procedure and collect the eluent: The eluent composition from 0 min to 2-3 min is 94-100% v / v buffer + balance acetonitrile; The eluent composition gradually changes until min 11-13, becoming buffer solution 50-60% v / v + acetonitrile balance; The eluent composition gradually changes until min 15-17, becoming acetonitrile 35-45% v / v + isopropanol balance; The eluent composition gradually changed from 17 to 19 min to 10-20% v / v acetonitrile + balance isopropanol, and remained unchanged until 27 to 29 min; S3: Perform diode array detection, series electro-fogging detection, or evaporative light scattering detection on the eluent.

[0034] In some specific embodiments, the whitening active ingredient includes a hydrophilic component and a lipophilic component; the hydrophilic component includes at least one of acetylglucosamine, tranexamic acid, and niacinamide; the lipophilic component includes at least one of phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate.

[0035] In some specific embodiments, the cosmetic is a lotion, cream, ointment, or oil.

[0036] In some specific embodiments, step S1 includes the following steps: adding dichloromethane to the sample to be tested, vortexing and mixing, then adding water, vortexing and mixing again, centrifuging, and filtering the top and bottom layers of liquid to obtain the extract; the filter membrane used in the filtration process has a pore size of 0.1~0.45μm; the vortex mixing time is 30~90s; the centrifugation speed is 5000~10000r / min, and the time is 4~10min.

[0037] In some specific embodiments, in step S2, the conditions for liquid chromatography separation are: HSS T3 column, column temperature 25~45℃, injection volume 1~5μL, and flow rate 0.1~0.5mL / min.

[0038] In some specific embodiments, during the elution process in step S2, after 27 to 29 minutes, the eluent composition gradually changes to 94 to 100% v / v buffer + the remainder acetonitrile.

[0039] Using the conventional representation of elution procedures in liquid chromatography, the above elution procedures can be summarized in Table 1.

[0040] Table 1 Elution Procedure

[0041] The specific meanings of the elution procedures shown in Table 1 are as follows (the elution procedures are also represented in the same way in the embodiments below): 1) At the beginning (0 min), the eluent used consists of the following components by volume percentage: 94~100% v / v buffer, with the balance being acetonitrile; the eluent composition remains unchanged until the 2nd~3rd min. 2) The composition of the eluent gradually changes until it becomes 50-60% v / v buffer solution, with the remainder being acetonitrile, around min 11-13. 3) The composition of the eluent gradually changes until it becomes 35-45% v / v acetonitrile, with the remainder being isopropanol solution, around the 15th to 17th minute. 4) The eluent composition gradually changes until it becomes 10-20% v / v acetonitrile, with the remainder being isopropanol solution, at 17-19 min; the eluent composition remains unchanged until 27-29 min. 5) The eluent composition gradually changes to: 94~100% v / v buffer solution, with the remainder being acetonitrile solution; the eluent composition remains unchanged thereafter, and the column enters the equilibrium state, with no limit to the equilibration time.

[0042] In some specific embodiments, in the elution process of step S2, the buffer solution is an aqueous solution of ammonium formate or ammonium acetate with a concentration of 0.1~0.2wt%, and the pH value is adjusted to 3.5~4.5 using formic acid or acetic acid.

[0043] In some specific implementations, in step S3: During the diode array detection process, the scanning range is 200~400nm; The conditions for the electro-fogging detection are: atomization temperature 30~50℃, acquisition frequency 2~20Hz, and filtration constant 3.6~5.0; or, the conditions for the evaporative light scattering detection are: gas pressure 35~50psi, drift tube temperature 40~60℃, gain 100~1000, and sprayer mode is cooling.

[0044] In some specific embodiments, the whitening active ingredients in the sample to be tested are qualitatively and / or quantitatively analyzed based on the detection results obtained in step S3, as follows: Qualitative analysis: The standard working solution was tested according to steps S2 and S3 to determine the retention time of each whitening active ingredient to be tested; based on the retention time of the chromatographic peaks in the test results obtained in step S3, it was determined whether the sample to be tested contained each whitening active ingredient to be tested. Quantitative analysis: The standard working solution was tested according to steps S2 and S3 to obtain the concentration-peak area standard curve of each whitening efficacy ingredient to be tested; based on the peak area of ​​the corresponding chromatographic peak in the test results obtained in step S3, combined with the standard curve, the content of each whitening efficacy ingredient to be tested in the sample was calculated.

[0045] In some specific embodiments, step S3 uses diode array detection in series electro-fogging to detect the eluent; the retention times for each whitening active ingredient to be tested are as follows: Acetyl glucosamine: 1.7~1.9 min; Tranexamic acid: 2.5~2.7 min; Nicotinamide: 3.6~3.9 min; Phenethyl resorcinol: 16.3~17.25 min; Glycyrrhizin: 17.3~18.2 min; Ascorbate tetraisopalmitate: 23.0~24.3 min.

[0046] The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0047] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0048] The following provides information on the main instruments and reagents used in the various embodiments and comparative examples, but the scope of protection of this invention is not limited thereto: (1) Information on the main instruments is shown in Table 2: Table 2 Main Instrument Information

[0049] (2) Main reagents and solutions: 1) Deionized water: Deionized water produced by the ELGA flex2 pure water system; 2) Dichloromethane: analytical grade; 3) Acetonitrile: chromatographic grade; 4) Ammonium acetate: chromatographic grade; 5) The CAS numbers of the six whitening ingredients are shown in Table 3.

[0050] Table 3 Information on Whitening Ingredients

[0051] Example 1: Detection method for the sample to be tested The following steps were followed to test the skin-whitening ingredients (acetylglucosamine, tranexamic acid, niacinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate) in the test samples: S1: Prepare single-standard stock solution Accurately weigh 100 mg of acetyl glucosamine, tranexamic acid, and nicotinamide standards into 50 mL volumetric flasks, dissolve them in deionized water, and dilute to the mark to obtain a 2 mg / mL single-standard stock solution.

[0052] Accurately weigh 50 mg of phenylethyl resorcinol and glycyrrhizin standard into 50 mL volumetric flasks, dissolve them in dichloromethane, and dilute to the mark to obtain a 1 mg / mL single standard stock solution.

[0053] Accurately weigh 100 mg of ascorbate tetraisopalmitate standard into a 50 mL volumetric flask, dissolve in dichloromethane, and dilute to the mark to obtain a 2 mg / mL single standard stock solution.

[0054] S2: Preparation of mixed working fluid Accurately pipette 200 μL of acetylglucosamine single-standard stock solution, 200 μL of tranexamic acid single-standard stock solution, and 250 μL of nicotinamide single-standard stock solution into a 1.5 mL vial, and dilute to 1000 μL with deionized water to obtain hydrophilic mixed working solution A, wherein the concentrations of acetylglucosamine, tranexamic acid, and nicotinamide are 400 μg / mL, 400 μg / mL, and 500 μg / mL, respectively.

[0055] Accurately pipette 50 μL of acetylglucosamine single-standard stock solution, 50 μL of tranexamic acid single-standard stock solution, and 50 μL of nicotinamide single-standard stock solution into a 1.5 mL vial, and dilute to 1000 μL with deionized water to obtain hydrophilic mixed working solution B, wherein the concentrations of acetylglucosamine, tranexamic acid, and nicotinamide are 100 μg / mL, 100 μg / mL, and 100 μg / mL, respectively.

[0056] Accurately pipette 200 μL of phenylethyl resorcinol single-standard stock solution, 200 μL of glycyrrhizin single-standard stock solution, and 500 μL of ascorbate tetraisopalmitate single-standard stock solution into a 1.5 mL vial, and dilute to 1000 μL with dichloromethane to obtain lipophilic mixed working solution A, wherein the concentrations of phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate are 200 μg / mL, 200 μg / mL, and 1000 μg / mL, respectively.

[0057] Accurately pipette 20 μL of phenylethyl resorcinol single-standard stock solution, 20 μL of glycyrrhizin single-standard stock solution, and 50 μL of ascorbate tetraisopalmitate single-standard stock solution into a 1.5 mL vial, and dilute to 1000 μL with dichloromethane to obtain lipophilic mixed working solution B, wherein the concentrations of phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate are 20 μg / mL, 20 μg / mL, and 100 μg / mL, respectively.

[0058] S3: Prepare standard working solution Hydrophilic mixed working solution A and hydrophilic mixed working solution B were diluted with deionized water, and lipophilic mixed working solution A and lipophilic mixed working solution B were diluted with dichloromethane to obtain a series of standard working solutions with varying concentrations. The concentrations of each whitening active ingredient in the standard working solutions are as follows: Acetyl glucosamine: 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL; Tranexamic acid: 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL; Nicotinamide: 10 μg / mL, 50 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL; Phenethyl resorcinol: 5 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL; Glycyrrhizin: 5 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL; Ascorbate tetraisopalmitate: 10 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL.

[0059] S4: Standard working solution testing Each standard working solution was analyzed by ultra-high performance liquid chromatography (UPLC). The autosampler of the UPLC system injected 1 μL of the standard working solution into the column for gradient elution. The eluent was then analyzed in the detection system to obtain a chromatogram. The UPLC conditions used in the above process are as follows: a) Column: Waters HSS T3 150×2.1mm 1.7μm; b) Column temperature: 35℃; c) Flow rate: 0.3 mL / min; d) Mobile phase: Mobile phase A is a 20mM ammonium acetate-formic acid aqueous solution with pH=4 (i.e., a 20mM ammonium acetate aqueous solution, with the pH adjusted to 4 using formic acid); Mobile phase B is acetonitrile; Mobile phase C is isopropanol; e) Elution method: gradient elution, the elution procedure is shown in Table 4.

[0060] Table 4 Elution Procedure

[0061] f) The eluent is detected using two detection systems: diode array detection-electro-fogging detection in series and diode array detection-evaporative light scattering detection in series. The diode array detection was performed using a photodiode array detector (PDA), with a scanning range of 200~400nm, and the 220nm, 260nm and 280nm were extracted. Electro-atomization detection was performed using an electro-spray detector (CAD), with an atomization temperature of 35℃, a sampling frequency of 5Hz, and a filtration constant of 5.0. Evaporative light scattering (ELSD) was performed using an evaporative light scattering detector (ELSD), with a gas pressure of 40 psi, a drift tube temperature of 50 °C, a gain of 250, and the sprayer mode set to cooling.

[0062] When using a diode array detection-electro-fogging detection tandem detection system, a typical chromatogram obtained from the standard working solution is as follows: Figure 1 As shown in Table 5 (the concentration of each whitening active ingredient is 50 μg / mL), peak 1 represents acetylglucosamine, peak 2 represents tranexamic acid, peak 3 represents nicotinamide, peak 4 represents phenylethyl resorcinol, peak 5 represents glycyrrhizin, and peak 6 represents tetraisopalmitate ascorbate. Based on the detection results of each standard working solution, the correspondence between each whitening active ingredient and retention time was determined as shown in Table 5 (allowable deviation within ±2.5%). A standard curve (linear regression equation) for each whitening active ingredient was established with concentration as the x-axis and peak area as the y-axis, and the results are shown in Table 6.

[0063] Table 5. Correspondence between various whitening active ingredients and their retention time

[0064] Table 6 Standard curves of various whitening ingredients

[0065] When a diode array detection-evaporative light scattering detection tandem detection system is used, nicotinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate are detected by diode array detection, and the chromatogram is approximately the same as... Figure 1Acetyl glucosamine and tranexamic acid were detected by evaporative light scattering, and typical chromatograms were obtained as follows: Figure 2 As shown (the concentration of each whitening ingredient is 50 μg / mL), peak 1 represents acetyl glucosamine and peak 2 represents tranexamic acid.

[0066] S5: Sample Preprocessing Accurately weigh 0.2g of the sample to be tested (accurate to 0.0001g) and add it to a 50mL plastic centrifuge tube. Use a 10mL pipette to accurately add 10mL of dichloromethane to the centrifuge tube and vortex for 1min to ensure that the sample is fully dispersed, dissolved, and extracted. Then, use a 10mL pipette to accurately add 10mL of deionized water to the centrifuge tube and vortex for 1min to ensure that the sample is fully dispersed, dissolved, and extracted. Centrifuge at 8000r / min for 5min. Filter the upper and lower layers of liquid through a 0.2μm filter membrane. The filtrate obtained is the extract and is ready for use.

[0067] S6: Sample Detection Following the method in step S4, the extract was subjected to ultra-high performance liquid chromatography (UHPLC) (the upper and lower layers of the filtered liquid were injected separately) to obtain a chromatogram. Based on the retention time of each chromatographic peak in the chromatogram, the corresponding whitening active ingredients were determined according to Table 5; based on the peak area corresponding to each whitening active ingredient in the chromatogram, and combined with the standard curve in Table 6, the content of each whitening active ingredient in the sample was calculated.

[0068] Example 2: The Influence of Column Selection on Detection Results Based on the structure and properties of the six whitening active ingredients, three chromatographic columns—Waters BEH C18, Waters HILIC, and Waters HSS T3—were selected as the subjects of investigation. The self-made cosmetic samples were tested according to the method in Example 1 (in this example, a diode array detection-electro-fogging detection tandem detection system was used). The formulas of the self-made cosmetic samples are shown in Table 7 (the formulas of the self-made cosmetic samples shown in Table 7 are only for verifying the feasibility of the whitening active ingredient detection method of this invention and are not intended as cosmetic formulas for production and sale).

[0069] Table 7. Self-made cosmetic sample formula

[0070] Comparing the detection results using three different chromatographic columns, we found that: (1) Acetyl glucosamine and tranexamic acid showed poor retention and low separation on Waters BEH C18 columns, such as Figure 3As shown (the concentrations of acetylglucosamine and tranexamic acid are both 100 μg / mL, peak 1 represents acetylglucosamine, and peak 2 represents tranexamic acid). The reason for this is that the C18 chromatographic column uses high-density bonded octadecyl (C18) ligands, and the surface of the stationary phase is covered by hydrophobic long chains. Polar sites (such as the polar parts of the siloxane skeleton) are masked, making it impossible to form effective interactions with the hydroxyl and carbonyl groups of strongly polar molecules. This results in extremely weak hydrophobic interactions between acetylglucosamine and tranexamic acid, which cannot be effectively retained.

[0071] (2) Acetyl glucosamine and tranexamic acid showed good peak shape and resolution on a Waters HILIC column, but phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate showed poor peak shape, such as... Figure 4 As shown (the concentrations of acetylglucosamine, tranexamic acid, phenylethyl resorcinol, and glycyrrhizin are all 100 μg / mL; peak 1 represents acetylglucosamine, peak 2 represents tranexamic acid, peak 3 represents phenylethyl resorcinol, and peak 4 represents glycyrrhizin), the reason for this is that the HILIC column, as a hydrophilic column, has the opposite retention properties to the C18 column. It has a stronger retention capacity for highly polar substances such as acetylglucosamine and tranexamic acid, and its separation effect is better than that of the reverse-phase C18 column, but its retention and separation of weakly polar substances are poor.

[0072] (3) In summary, Waters HSS T3 exhibits good retention and separation effects on the six whitening active ingredients, such as... Figure 1 As shown. The reasons are as follows: First, the HSS T3 column actively exposes polar sites through a low ligand density design. By reducing the coverage of the octadecyl chain, the polar groups of siloxanes in the stationary phase framework are fully exposed, allowing them to form directional hydrogen bonds and dipole-dipole interactions with the polar groups of strongly polar molecules. This polar interaction directly compensates for the weak hydrophobicity of strongly polar substances, effectively extending their retention time and providing a basis for separation. Second, the HSS T3 column uses short-chain silanes to block unbonded silanol groups. This reduces the non-specific binding of strongly polar molecules to free silanol groups (avoiding peak tailing) without excessively weakening the polar characteristics of the stationary phase, achieving a balance between effective retention and peak shape optimization. Third, HSS... The T3 column has the advantage of 100% aqueous phase compatibility. Under 100% aqueous phase conditions, the hydrophobic long chains of the traditional C18 column will spontaneously aggregate due to "hydrophobic interactions", which will cause the overall structure of the stationary phase to shrink, the pore size to become smaller, or even the pores to close, resulting in stationary phase collapse, which will lead to a decrease in column efficiency or retention time drift. The T3 column, by reducing the density of the bonded phase and end-capping treatment, allows water to enter the pores of the packing material more easily, ensuring stable operation under pure aqueous phase conditions.

[0073] Example 3: The Influence of Mobile Phase Selection on Detection Results A 0.1 wt% formic acid aqueous solution and a 20 mM ammonium acetate-formic acid aqueous solution with pH=4 were used as mobile phase A, respectively, to detect the self-made cosmetic samples according to the method in Example 1 (using a diode array detection-electro-fogging detection tandem detection system; the only difference from Example 1 is the selection of mobile phase A in step S6). Comparing the detection results using the two mobile phases A, it was found that: When a 0.1 wt% formic acid aqueous solution is used as mobile phase A, the separation effect of acetylglucosamine and tranexamic acid is poor, and the peak shape is also poor. Figure 5 As shown (the concentrations of acetylglucosamine and tranexamic acid are both 100 μg / mL, peak 1 represents acetylglucosamine, and peak 2 represents tranexamic acid); when a 20 mM ammonium acetate-formic acid aqueous solution at pH=4 is used as mobile phase A, the separation effect of acetylglucosamine and tranexamic acid is better, and the peak shape is better, such as... Figure 1 As shown. The reason for this is that although a 0.1 wt% formic acid aqueous solution can adjust the pH of the mobile phase through formic acid ionization, formic acid is a weak acid with extremely low buffer capacity. Small changes in the sample matrix or environment during the experiment (such as temperature fluctuations or trace amounts of acid or alkali introduced by the sample) will cause fluctuations in the actual pH of the mobile phase, making the ionization degree of acetylglucosamine and tranexamic acid unstable. This fluctuation in ionization state will lead to unstable polar interaction strength between the two and the stationary phase, reducing the difference in retention time and ultimately decreasing the separation degree. In contrast, the buffer system of 20 mM ammonium acetate-formic acid aqueous solution is more stable. At pH=4, the protonation degree of the two substances may differ, and this difference may precisely amplify the difference in retention behavior, optimizing the separation and peak shape of acetylglucosamine and tranexamic acid.

[0074] In the organic phase of the eluent, acetonitrile + isopropanol (i.e., according to the elution procedure in Table 4) and acetonitrile (i.e., in the elution procedure in Table 4, only mobile phase A and mobile phase B are used, where the volume fraction of mobile phase A is the same as in Table 4, and the volume fraction of mobile phase B is the sum of the volume fractions of mobile phases B and C in Table 4) were used respectively. The self-made cosmetic samples were tested according to the method in Example 1 (using a diode array detection-electro-fogging detection tandem detection system; the only difference from Example 1 is the proportion of mobile phases B and C in step S6). Comparing the detection results under the two schemes, it was found that: When using acetonitrile alone, ascorbic acid tetraisopalmitate cannot be eluted from the column. Using acetonitrile + isopropanol solves this problem. The reason is that ascorbic acid reacts with four palmitic acids, and its relatively weak polarity results in strong retention in a Waters HSS T3 column. Using acetonitrile + isopropanol as the organic phase, due to the weak polarity of isopropanol and its stronger eluting power, disrupts the hydrophobic interaction between ascorbic acid tetraisopalmitate and the column, making it easier to elute.

[0075] Example 4: The effect of elution procedure on detection results The self-made cosmetic samples were tested using the elution procedures shown in Tables 4, 8, and 9, respectively, following the method described in Example 1 (using a diode array detection-electro-fogging detection tandem detection system; the only difference from Example 1 is the elution procedure in step S6). Comparing the test results using the three elution procedures, the following was found: (1) When the elution procedure in Table 8 is used, the separation effect and peak shape of acetylglucosamine and tranexamic acid are relatively poor, such as Figure 6 As shown (the concentrations of acetylglucosamine and tranexamic acid are both 100 μg / mL, peak 1 represents acetylglucosamine, and peak 2 represents tranexamic acid); and ascorbic acid tetraisopalmitate could not be effectively eluted from the column. The reason for this is that: 95% v / v mobile phase A + 5% v / v mobile phase B still has a relatively strong eluting ability for both acetylglucosamine and tranexamic acid, resulting in a relatively weak separation effect; 35% v / v mobile phase B + 65% v / v mobile phase C has insufficient eluting ability against ascorbic acid tetraisopalmitate.

[0076] (2) When using the elution procedure in Table 9, the matrix interference in the chromatograms of phenylethyl resorcinol and glycyrrhizin is strong, making analysis difficult, such as Figure 7 As shown (the concentrations of phenylethyl resorcinol and glycyrrhizin are both 100 μg / mL, peak 4 represents phenylethyl resorcinol, and peak 5 represents glycyrrhizin). The reason for this is that the abrupt change in the gradient at 12-14 min triggered co-elution of phenylethyl resorcinol and glycyrrhizin with other interfering components in the cosmetic.

[0077] (3) When the elution procedure in Table 4 is used, the elution and separation of the six whitening active ingredients are better, such as Figure 1As shown in Table 4, the initial conditions of 100% v / v mobile phase A in Table 9 were continued, but the high aqueous phase retention time was adjusted to 2.5 min to ensure sufficient retention of acetylglucosamine and tranexamic acid. Secondly, the 12th to 16th min period was set as a gentle gradient transition zone, with the mobile phase gradually switching from 55% v / v A + 45% v / v B to 0% v / v A + 40% v / v B + 60% v / v C. The gradient change rate was reduced to 13.75% / min (only 50% of that in Table 9), providing a differentiated retention window for moderately polar components. Thirdly, considering the strong lipophilicity of ascorbic acid tetraisopalmitate, Table 4 later used 15% v / v B + 85% v / v B. A high-proportion isopropanol system, maintained for 28 minutes, allows the strong hydrophobic effect of isopropanol to effectively compete for the C18 ligand binding sites on the stationary phase, completely desorbing residual lipid-soluble molecules. Extending the elution time (28 minutes) with a high proportion of isopropanol ensures no component residue, avoids cross-contamination in subsequent runs, and produces sharp, symmetrical peaks. In summary, through slow transitions and precise switching of the elution procedure, the six whitening components (strong polarity → moderate polarity → lipid-soluble) were eluted in an orderly manner according to theoretical retention rules, ultimately achieving a resolution >1.5 and a peak symmetry factor of 0.9–1.1.

[0078] Table 8 Elution Procedure

[0079] Table 9 Elution Procedure

[0080] Example 5: The effect of the choice of pretreatment extractant on the detection effect The self-made cosmetic samples were analyzed using three extraction solvents: a 1:1 dichloromethane:water mixture (as per Example 1), a 1:1 methanol:water mixture (replacing the dichloromethane in step S5 of Example 1 with an equal volume of methanol), and anhydrous ethanol (replacing both dichloromethane and deionized water in step S5 of Example 1 with an equal volume of anhydrous ethanol). The analysis was performed using the method described in Example 1 (a diode array detection-electro-fogging detection tandem detection system was employed; the only difference from Example 1 was the choice of extraction solvent in step S5). The recoveries using the three extraction solvents are shown in [Figure 1]. Figure 8 From this, we can see that: (1) When a 1:1 dichloromethane:water ratio was used as the extractant, the recovery rates of the six whitening ingredients were all above 95% (among which acetylglucosamine, tranexamic acid and nicotinamide were analyzed by injection into the upper aqueous phase solution, while phenylethyl resorcinol, glycyrrhizin and ascorbate tetraisopalmitate were analyzed by injection into the lower dichloromethane solution).

[0081] (2) When a 1:1 methanol:water ratio was used as the extractant, the recovery rates of acetylglucosamine, tranexamic acid, and nicotinamide were above 95%, but the recovery rates of phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate were poor. The reason for this is that phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate are weakly polar and have low solubility in methanol-water solution.

[0082] (3) When anhydrous ethanol is used as the extractant, the overall recovery rate is not high. The reasons are as follows: for strongly polar substances, the solvent effect may lead to poor chromatographic peak shape, which in turn affects the recovery rate; for weakly polar substances, the recovery rate is low, which may be related to the solubility of the compound in ethanol.

[0083] Example 6: Sensitivity Test The sensitivity was verified using the detection method in Example 1 (using a diode array detection-electro-fog detection series detection system), and the detection limits and quantitation limits of each whitening ingredient were obtained as shown in Table 10.

[0084] Table 10 Sensitivity Test Results

[0085] Example 7: Spiked Recovery Test The detection method described in Example 1 (using a diode array detection-electro-fog detection tandem detection system) was used for spiked recovery verification. The specific operation steps are as follows: Three types of commercially available cosmetic samples (lotion, cream, and oil) that tested negative (without detection of acetylglucosamine, tranexamic acid, niacinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate) were used as the matrix for spiked analysis. The spiked amounts of acetylglucosamine, tranexamic acid, and niacinamide were 0.25 μg / g, 0.5 μg / g, and 2.5 μg / g, respectively. The spiked amounts of phenylethyl resorcinol, glycyrrhizin, and ascorbate tetraisopalmitate were 0.125 μg / g, 0.25 μg / g, and 1.25 μg / g, respectively. The spiked cosmetics were used as test samples and tested according to the method in Example 1. Six parallel tests were conducted, and the spiked recovery rate and relative deviation (RSD) of each whitening ingredient were calculated. The results are shown in Table 11. The low, medium and high spiked recovery rates of the six whitening ingredients were between 93% and 110%, and the relative deviation of the six parallel tests was between 0.10% and 5.45%.

[0086] Table 11 Spiked Recovery Test Results

[0087] Example 8: Precision Test The precision was verified using the detection method in Example 1 (using a diode array detection-electro-fog detection tandem detection system). The specific operation steps are as follows: A mixed standard solution containing acetyl glucosamine, tranexamic acid, nicotinamide, phenylethyl resorcinol, glycyrrhizin, and ascorbic acid tetraisopalmitate was prepared, wherein the concentration of each whitening active ingredient was 50 μg / mL. This solution was used as the test sample and detected according to the method in Example 1. Six consecutive injections were performed, and the relative deviation (RSD) was calculated. The results are shown in Table 12. The relative deviation ranged from 0.92% to 2.21%, indicating that the method of the present invention has good stability.

[0088] Table 12 Precision Test Results

[0089] Example 9: Actual Sample Testing Following the method described in Example 1 (using a diode array detection-electro-fog detection series detection system), commercially available emulsion, cream, and oil-based cosmetics (sunscreen, essential oil, serum, and facial mask) were tested. The test results of the whitening ingredients contained in the back labels of the cosmetics are shown in Table 13.

[0090] Table 13 Actual Sample Test Results

Claims

1. A method for detecting multiple whitening ingredients in cosmetics, characterized in that the steps include... include: S1: Add dichloromethane and water to the sample to be tested in sequence for extraction, and take the uppermost and lowermost liquids to obtain the extract; S2: Perform liquid chromatography separation of the extract according to the following elution procedure and collect the eluent: The eluent composition from 0 min to 2-3 min is 94-100% v / v buffer + balance acetonitrile; The eluent composition gradually changes until min 11-13, becoming buffer solution 50-60% v / v + acetonitrile balance; The eluent composition gradually changes until min 15-17, becoming acetonitrile 35-45% v / v + isopropanol balance; The eluent composition gradually changed from 17 to 19 min to 10-20% v / v acetonitrile + balance isopropanol, and remained unchanged until 27 to 29 min; S3: Perform diode array detection, series electro-fogging detection, or evaporative light scattering detection on the eluent.

2. The detection method according to claim 1, characterized in that, The whitening ingredients include hydrophilic and lipophilic components; the hydrophilic components include at least one of acetylglucosamine, tranexamic acid, and niacinamide; the lipophilic components include at least one of phenylethyl resorcinol, glycyrrhizin, and tetraisopalmitate ascorbate.

3. The detection method according to claim 1, characterized in that, In step S2, the conditions for liquid chromatography separation are: HSS T3 column, column temperature 25~45℃, injection volume 1~5μL, and flow rate 0.1~0.5mL / min.

4. The detection method according to claim 1, characterized in that, In step S3: During the diode array detection process, the scanning range is 200~400nm; The conditions for the electro-fogging detection are: atomization temperature 30~50℃, acquisition frequency 2~20Hz, and filtration constant 3.6~5.0; or, the conditions for the evaporative light scattering detection are: gas pressure 35~50psi, drift tube temperature 40~60℃, gain 100~1000, and sprayer mode is cooling.

5. The detection method according to claim 1, characterized in that, In the elution procedure of step S2, after 27 to 29 minutes, the eluent composition gradually changes to 94 to 100% v / v buffer + the remainder acetonitrile.

6. The detection method according to claim 1 or 5, characterized in that, In the elution process of step S2, the buffer solution is an aqueous solution of ammonium formate or ammonium acetate with a concentration of 0.1~0.2wt%, and the pH value is adjusted to 3.5~4.5 using formic acid or acetic acid.

7. The detection method according to claim 1, characterized in that, Based on the detection results obtained in step S3, qualitative and / or quantitative analysis of the whitening efficacy components in the sample to be tested is performed, as follows: Qualitative analysis: The standard working solution was tested according to steps S2 and S3 to determine the retention time of each whitening active ingredient to be tested; based on the retention time of the chromatographic peaks in the test results obtained in step S3, it was determined whether the sample to be tested contained each whitening active ingredient to be tested. Quantitative analysis: The standard working solution was tested according to steps S2 and S3 to obtain the concentration-peak area standard curve of each whitening efficacy ingredient to be tested; based on the peak area of ​​the corresponding chromatographic peak in the test results obtained in step S3, combined with the standard curve, the content of each whitening efficacy ingredient to be tested in the sample was calculated.

8. The detection method according to claim 7, characterized in that, In step S3, the eluent is detected using a diode array followed by a series electro-fogging method; the retention times for each whitening active ingredient to be tested are as follows: Acetyl glucosamine: 1.7~1.9 min; Tranexamic acid: 2.5~2.7 min; Nicotinamide: 3.6~3.9 min; Phenethyl resorcinol: 16.3~17.25 min; Glycyrrhizin: 17.3~18.2 min; Ascorbate tetraisopalmitate: 23.0~24.3 min.

9. The detection method according to claim 1, characterized in that, The specific process of step S1 includes: adding dichloromethane to the sample to be tested, vortexing and mixing, then adding water, vortexing and mixing again, centrifuging, and filtering the top and bottom layers of liquid to obtain the extract; during the filtration process, the filter membrane pore size is 0.1~0.45μm; the vortex mixing time is 30~90s; the centrifugation speed is 5000~10000r / min, and the time is 4~10min.

10. The detection method according to claim 1, characterized in that, The cosmetic product is a lotion, cream, ointment, or oil.

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