Novel method for quantifying whitening and freckle-removing component peptide anmidol

Abstract

The invention relates to the field of analytical testing, and particularly discloses a novel whitening and freckle-removing component peptomidodol quantitative method which comprises the following steps: (1) drawing a peptomidodol standard curve; (2) preparing a sample solution; and (3) high performance liquid chromatography detection and analysis. According to the present invention, with the liquid chromatography external standard method and the specific pretreatment means, the peptomidogen content in the cream sample can be simply, rapidly and efficiently determined, such that the peak interference problem of various substances is solved, and the quantitative error of the peptomidogen is reduced.

Description

Technical Field

[0001] The invention belongs to the field of analysis and testing, and specifically relates to a quantitative method for a novel whitening and freckle-removing ingredient peptide Anmido. Background Art

[0002] In recent years, with the demand for "whitening", the research on the whitening and freckle removal mechanism, whitening and freckle removal ingredients, whitening and freckle removal effects and the detection of their effective ingredients has been continuously deepened. At present, whitening and freckle removal cosmetics have become an important type of skin care cosmetics. At present, the commonly used whitening active ingredients on the market mainly include those that inhibit melanin production (linoleic acid, glycyrrhizin, resveratrol, kojic acid and its derivatives, arbutin and its derivatives, phenylethyl resorcinol), reduce melanin (vitamin C and its derivatives, oligomeric proanthocyanidins), inhibit melanin operation (niacinamide) and accelerate skin metabolism and stratum corneum shedding (fruit acid, retinoic acid), etc. In order to seize market share, many cosmetics companies at home and abroad attach great importance to the development of whitening and freckle removal cosmetics, and constantly innovate in this field.

[0003] Isobutylamidothiazolylresorcinol, also known as peptide Anmido, is the strongest tyrosinase inhibitor known so far. It can inhibit the formation of pigments from the source, thereby achieving a strong whitening and freckle-removing effect. In my country, freckle-removing and whitening cosmetics are special cosmetics that must be registered with the State Council's drug supervision and administration department before they can be produced and imported. Whether a product belongs to the whitening category is mainly determined by whether the relevant information provided at the time of registration can prove that it has a whitening effect. However, some freckle-removing and whitening products on the market are suspected of false additives, and consumers have doubts about the true content of their active ingredients. Therefore, it is necessary to provide a targeted method to detect the content of peptide Anmido.

[0004] The current national whitening ingredient testing standard is GB / T35954-2018 "Determination of 10 Whitening and Anti-Freckle Agents in Cosmetics-High Performance Liquid Chromatography". Its determination range includes magnesium ascorbyl phosphate, ascorbyl glucoside, β-arbutin, kojic acid, niacinamide, 3-O-ethyl ascorbic acid, potassium methoxysalicylate, raspberry ketone glucoside, dipotassium glycyrrhizate, and 4-butylresorcinol. The determination of peptide Anmido is not mentioned, and there is no clear detection method in the existing literature and patents to determine the content of peptide Anmido.

[0005] At present, major testing institutions mostly use nuclear magnetic resonance internal standards to quantify peptide Anmido, but for peptide Anmido, the detection limit of nuclear magnetic resonance internal standards is relatively high. If the content of peptide Anmido is too low, the test results will be inaccurate; and the various substances contained in skin care products have peak interference during nuclear magnetic resonance quantification, resulting in large errors in the quantification of peptide Anmido; in addition, there is also the question of whether the peptide Anmido in skin care products can be completely dissolved in the solvent. Therefore, there is an urgent need for a quantitative method for peptide Anmido on the market, especially for the quantification of peptide Anmido in cream samples. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a method for quantifying a novel whitening and anti-freckle ingredient, peptide Anmido, which can simply, quickly and efficiently measure the content of peptide Anmido in cream samples through liquid chromatography external standard method and specific pretreatment means.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention provides a quantitative method for a novel whitening and freckle-removing peptide, which specifically comprises the following steps:

[0009] (1) Drawing a standard curve of peptide Anmido: dissolving peptide Anmido with a solvent, passing through a microporous filter membrane to obtain a peptide Anmido standard solution, performing high performance liquid chromatography detection and making a standard curve;

[0010] (2) Preparation of sample solution: vortex the cream sample with the extraction enhancer, saturated sodium chloride solution and n-hexane on a vortex mixer, add methanol after reaching a demulsified state, continue vortexing for 2 to 4 minutes, then ultrasonicate for 15 to 30 minutes, centrifuge at a speed of 9000 to 11000 r / min for 4 to 8 minutes, extract the intermediate layer of clarified liquid, transfer it to an ammonia blowpipe, blow it with nitrogen at 30 to 50°C until it is nearly dry, dissolve it with a solvent, vortex it, and pass it through a microporous filter membrane to obtain a sample solution;

[0011] (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) is dissolved in a mobile phase for elution, and then subjected to high performance liquid chromatography detection to obtain a chromatogram and perform analysis.

[0012] The present invention quantifies the amount of peptide Anmito in cream samples by high performance liquid chromatography. The method has strong applicability and high accuracy and can be applied to the common cream sample systems in the prior art. First, the peptide Anmito is dissolved by a solvent and passed through a microporous filter membrane to obtain a peptide Anmito standard solution, which is then measured on a machine to obtain a peptide Anmito standard curve.

[0013] When preparing the sample solution, the present invention adds an extraction enhancer, combines a saturated sodium chloride solution and n-hexane, destroys the original system of the cream sample, completely separates the water phase and the oil phase, and then adds methanol for vortexing, ultrasound, and centrifugation, so that the peptide Anmido is completely dissolved, thereby reducing the analytical error of the peptide Anmido content in the sample and improving the 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 the methanol to ultrapure water is 1:1.

[0016] The solvent selected in the present invention is a composite solvent, methanol and ultrapure water are specifically selected, and the volume ratio is controlled so that the peak shape of the obtained peptide Anmido is stable and will not shift with changes in concentration, thereby improving data accuracy and obtaining 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:(0.13-0.28).

[0018] The invention improves the extraction effect of peptide Anmido by controlling the mass ratio of cream samples and extraction enhancers, so as to improve 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 tolerant to solvents and has a good filtering effect during the treatment process of the present invention, while having a large flow rate and reducing time costs.

[0023] In some embodiments, in step (2), the preparation method of the extraction enhancer is as follows:

[0024] OP-10 is dissolved in deionized water, and an ammonium persulfate solution is added at 60-80° C., and after stirring for 5-15 minutes, a mixture of a reaction monomer and a cross-linking agent is added, and the mixture is kept warm for 2-5 hours, naturally cooled to room temperature, centrifuged, washed, and vacuum-freeze-dried to constant weight to obtain the extraction enhancer.

[0025] The extraction enhancer provided by the present invention is an emulsion-like substance containing polar groups such as amide and carboxyl, which increases the destructiveness to the original system of cream samples, and is combined with saturated sodium chloride solution and n-hexane to ensure that the intermediate layer clear liquid contains all the peptide Anmido in the sample.

[0026] In some embodiments, the mass ratio of OP-10 to reactive monomer is 1:(45-55).

[0027] OP-10 is an emulsifier in the process of preparing the extraction enhancer. By controlling its mass ratio with the reaction monomer, the formation of a uniform emulsion state is ensured.

[0028] In some embodiments, the reactive monomers include ethyl acrylate, methacrylic acid, and acrylamide.

[0029] Preferably, based on 100 wt%, the reactive monomers include 3 to 7 wt% of ethyl acrylate, 74 to 90 wt% of methacrylic acid and 7 to 20 wt% of acrylamide.

[0030] The present invention ensures the content of polar groups such as amide and carboxyl by controlling the mass ratio of ethyl acrylate, methacrylic acid and acrylamide.

[0031] In some embodiments, in step (3), the mobile phase for on-machine detection comprises potassium dihydrogen phosphate-phosphate buffer solution and methanol.

[0032] Preferably, the pH value of the potassium dihydrogen phosphate-phosphate buffer solution is 2-3.

[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 mode of the HPLC detection is isocratic elution.

[0035] The present invention controls the ratio of the composite mobile phase to facilitate isocratic elution, thereby ensuring that the peptide Anmito in the cream sample of the present invention can sensitively emit a peak.

[0036] In some embodiments, in step (3), the conditions for the HPLC detection are as follows: the chromatographic column is a C18 column, the column temperature is 30-40° C., the injection volume is 40-60 μL, and the flow rate is 0.5-1.5 mL / min.

[0037] In some embodiments, in step (3), the HPLC detection uses a DAD detector with a wavelength of 220 to 260 nm.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention provides a method for quantifying a novel whitening and freckle-removing ingredient, peptide Anmido, which realizes the quantification of peptide Anmido in cream samples by high performance liquid chromatography. Compared with the internal standard error existing in the nuclear magnetic internal standard method, this method is simple, fast, efficient, accurate, and has comprehensive application and strong applicability. It can be applied to the common cream sample systems in the prior art.

[0040] 2. The present invention uses a demulsifier, a saturated sodium chloride solution and n-hexane to pre-treat cream samples, thereby achieving complete extraction of Peptide Anmido. Compared with the integrated area error caused by the overlap of multiple substance peaks during nuclear magnetic resonance quantification, this method solves the peak interference caused by multiple substance components contained in skin care products and reduces the quantitative error of Peptide Anmido.

[0041] 3. The present invention adopts a composite mobile phase of methanol and potassium dihydrogen phosphate for isocratic elution, which helps to increase the accuracy of quantitative detection by high performance liquid chromatography and realizes the qualitative and quantitative simultaneous analysis of multiple components of peptide Anmi. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the HPLC spectrum of 10 ppm peptide Anmido standard solution;

[0043] Figure 2 This is the HPLC spectrum of 50ppm peptide Anmido standard solution;

[0044] Figure 3 This is the HPLC spectrum of 100 ppm peptide Anmido standard solution;

[0045] Figure 4 is the peptide Amito standard curve;

[0046] Figure 5 is the HPLC spectrum of the sample;

[0047] Figure 6 This is the hydrogen NMR spectrum of the sample. DETAILED DESCRIPTION

[0048] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Without departing from the spirit or scope of the present invention, other combinations and various improvements within the concept of the present invention may be carried out. It is worth noting that if the raw materials and manufacturers of the embodiments and comparative examples are not specifically stated, they are 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: Rotary evaporator desktop rotary evaporator R3001;

[0051] HPLC parameters: the chromatographic column was a C18 column, the column temperature was 30 °C, the injection volume was 50 μL, and the elution flow rate was 1 mL / min;

[0052] NMR parameters: H NMR spectrum, radio frequency 600M;

[0053] Preparation Example

[0054] The steps for preparing the extraction enhancer are as follows:

[0055] 0.20g OP-10 was dissolved in 70g deionized water, 10mL of 0.01g / mL ammonium persulfate solution was added at 70°C, and after stirring for 10min, a mixture of 0.5g ethyl acrylate, 8.0g methacrylic acid, 1.25g acrylamide and 4.7g divinylbenzene was added, and the mixture was kept warm for 3h, naturally cooled to room temperature, centrifuged, washed with deionized water, and vacuum freeze-dried to constant weight to obtain an extraction enhancer.

[0056] Example 1

[0057] (1) Draw a standard curve of peptide anmido: dissolve peptide anmido in a mixed solution of methanol and ultrapure water in a volume ratio of 1:1, pass through a 0.2 μm polytetrafluoroethylene microporous filter membrane to obtain peptide anmido standard solutions with concentrations of 10 ppm, 50 ppm and 100 ppm, respectively, and perform high performance liquid chromatography detection to obtain Figure 1 , Figure 2 and Figure 3 ,right Figures 1 to 3 The obtained spectral data were integrated to obtain the standard curve, see Figure 4 ;

[0058] (2) Preparation of sample solution: 5 g of cream sample (commercially available) was mixed with 1 g of extraction enhancer, 50 mL of saturated sodium chloride solution, and 100 mL of n-hexane on a vortex mixer and vortexed. After reaching a demulsified state, 150 mL of methanol was added and vortexed for 3 min. Then, ultrasonication was performed for 20 min, and centrifugation was performed at a speed of 10,000 r / min for 6 min. The intermediate layer of clarified liquid was extracted and transferred to an ammonia blowpipe. Nitrogen was blown to near dryness at 40°C, and dissolved in a mixed solution of methanol and ultrapure water in a volume ratio of 1:1. The mixture was vortexed and filtered through 0.2 μm polytetrafluoroethylene to obtain a sample solution.

[0059] (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) was dissolved in a mobile phase containing 100 mL of potassium dihydrogen phosphate-phosphate buffer solution with a pH value of 2.5 and 300 mL of methanol for elution, and high performance liquid chromatography was performed to obtain a chromatogram, see Figure 5 .

[0060] Depend on Figures 1 to 3 It can be seen from the data that the peak positions of Peptide Anmido are 3.907min, 3.903min and 3.907min respectively, indicating that the solvent selected in the present invention makes the peak model of Peptide Anmido stable and will not shift with changes in concentration, thereby improving data accuracy.

[0061] At the same time, the actual concentrations of 10ppm, 50ppm and 100ppm peptide Anmido standard solutions were 9.6767ppm, 49.3091ppm and 97.1543ppm, respectively. From the corresponding differences, it can be seen that the detection method of the present application is accurate for detecting ultra-low concentrations of peptide Anmido. Figures 1 to 3 Based on Figure 4 The standard curve shown is more accurate.

[0062] Provided by the sample solution Figure 5 It can be seen that the peak position of peptide Anmido is 3.903min, combined with Figures 1 to 3 The peak position in the figure shows that the peak position of Peptide Anmido tested 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: 5 g of cream sample (commercially available) was mixed with 1 g of extraction enhancer, 50 mL of saturated sodium chloride solution, and 100 mL of n-hexane on a vortex mixer and vortexed. After reaching a demulsified state, 150 mL of methanol was added and vortexed for 3 min. Then, ultrasonication was performed for 20 min, and centrifugation was performed at a speed of 10,000 r / min for 6 min. The intermediate layer of clarified liquid was extracted and transferred to an ammonia blowpipe. Nitrogen was blown to near dryness at 40°C, and dissolved in a mixed solution of methanol and ultrapure water in a volume ratio of 1:1. The mixture was vortexed and filtered through 0.2 μm polytetrafluoroethylene to obtain a sample solution.

[0065] (2) Detection and analysis by nuclear magnetic internal standard method: The sample solution obtained in step (1) was dissolved in a mobile phase, the mobile phase was deuterated methanol phase, and nuclear magnetic internal standard method detection was performed at a radio frequency of 600M to obtain a chromatogram, see Figure 6 .

[0066] Depend on Figure 6 It can be seen that when the NMR internal standard method is used for integrated area, there is an error in the size of the integrated area due to the overlap of peaks of multiple substances. Therefore, compared with the NMR internal standard method for qualitative and quantitative analysis, the HPLC method is more accurate in the qualitative and quantitative analysis of peptide Anmido.

[0067] The embodiments described above do not impose any form of limitation on the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, 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 still falls within the scope of the technical solution of the present invention.

Claims

1. A quantitative method for a novel whitening and freckle-removing peptide, characterized in that: The specific steps include: (1) Drawing a standard curve of peptide Anmido: dissolving peptide Anmido with a solvent, passing through a microporous filter membrane to obtain a peptide Anmido standard solution, performing high performance liquid chromatography detection and making a standard curve; (2) Preparation of sample solution: vortex the cream sample with the extraction enhancer, saturated sodium chloride solution and n-hexane on a vortex mixer, add methanol after reaching a demulsified state, continue vortexing for 2 to 4 minutes, then ultrasonicate for 15 to 30 minutes, centrifuge at a speed of 9000 to 11000 r / min for 4 to 8 minutes, extract the intermediate layer of clarified liquid, transfer it to an ammonia blowpipe, blow it with nitrogen at 30 to 50°C until it is nearly dry, dissolve it with a solvent, vortex it, and pass it through a microporous filter membrane to obtain a sample solution; (3) High performance liquid chromatography detection and analysis: The sample solution obtained in step (2) is dissolved in a mobile phase for elution, and then subjected to high performance liquid chromatography detection 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 in 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 acetate fiber.

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° C., stir for 5-15 minutes, then add a mixture of the reaction monomer and the cross-linking agent, keep warm for 2-5 hours, naturally cool to room temperature, centrifuge, wash, and vacuum freeze-dry to constant weight to obtain the extraction enhancer.

6. The quantitative method according to claim 5, characterized in that The mass ratio of the OP-10 to the reaction 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 mode of 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 the HPLC detection are as follows: the chromatographic column is a C18 column, the column temperature is 30-40°C, the injection volume is 40-60 μL, and the flow rate is 0.5-1.5 mL / min.

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