A method for detecting copper peptides, hexapeptide-9 and biotinoyl tripeptide-1 in cosmetics

Abstract

The application discloses a method for detecting copper peptide, hexapeptide-9 and biotinoyl tripeptide-1 in cosmetics, which comprises the following steps: step one, weighing three standard samples, respectively dissolving in water, and preparing standard stock solutions; weighing a blank sample, dissolving in water, and preparing a blank matrix extract solution; preparing a matrix standard intermediate solution; preparing a matrix standard series solution; step two, precisely weighing a sample, and preparing a sample solution; step three, detecting by using a high performance liquid chromatography-mass spectrometry system. The application first proposes a method for simultaneously detecting copper peptide, hexapeptide-9 and biotinoyl tripeptide-1 in cosmetics, and can detect copper peptide, hexapeptide-9 and biotinoyl tripeptide-1 raw materials in cream, emulsion, water agent, gel, oil base and freeze-dried base cosmetics, and the method is simple in operation, rapid in analysis, strong in specificity, high in separation degree, and is beneficial to quickly responding to supervision of copper peptide, hexapeptide-9 and biotinoyl tripeptide-1 component addition in existing market products.

Description

Technical Field

[0001] This invention relates to the field of detection technology for copper peptides, hexapeptide-9, and biotinylate tripeptide-1, and particularly to a method for detecting copper peptides, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics. Background Technology

[0002] Peptides are a class of compounds formed by amino acids linked by peptide bonds; they are protein fragments with biological functions. Due to the diversity of amino acid types and the high degree of freedom in the spatial conformation of peptide chains, peptides possess a wide range of biological effects. The development and application of bioactive peptides have opened up a new field for personal care products.

[0003] Copper glycyl-histidine-tripeptide (GHK-Cu) is a compound formed by the combination of glycyl histidine tripeptide (GHK) and divalent copper. Its aqueous solution is blue, hence its other name, "blue copper peptide." GHK is an active tripeptide, first isolated from human plasma by Pickart and his research team in 1973. Further research revealed a strong affinity between the tripeptide structure and copper, readily forming the complex GHK-Cu. It was found that the GHK-Cu complex exhibits better bioactivity than the single GHK structure. The biggest application of blue copper peptide in skincare is anti-aging. It can promote the regeneration of collagen and elastin, firm the skin, increase elasticity, and reduce wrinkles. In addition, copper peptide also has good anti-hair loss properties, playing an important role in anti-hair loss cosmetics. It works by stimulating the production of vascular endothelial growth factor, which enlarges blood vessels near hair follicles, thereby increasing nutrient supply to the hair, promoting hair growth, thickening hair roots, and increasing hair thickness. Additionally, copper peptides can inhibit the production of transforming growth factor β1, preventing hair follicles from prematurely transitioning from the anagen to catagen phase, thus preventing hair loss. Simultaneously, copper peptides can stimulate the production of basic fibroblast growth factor, which can stimulate angiogenesis and promote hair growth.

[0004] Hexapeptide-9, composed of six amino acids, is a very stable collagen peptide. It enhances dermal protein synthesis, improves the dermal-epidermal junction structure and epidermal component damage, and promotes fibroblast production, thereby achieving comprehensive and significant anti-wrinkle and repair effects. It also has a significant effect on the repair of acne scars. In addition, hexapeptide-9 also has the effect of removing wrinkles around the eyes.

[0005] Biotinyl tripeptide-1 is a tripeptide that combines vitamin H and the Matrikine series signal peptide GHK. Biotinyl tripeptide-1 increases the synthesis of extracellular matrix components such as collagen IV and laminin 5, delays hair follicle aging, and improves hair follicle structure; it also helps hair adhere to the dermal hair follicle, thus achieving the effect of preventing hair loss.

[0006] Currently, there are no literature reports on the detection methods for copper peptides, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for detecting copper peptides, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics, using a high-performance liquid chromatography-tandem mass spectrometry system.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for detecting copper peptide, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics is provided, comprising the following steps:

[0010] Step 1: Accurately weigh copper peptide, hexapeptide-9, and biotinylate tripeptide-1 standards, dissolve them in water respectively, and prepare standard stock solutions.

[0011] Accurately weigh the blank sample, dissolve it in water, and prepare the blank matrix extraction solution;

[0012] The standard stock solution was diluted with the blank matrix extract to prepare an intermediate matrix standard solution;

[0013] Precisely measure the intermediate matrix standard solution and dilute it with the blank matrix extract to prepare a series of matrix standard solutions;

[0014] Step 2: Accurately weigh the sample, add water, vortex to disperse evenly, extract by sonication, let it cool to room temperature, dilute to the mark with water, centrifuge, filter, and use as the test solution for later use;

[0015] Step 3: Detection is performed using a high-performance liquid chromatography-mass spectrometry system.

[0016] Furthermore, the blank sample is one of the following: blank cream matrix sample, blank oil-based matrix sample, blank gel matrix sample, and blank water matrix sample.

[0017] Furthermore, the specific process for preparing the blank matrix extract is as follows: accurately weigh 0.2-0.5g of blank sample, place it in a stoppered colorimetric tube, add 15-30mL of water, vortex for 30-60s to disperse evenly, extract by ultrasonication for 20-30min, let it cool to room temperature, dilute to the mark with water, centrifuge at 10000-15000r / min for 5-10min, and filter through a 0.22μm filter membrane.

[0018] Further, step two specifically involves: weighing 0.2-0.5g of the sample, placing it in a stoppered colorimetric tube, adding 15-30mL of water, vortexing for 30-60s to disperse evenly, ultrasonically extracting for 20-30min, allowing it to reach room temperature, diluting to the mark with water, centrifuging at 10000-15000r / min for 5-10min, and filtering through a 0.22μm filter membrane to obtain the test solution for later use.

[0019] Furthermore, in step three, the chromatographic conditions are as follows:

[0020] Chromatographic column: CAPCELLPAKADME column, 150 mm × 2.1 mm, 2.0 μm; mobile phase: A is 0.1 v / v% formic acid aqueous solution, B is acetonitrile; flow rate: 0.15-0.25 mL / min; column temperature: 25-30℃; injection volume: 1-5 μL; gradient elution.

[0021] Furthermore, the gradient elution procedure is as follows:

[0022]

[0023] Furthermore, in step three, the mass spectrometry conditions are as follows:

[0024] Ion source: Electrospray ionization source (ESI source); Capillary voltage: 3500V; Decomposition voltage: 380V; Curtain gas temperature: 250℃; Curtain gas flow rate: 11L / min; Nebulizer pressure: 20psi; Sheath gas temperature: 250℃; Sheath gas flow rate: 14L / min; Scanning method: Positive ion scan; Monitoring mode: Positive ion multiple reaction monitoring mode (MRM).

[0025] Furthermore, ions with high abundance and minimal interference are used as quantitative ions, while the rest are used as qualitative ions:

[0026]

[0027] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0028] This invention proposes for the first time a method for the simultaneous determination of copper peptides, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics. This method can be used to determine the presence of copper peptides, hexapeptide-9, and biotinylate tripeptide-1 in creams, lotions, aqueous solutions, gels, oil-based cosmetics, and lyophilized base cosmetics. The method is simple to operate, rapid in analysis, highly specific, and has high separation, which is beneficial for quickly addressing the regulation of the addition of copper peptides, hexapeptide-9, and biotinylate tripeptide-1 to existing commercially available products. Attached Figure Description

[0029] Figure 1 This is a multiple reaction monitoring chromatogram of a copper peptide standard solution.

[0030] Figure 2 This is a multiple reaction monitoring chromatogram of the hexapeptide-9 standard solution;

[0031] Figure 3 This is a multiple reaction monitoring chromatogram of a biotinylate tripeptide-1 standard solution. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0033] Example 1

[0034] This embodiment provides a high-performance liquid chromatography-mass spectrometry method for the detection of copper peptide, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics:

[0035] 1. Reagents and Materials

[0036] Unless otherwise specified, all reagents used in this embodiment are of analytical grade or higher, and the water is Grade I water conforming to GB / T6682.

[0037] 1.1 Acetonitrile, chromatographic grade, Merk GmbH, Germany.

[0038] 1,2-Formic acid, chromatographic grade, Merk GmbH, Germany.

[0039] 1.30.1 v / v% formic acid solution: Take 1.0 mL of formic acid (1.2), add water to 1000 mL, mix well, and the solution is ready.

[0040] 1.4 Standards: Copper peptide standard, purity 99.3% (mass fraction, m / m, Bioder Pharmaceuticals); Hexapeptide-9 standard, purity 99.91% (mass fraction, m / m, Chengdu Yunxi Chemical Co., Ltd.); Biotinylate Tripeptide-1 standard 95% (mass fraction, m / m, Maclean's). The Chinese name, English name, CAS number, molecular formula, relative molecular mass, and structural formula of the standards are detailed in Table 1 below.

[0041] Table 1

[0042]

[0043]

[0044] 1.5 Standard Stock Solutions: Weigh 10 mg each of copper peptide, hexapeptide-9, and biotinylate tripeptide-1 standards (accurate to 0.00001 g), place them in a 10 mL amber volumetric flask, dissolve in water, and dilute to the mark. The mass concentration of each standard stock solution is 1000 mg / L. Store at -18°C protected from light.

[0045] It should be noted that, after extensive testing, water was found to be the best solvent for the standard, resulting in good solubility of the target compound, symmetrical peak shape, and high response.

[0046] 2. Instruments and Equipment

[0047] Agilent 6495 high-performance liquid chromatography-mass spectrometry system (Agilent Technologies, USA); Sartorius CP224S and 225D-1CN electronic balances (Sartorius, Germany), with sensitivities of 0.0001 g and 0.00001 g respectively; 5800 ultrasonic instrument (Branson, USA); MS3 vortex mixer (IKA, Germany); 5810R benchtop centrifuge (Eppendof, Germany); Milli-Q Reference A+ ultrapure water system (Millipore, USA); 0.22 μm filter membrane.

[0048] 3. Sample preparation and preservation

[0049] Samples should be stored according to the storage conditions indicated on the label. Before sampling, check the integrity of the seal, observe the properties and characteristics of the sample, and ensure the sample is thoroughly mixed. After opening the packaging, remove the portion to be analyzed as quickly as possible. After sampling, seal the sample for preservation.

[0050] 4. Analysis Steps

[0051] 4.1 Blank matrix extract

[0052] Weigh 0.2 g of blank sample (accurate to 0.0001 g) and place it in a 20 mL stoppered colorimetric tube. Starting from “add 15 mL of water”, process it in the same way as the sample (4.4) to obtain blank matrix extract.

[0053] 4.2 Matrix Standard Intermediate Solution

[0054] Accurately measure 0.1 mL each of copper peptide, hexapeptide-9 and biotinylate tripeptide-1 standard stock solutions (1.5), place them in a 10 mL brown volumetric flask, dilute to the mark with blank matrix extract (4.1), shake well, and prepare a matrix standard intermediate solution with a concentration of 10 mg / L.

[0055] 4.3 Matrix Standard Series Solutions

[0056] Accurately measure appropriate amounts of the intermediate matrix standard solution (4.2) and prepare a series of matrix standard solutions of 50, 100, 150, 200, 250, 500, and 1000 μg / L using blank matrix extract (4.1). (The concentration range can be adjusted according to the actual situation.) The matrix standard solutions should be prepared fresh for each use.

[0057] 4.4 Sample Preparation

[0058] Weigh 0.2 g of sample (accurate to 0.0001 g), place it in a 20 mL stoppered colorimetric tube, add 15 mL of water, vortex for 30 s to disperse evenly, extract by sonication for 20 min, let it cool to room temperature, dilute to the mark with water, centrifuge at 10000 r / min for 5 min, filter through a 0.22 μm filter membrane, and use it as the test solution for later use (the test solution can be appropriately diluted according to the actual concentration).

[0059] It should be noted that:

[0060] When testing the extraction solutions for cosmetic samples, a large number of commonly used matrix types in cosmetics were investigated: creams, lotions, aqueous solutions, gels, oil-based solutions, and lyophilized matrices. It was found that using water as the extraction solvent, the extraction effect met the requirements, and the recovery rate could reach 85%-115%.

[0061] When testing the extraction solution for cosmetic samples, we compared two extraction methods: dissolving the oil matrix in dichloromethane, extracting twice with water, and then diluting the extract to 20 mL. We also compared the method of direct water extraction. The results showed that both methods had good recovery rates, and we ultimately chose the simple and easy-to-implement method of direct water extraction.

[0062] 5. Detection was performed using a high-performance liquid chromatography-mass spectrometry system.

[0063] 5.1 Chromatographic conditions

[0064] Chromatographic column: Shiseido CAPCELL PAKADME column (150mm × 2.1mm, 2.0μm);

[0065] Mobile phase: A is 0.1 v / v formic acid aqueous solution, B is acetonitrile;

[0066] Flow rate: 0.15 mL / min;

[0067] Column temperature: 25℃;

[0068] Injection volume: 1 μL;

[0069] Gradient elution: The gradient elution procedure is shown in Table 2.

[0070] Table 2

[0071]

[0072] It should be noted that:

[0073] Extensive testing in the selection of mobile phases revealed that using 0.1 v / v% formic acid aqueous solution and acetonitrile as mobile phases resulted in symmetrical peaks and stable baselines for the three bioactive peptides.

[0074] 5.2 Mass Spectrometry Conditions

[0075] Ion source: AJS ESI; capillary voltage: 3500V; pyrolysis voltage: 380V; curtain gas temperature: 250℃; curtain gas flow rate: 11L / min; nebulizer pressure: 20psi; sheath gas temperature: 250℃; sheath gas flow rate: 14L / min; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring (MRM). Ions with high abundance and few interferences were used as quantitative ions, and the rest were used as qualitative ions (Table 3).

[0076] Table 3

[0077]

[0078] Note: * indicates quantitative ions.

[0079] 6. Linear regression analysis was performed with peak area as the ordinate (y) and concentration as the abscissa (x, μg / L) to obtain the linear equation.

[0080] 6.1 The standard curves for the cream bases of copper peptide, hexapeptide-9, and biotinylate tripeptide-1 are shown in Table 4 below:

[0081] Table 4

[0082]

[0083] 6.2 The standard curves for copper peptide, hexapeptide-9, and biotinylate tripeptide-1 oil-based matrices are shown in Table 5 below:

[0084] Table 5

[0085]

[0086] 6.3 The standard curves for the gel matrix of copper peptide, hexapeptide-9, and biotinylate tripeptide-1 are shown in Table 6 below:

[0087] Table 6

[0088]

[0089]

[0090] 6.4 The standard curves for copper peptide, hexapeptide-9, and biotinylate tripeptide-1 in aqueous matrix are shown in Table 7 below:

[0091] Table 7

[0092]

[0093] Example 2

[0094] Spiking determination of positive ointment samples: Select positive cosmetic samples containing copper peptides (matrix type: ointment), perform positive spiking test on them, add an amount of the analyte equivalent to the sample, and determine the recovery rate.

[0095] Positive sample spike recovery rate; sample solution processing:

[0096] Weigh 0.1 g (accurate to 0.0001 g) of the copper peptide positive sample (containing 2.3896 mg / g of copper peptide, with no detection of hexapeptide-9 and biotinylate-1) into six portions. Add 0.2 mL of the standard stock solution (1 mg / mL) of copper peptide, hexapeptide-9, and biotinylate-1 to each portion into a 20 mL stoppered colorimetric tube. Add 15 mL of water, vortex for 30 s to disperse evenly, and sonicate for 20 min. Allow to cool to room temperature, then dilute to the mark with water. Centrifuge at 10000 rpm for 5 min. Accurately pipette 0.1 mL of the supernatant into a 10 mL volumetric flask, add blank matrix extract to the mark, shake well, and filter through a 0.22 μm filter membrane. This recovered sample solution is used for testing. The results are shown in Table 8.

[0097] The result is calculated according to formula (1):

[0098]

[0099] In the formula:

[0100] ω—mass fraction of the three components, including copper peptide, in the sample, in mg / kg;

[0101] ρ—mass concentration of the three components, including copper peptide, in the test solution, in μg / L;

[0102] V—Sample volume at final volume, mL;

[0103] m—sample volume, g;

[0104] D—Dilution factor (1 if undiluted).

[0105] The absolute difference between two independent measurements obtained under the same conditions shall not exceed 10% of the arithmetic mean.

[0106] Table 8

[0107]

[0108] Example 3

[0109] Blank oil-based sample spiked determination: A blank oil matrix sample without copper peptide, hexapeptide-9 and biotinylate tripeptide-1 was taken and subjected to a blank spiked test to determine the recovery rate.

[0110] Positive sample spike recovery rate; sample solution processing:

[0111] Weigh 0.1 g (accurate to 0.0001 g) of blank oil-based sample (without detection of copper peptide, hexapeptide-9, and biotinylate tripeptide-1) into 18 portions, with six portions as a group. Accurately add 0.2, 0.4, and 0.5 mL of intermediate standard solutions (10 mg / L) of copper peptide, hexapeptide-9, and biotinylate tripeptide-1 to 20 mL stoppered colorimetric tubes, respectively. Add water to 15 mL, vortex for 30 s to disperse evenly, and sonicate for 20 min. Allow to stand at room temperature, dilute to the mark with water, centrifuge at 10000 r / min for 5 min, shake well, and filter through a 0.22 μm filter membrane. The recovered sample solution is used for later use. The test results are shown in Table 9.

[0112] The result is calculated according to formula (1):

[0113]

[0114] In the formula:

[0115] ω—mass fraction of the three components, including copper peptide, in the sample, in mg / kg;

[0116] ρ—mass concentration of the three components, including copper peptide, in the test solution, in μg / L;

[0117] V—Sample volume at final volume, mL;

[0118] m—sample volume, g;

[0119] D—Dilution factor (1 if undiluted).

[0120] The absolute difference between two independent measurements obtained under the same conditions shall not exceed 10% of the arithmetic mean.

[0121] Table 9

[0122]

[0123] Example 4

[0124] The levels of copper peptide, hexapeptide-9, and biotinylate-1 were determined in 50 batches of ordinary skin care cosmetics (base types: 25 batches of cream, 15 batches of gel, 5 batches of aqueous solution, and 5 batches of oil-based solution) using the method in Example 1.

[0125] In this test, copper peptides were detected in 7 out of 50 samples, with contents ranging from 0.0015% to 0.759%, showing a significant difference. Hexapeptide-9 and biotinylate tripeptide-1 were not detected.

[0126] Of the 50 batches of samples, 10 batches were labeled with hexapeptide-9, but hexapeptide-9 was not detected in any of them. There was a discrepancy between the actual detection and the labeling, with a discrepancy rate of 100%. This should attract the attention of cosmetic regulatory authorities, who should strengthen the management of cosmetic product labeling.

[0127] Biotinyl tripeptide-1 is not listed in the "List of Used Cosmetic Ingredients" (2021 edition), but there are literature reports that some manufacturers add this active peptide to cosmetics. However, in this test, biotinyl tripeptide-1 was not detected in 50 batches of samples.

[0128] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting copper peptide, hexapeptide-9, and biotinylate tripeptide-1 in cosmetics, characterized in that, Includes the following steps: Step 1: Accurately weigh copper peptide, hexapeptide-9, and biotinylate tripeptide-1 standards, dissolve them in water respectively, and prepare standard stock solutions. Accurately weigh the blank sample, dissolve it in water, and prepare the blank matrix extraction solution; The standard stock solution was diluted with the blank matrix extract to prepare a matrix standard intermediate solution; Precisely measure the intermediate matrix standard solution and dilute it with the blank matrix extract to prepare a series of matrix standard solutions; Step 2: Accurately weigh the sample, add water, vortex to disperse evenly, extract by sonication, let it cool to room temperature, dilute to the mark with water, centrifuge, filter, and use as the test solution for later use; Step 3: Detection is performed using a high-performance liquid chromatography-mass spectrometry system; The chromatographic conditions are as follows: Chromatographic column: CAPCELL PAK ADME column, 150 mm × 2.1 mm, 2.0 μm; Mobile phase: A is 0.1 v / v% formic acid aqueous solution, B is acetonitrile; Flow rate: 0.15-0.25 mL / min; Column temperature: 25-30℃; Injection volume: 1-5 μL; Gradient elution; Mass spectrometry conditions include: Ion source: Electrospray ionization source; Scanning method: Positive ion scanning; Monitoring mode: Positive ion multiple reaction monitoring mode; Ion pair and collision energy of the detected component: 。 2. The detection method according to claim 1, characterized in that, The blank sample is one of the following: blank cream matrix sample, blank oil-based matrix sample, blank gel matrix sample, and blank water matrix sample.

3. The detection method according to claim 1, characterized in that, The specific process for preparing the blank matrix extract is as follows: accurately weigh 0.2-0.5g of blank sample, place it in a stoppered colorimetric tube, add 15-30mL of water, vortex for 30-60s to disperse evenly, extract by ultrasonication for 20-30min, let it cool to room temperature, dilute to the mark with water, centrifuge at 10000-15000r / min for 5-10min, and filter through a 0.22μm filter membrane.

4. The detection method according to claim 1, characterized in that, Step two specifically involves: weighing 0.2-0.5g of the sample, placing it in a stoppered colorimetric tube, adding 15-30mL of water, vortexing for 30-60s to disperse evenly, ultrasonically extracting for 20-30min, allowing it to reach room temperature, diluting to the mark with water, centrifuging at 10000-15000r / min for 5-10min, and filtering through a 0.22μm filter membrane to obtain the test solution for later use.

5. The detection method according to claim 1, characterized in that, Step three, the mass spectrometry conditions also include: Capillary voltage: 3500V; pyrolysis voltage: 380V; curtain gas temperature: 250℃; curtain gas flow rate: 11L / min; nebulizer pressure: 20psi; sheath gas temperature: 250℃; sheath gas flow rate: 14L / min.

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