Novel quantitative method for whitening and freckle-removing ingredient thiamidol
By using external standard liquid chromatography and specific pretreatment methods, the error problem in the quantitative determination of peptide amantadine in creams and ointments was solved, achieving efficient and accurate detection of peptide amantadine.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Filing Date
- 2025-04-22
- Publication Date
- 2026-07-09
AI Technical Summary
Existing technologies lack effective methods for quantifying peptides, especially for detecting peptide content in creams and ointments. The detection limit of NMR internal standard method is high and has errors, and the peaks of various substances cause serious interference.
The peptide Amidato was detected by high performance liquid chromatography using external standard method combined with specific pretreatment techniques, including extraction enhancer, saturated sodium chloride solution and n-hexane demulsification, and isocratic elution with a composite mobile phase of methanol and potassium dihydrogen phosphate.
This method enables efficient and accurate quantification of peptide amectoide in creams and ointments, reducing errors and improving the accuracy and applicability of the detection.
Smart Images
Figure CN2025090502_09072026_PF_FP_ABST
Abstract
Description
A quantitative method for a novel skin-whitening and spot-removing ingredient, peptide Anmido. Technical Field
[0001] This invention belongs to the field of analytical testing, specifically relating to a quantitative method for a novel skin-whitening and spot-removing ingredient, peptide Anmido. Background Technology
[0002] In recent years, with the increasing demand for "skin whitening," research on the mechanisms, ingredients, effects, and efficacy of skin whitening and freckle-removing products has been deepening. Currently, skin whitening and freckle-removing cosmetics have become an important category of skincare products. Commonly used skin whitening active ingredients on the market include those that inhibit melanin production (linoleic acid, glycyrrhizin, resveratrol, kojic acid and its derivatives, arbutin and its derivatives, phenylethyl resorcinol), those that reduce melanin (vitamin C and its derivatives, oligomeric proanthocyanidins), those that inhibit melanin transport (niacinamide), and those that accelerate skin metabolism and stratum corneum exfoliation (fruit acids, retinoic acid). To seize market share, many domestic and international cosmetic companies highly value the development of skin whitening and freckle-removing cosmetics, constantly innovating in this field.
[0003] Isobutylamidothiazolysorcinol, also known as Peptide Anmido, is currently the most potent known tyrosinase inhibitor. It can inhibit pigment formation at its source, thus achieving a strong whitening and spot-fading effect. In my country, whitening and spot-fading cosmetics are classified as special cosmetics and must be registered with the State Council's drug regulatory department before they can be produced or imported. Whether a product qualifies as a whitening product is primarily determined by whether the relevant information provided during registration can prove its whitening effect. However, some whitening and spot-fading products on the market are suspected of containing false additives, and consumers have doubts about the true content of their effective ingredients. Therefore, a targeted method is needed to test the content of Peptide Anmido.
[0004] The current national standard for testing whitening ingredients is GB / T35954—2018 "Determination of 10 Whitening and Spot-Removing Agents in Cosmetics - High Performance Liquid Chromatography". Its testing range includes magnesium ascorbate phosphate, ascorbate glucoside, β-arbutin, kojic acid, nicotinamide, 3-O-ethyl ascorbic acid, potassium methoxysalicylate, raspberry ketone glucoside, dipotassium glycyrrhizate, and 4-butylresorcinol. However, it does not mention the determination of peptide amido, and there is no clear detection method in existing literature and patents to determine the content of peptide amido.
[0005] Currently, most testing institutions use NMR internal standards for quantifying peptide amantadine. However, the detection limit of NMR internal standards for peptide amantadine is relatively high. If the peptide amantadine content is too low, the accuracy of the test results will be poor. Furthermore, various components in skincare products can cause peak interference during NMR quantification, leading to significant errors in the quantification of peptide amantadine. In addition, there is the issue of whether peptide amantadine in skincare products can be completely dissolved in solvents. Therefore, there is an urgent need for a quantitative method for peptide amantadine, especially for quantifying peptide amantadine in creams and ointments. Summary of the Invention
[0006] To overcome the shortcomings of the prior art, this invention provides a novel quantitative method for peptide Anmido, a whitening and freckle-removing ingredient. By using liquid chromatography with external standard method and specific pretreatment techniques, the content of peptide Anmido in cream samples can be determined simply, quickly, and efficiently.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] This invention provides a quantitative method for a novel skin-whitening and spot-removing ingredient, peptide Anmido, which specifically includes the following steps:
[0009] (1) Peptidone standard curve: Peptidone was dissolved in solvent, filtered through a microporous membrane to obtain a Peptidone standard solution, and detected by high performance liquid chromatography to prepare a standard curve;
[0010] (2) Preparation of sample solution: The cream sample, extractant, saturated sodium chloride solution and n-hexane are vortexed on a vortex mixer. After the demulsification state is reached, methanol is added and vortexing is continued for 2-4 min. Then, the mixture is sonicated for 15-30 min and centrifuged at 9000-11000 r / min for 4-8 min. The intermediate clear liquid is extracted, transferred to an ammonia blow-off tube, and blown with nitrogen at 30-50℃ until nearly dry. The liquid is dissolved in solvent, vortexed, and filtered through a microporous membrane to obtain the sample solution.
[0011] (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) is dissolved in the mobile phase and eluted, and high performance liquid chromatography is performed to obtain a chromatogram and perform analysis.
[0012] This invention quantifies peptide amantadine in creams and ointments using high performance liquid chromatography (HPLC). This method is highly applicable and accurate, and can be used for common cream and ointment sample systems in the prior art. First, peptide amantadine is dissolved in a solvent and filtered through a microporous membrane to obtain a peptide amantadine standard solution. Then, it is analyzed by HPLC to obtain a peptide amantadine standard curve.
[0013] In preparing the sample solution, this invention adds an extraction enhancer, combined with saturated sodium chloride solution and n-hexane, to disrupt the original system of cream-type samples, completely separating the aqueous and oil phases. Then, methanol is added for vortexing, sonication, and centrifugation, ensuring complete dissolution of peptide amantadine. This reduces analytical errors in the peptide amantadine content of the sample and improves data accuracy.
[0014] In some embodiments, in steps (1) and (2), the solvent is a mixed solution of methanol and ultrapure water in a volume ratio of 1:(0.25-3).
[0015] Preferably, the volume ratio of methanol to ultrapure water is 1:1.
[0016] The solvent used in this invention is a composite solvent, specifically methanol and ultrapure water, and the volume ratio is controlled to ensure that the peak shape of the obtained peptide Ammido is stable and does not shift with changes in concentration, thereby improving data accuracy and determining the most suitable peak ratio.
[0017] In some embodiments, in step (2), the mass ratio of the cream sample to the extraction enhancer is 1:(0.13 to 0.28).
[0018] This invention improves the extraction effect of peptide antacid by controlling the mass ratio of cream samples to extraction enhancers, thereby improving data accuracy.
[0019] In some embodiments, in step (2), the volume ratio of the saturated sodium chloride solution, n-hexane, and methanol is 1:(0.5-4):(1-4).
[0020] In some embodiments, the material of the microporous filter membrane is any one of polyethersulfone, polytetrafluoroethylene, and cellulose acetate.
[0021] Preferably, the material of the microporous filter membrane is polytetrafluoroethylene.
[0022] The present invention preferably uses polytetrafluoroethylene as the filter membrane material, which is resistant to solvents and has a good filtration effect in the process of the present invention, while having a large flow rate and reducing time costs.
[0023] In some embodiments, the preparation method of the extraction enhancer in step (2) is as follows:
[0024] Dissolve OP-10 in deionized water, add ammonium persulfate solution at 60-80℃, stir for 5-15 min, then add a mixture of reactant monomer and crosslinking agent, keep warm for 2-5 h, cool naturally to room temperature, centrifuge, wash, and freeze dry under vacuum to constant weight to obtain the extraction enhancer.
[0025] The extraction enhancer provided by this invention is an emulsion containing polar groups such as amide and carboxyl groups, which increases its destructive effect on the original system of cream-type samples. Combined with saturated sodium chloride solution and n-hexane, it ensures that the intermediate clear liquid contains all the peptides of the sample.
[0026] In some embodiments, the mass ratio of OP-10 to the reactant monomer is 1:(45-55).
[0027] OP-10 is an emulsifier used in the preparation of extraction enhancers. By controlling its mass ratio with the reactants, a uniform emulsion state is ensured.
[0028] In some embodiments, the reactive monomers comprise ethyl acrylate, methacrylic acid, and acrylamide.
[0029] Preferably, the reactive monomers comprise 3-7 wt% ethyl acrylate, 74-90 wt% methacrylic acid, and 7-20 wt% acrylamide, based on 100 wt%.
[0030] This invention ensures the content of polar groups such as amide and carboxyl groups by controlling the mass ratio of ethyl acrylate, methacrylic acid and acrylamide.
[0031] In some embodiments, in step (3), the mobile phase for the instrumental test comprises potassium dihydrogen phosphate-phosphate buffer solution and methanol.
[0032] Preferably, the pH value of the potassium dihydrogen phosphate-phosphate buffer solution is 2 to 3.
[0033] Preferably, the volume ratio of the potassium dihydrogen phosphate-phosphate buffer solution to methanol is 1:(2-4).
[0034] In some embodiments, the elution method for the high performance liquid chromatography detection is isocratic elution.
[0035] This invention facilitates isocratic elution by controlling the ratio of the composite mobile phase, ensuring that the peptides in the creams and ointments described in this invention can be sensitively eluted.
[0036] In some embodiments, the conditions for high performance liquid chromatography detection in step (3) are as follows: the chromatographic column is a C18 column, the column temperature is 30-40℃, the injection volume is 40-60μL, and the flow rate is 0.5-1.5mL / min.
[0037] In some embodiments, in step (3), the high performance liquid chromatography detection is performed using a DAD detector with a wavelength of 220–260 nm.
[0038] Compared with the prior art, the present invention has the following beneficial effects:
[0039] 1. This invention provides a novel quantitative method for peptide Anmido, a whitening and freckle-removing ingredient. The method achieves quantitative analysis of peptide Anmido in cream samples using high performance liquid chromatography. Compared with the internal standard method of nuclear magnetic resonance, which has errors due to the addition of internal standard, this method is simple, fast, efficient, accurate, and has comprehensive applications and strong applicability. It can be applied to common cream sample systems in the prior art.
[0040] 2. This invention uses demulsifier, saturated sodium chloride solution and n-hexane to pretreat cream samples, achieving complete extraction of peptide antacid. Compared with the integration area error caused by the overlap of multiple substance peaks when performing quantitative NMR, this method solves the peak interference caused by multiple substances in skin care products and reduces the quantitative error of peptide antacid.
[0041] 3. This invention uses a composite mobile phase of methanol and potassium dihydrogen phosphate for isocratic elution, which helps to increase the accuracy of high performance liquid chromatography detection and quantification, and realizes the qualitative and simultaneous quantitative analysis of multiple components of peptide antacid. Attached Figure Description
[0042] Figure 1 shows the HPLC chromatogram of a 10 ppm peptide ampicillin standard solution;
[0043] Figure 2 shows the HPLC chromatogram of a 50 ppm peptide ampicillin standard solution;
[0044] Figure 3 shows the HPLC chromatogram of a 100 ppm peptide ampicillin standard solution;
[0045] Figure 4 shows the standard curve of peptide Amex;
[0046] Figure 5 shows the HPLC chromatogram of the sample;
[0047] Figure 6 shows the hydrogen nuclear magnetic resonance spectrum of the sample. Detailed Implementation
[0048] The present invention will be described below with reference to specific implementation schemes. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope. It is worth noting that unless otherwise specified, the raw materials and manufacturers used in the embodiments and comparative examples represent those purchased from any commercially available manufacturer.
[0049] In the description of the embodiments and comparative examples of the present invention, the instruments involved are described as follows:
[0050] Rotary evaporator parameters: R3001 benchtop rotary evaporator;
[0051] HPLC parameters: C18 column, column temperature 30℃, injection volume 50μL, elution flow rate 1mL / min;
[0052] NMR parameters: 1H NMR spectrum, radio frequency 600M.
[0053] Preparation Example
[0054] The preparation steps of the extraction enhancer are as follows:
[0055] Dissolve 0.20g of OP-10 in 70g of deionized water, add 10mL of 0.01g / mL ammonium persulfate solution at 70℃, stir for 10min, then add a mixture of 0.5g of ethyl acrylate, 8.0g of methacrylic acid, 1.25g of acrylamide and 4.7g of divinylbenzene, keep warm for 3h, cool naturally to room temperature, centrifuge, wash with deionized water, and freeze dry under vacuum to constant weight to obtain the extraction enhancer.
[0056] Example 1
[0057] (1) Peptidone standard curve: Peptidone was dissolved in a 1:1 mixture of methanol and ultrapure water and filtered through a 0.2 μm polytetrafluoroethylene microporous membrane to obtain Peptidone standard solutions with concentrations of 10 ppm, 50 ppm and 100 ppm. High performance liquid chromatography was performed to detect the solutions, and Figures 1, 2 and 3 were obtained respectively. The spectral data obtained in Figures 1 to 3 were integrated to obtain the standard curve, as shown in Figure 4.
[0058] (2) Preparation of sample solution: 5g of ointment sample (commercially available), 1g of extraction enhancer, 50mL of saturated sodium chloride solution, and 100mL of n-hexane were vortexed on a vortex mixer until the emulsion was broken. Then 150mL of methanol was added and vortexed for 3 min. Then, the mixture was sonicated for 20 min and centrifuged at 10000 r / min for 6 min. The clear intermediate layer was extracted and transferred to an ammonia blow-off tube. The mixture was blown with nitrogen at 40℃ until nearly dry. The solution was dissolved in a 1:1 mixture of methanol and ultrapure water, vortexed, and passed through 0.2μm polytetrafluoroethylene to obtain the sample solution.
[0059] (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) was dissolved in the mobile phase for elution. The mobile phase contained 100 mL of potassium dihydrogen phosphate-phosphate buffer solution with a pH of 2.5 and 300 mL of methanol. High performance liquid chromatography detection was performed to obtain the chromatogram, as shown in Figure 5.
[0060] As shown in Figures 1-3, the peak positions of peptide amantadine are 3.907 min, 3.903 min, and 3.907 min, respectively. This indicates that the solvent used in this invention makes the peak pattern of the obtained peptide amantadine stable and does not shift with changes in concentration, thus improving the accuracy of the data.
[0061] Meanwhile, the actual concentrations of the 10ppm, 50ppm and 100ppm peptide-amidad standard solutions were 9.6767ppm, 49.3091ppm and 97.1543ppm, respectively. The corresponding differences show that the detection method of this application is accurate for detecting ultra-low concentrations of peptide-amidad. Therefore, the standard curve shown in Figure 4, which is based on Figures 1 to 3, is more accurate.
[0062] As shown in Figure 5 provided by the sample solution, the peak position of Peptide Anmido is 3.903 min. Combined with the peak positions in Figures 1-3, this indicates that the peak position of Peptide Anmido obtained by this method is stable and completely extracted, which solves the peak interference caused by various substances contained in skin care products and reduces the quantitative error of Peptide Anmido.
[0063] Example 2
[0064] (1) Preparation of sample solution: 5g of cream sample (commercially available), 1g of extraction enhancer, 50mL of saturated sodium chloride solution, and 100mL of n-hexane were vortexed on a vortex mixer until the emulsion was broken. Then 150mL of methanol was added and vortexed for 3 min. Then, the mixture was sonicated for 20 min and centrifuged at 10000 r / min for 6 min. The intermediate clear liquid was extracted and transferred to an ammonia blow-off tube. The mixture was blown with nitrogen at 40℃ until nearly dry. The solution was dissolved in a 1:1 mixture of methanol and ultrapure water, vortexed, and passed through 0.2μm polytetrafluoroethylene to obtain the sample solution.
[0065] (2) NMR internal standard method detection and analysis: The sample solution obtained in step (1) was dissolved in the mobile phase for elution. The mobile phase was deuterated methanol. The NMR internal standard method was used for detection. The radio frequency was 600M. The chromatogram was obtained, as shown in Figure 6.
[0066] As shown in Figure 6, when integrating the area using the NMR internal standard method, the overlapping of peaks from multiple substances leads to errors in the size of the integrated area. Therefore, compared to the NMR internal standard method for qualitative and quantitative analysis, the HPLC method is more accurate for qualitative and quantitative analysis of peptide Ammido.
[0067] The embodiments described above do not limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A quantitative method for a novel skin-whitening and freckle-removing ingredient, peptide Anmido, characterized in that, Specifically, it includes the following steps: (1) Peptidone standard curve: Peptidone was dissolved in solvent, filtered through a microporous membrane to obtain a Peptidone standard solution, and detected by high performance liquid chromatography to prepare a standard curve; (2) Preparation of sample solution: The cream sample, extractant, saturated sodium chloride solution and n-hexane are vortexed on a vortex mixer. After the demulsification state is reached, methanol is added and vortexing is continued for 2-4 min. Then, the mixture is sonicated for 15-30 min and centrifuged at 9000-11000 r / min for 4-8 min. The intermediate clear liquid is extracted, transferred to an ammonia blow-off tube, and blown with nitrogen at 30-50℃ until nearly dry. The liquid is dissolved in solvent, vortexed, and filtered through a microporous membrane to obtain the sample solution. (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) is dissolved in the mobile phase and eluted, and high performance liquid chromatography is performed to obtain a chromatogram and perform analysis.
2. The quantitative method according to claim 1, characterized in that, In steps (1) and (2), the solvent is a mixed solution of methanol and ultrapure water with a volume ratio of 1:(0.25~3).
3. The quantitative method according to claim 1, characterized in that, In step (2), the mass ratio of the cream sample to the extraction enhancer is 1:(0.13-0.28).
4. The quantitative method according to claim 1, characterized in that, In steps (1) and (2), the material of the microporous filter membrane is any one of polyethersulfone, polytetrafluoroethylene, and cellulose acetate.
5. The quantitative method according to claim 1, characterized in that, In step (2), the preparation method of the extraction enhancer is as follows: Dissolve OP-10 in deionized water, add ammonium persulfate solution at 60-80℃, stir for 5-15 min, add a mixture of reaction monomer and crosslinking agent, keep warm for 2-5 h, cool naturally to room temperature, centrifuge, wash, and freeze dry under vacuum to constant weight to obtain the extraction enhancer.
6. The quantitative method according to claim 5, characterized in that, The mass ratio of OP-10 to the reactant monomer is 1:(45-55).
7. The quantitative method according to claim 5, characterized in that, The reactive monomers include ethyl acrylate, methacrylic acid, and acrylamide.
8. The quantitative method according to claim 1, characterized in that, In step (3), the mobile phase comprises potassium dihydrogen phosphate buffer solution and methanol.
9. The quantitative method according to claim 1, characterized in that, The elution method used for the high-performance liquid chromatography detection is isocratic elution.
10. The quantitative method according to claim 1, characterized in that, In step (3), the conditions for high performance liquid chromatography detection are as follows: the chromatographic column is a C18 column, the column temperature is 30-40℃, the injection volume is 40-60μL, and the flow rate is 0.5-1.5mL / min.