Method for detecting arginine / lysine polypeptide in cosmetics and application thereof

Abstract

The invention relates to the technical field of detection, in particular to a method for detecting arginine / lysine polypeptide in cosmetics and application of the method. The method optimizes a high performance liquid chromatography-tandem mass spectrometry method, has the detection advantages of simple operation, accurate quantification, stable recovery rate, high sensitivity, high precision, strong specificity and high throughput, is suitable for determination of arginine / lysine polypeptide in cosmetics with different matrixes, and can detect the real condition of the product; according to the application, the defects of the existing arginine / lysine polypeptide detection method are overcome, and a quality control basis can be provided for the application of the arginine / lysine polypeptide in cosmetics.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a method for detecting arginine / lysine polypeptides in cosmetics and its application. Background Art

[0002] Arginine / lysine peptide is also called μ-conotoxin peptide, with a molecular weight of 2375.7, containing 22 amino acids and a structure of

[0003] The peptide of pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Al a-Arg-Cys-Cys-amide contains three disulfide bonds in its molecule, and the disulfide bonds are located at Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6. Arginine / lysine peptides have good anti-wrinkle, repair and moisturizing effects. In vitro experiments show that the effective concentration is 0.01-10ppm. It is widely used in anti-wrinkle products. It can specifically block current conduction and inhibit muscle contraction, thereby achieving the purpose of reducing wrinkles. According to the "2022-2027 China Conotoxin Peptide Industry Market In-depth Research and Development Prospects Forecast Report" released by the Xin Shijie Industry Research Center, arginine / lysine peptide raw materials are superior to botulinum toxin, acetyl hexapeptide-8 and other anti-wrinkle raw materials in terms of environmental protection, convenience, controllability and low side effects, and have broad future development space.

[0004] In recent years, due to the excellent skin care effects of arginine / lysine peptides, many products containing arginine / lysine peptides have appeared on the market, all of which use them as the focus of efficacy promotion. However, the price of arginine / lysine peptide raw materials is high, and the effective concentration is low. It is difficult to tell whether it is added in cosmetic formulas, and a detection solution is urgently needed to verify whether it is effectively added.

[0005] Moreover, natural arginine / lysine polypeptides construct their complex three-dimensional spatial configuration through three specific disulfide bonds (Cys3-Cys15, Cys4-Cys21, Cys10-Cys22). However, this configuration does not manifest as a thermodynamically stable state, but relies on the unique biosynthetic mechanism in cone snail cells to synthesize this complex spatial structure. In the process of synthesizing arginine / lysine standards, even under optimized disulfide bond formation conditions, the yield of the product decreases significantly with the increase in the number of disulfide bonds. Taking the synthesis amount of linear peptides as 100% as the benchmark, the yield of disulfide bond products that can be correctly folded to form the correct spatial structure is only about 1%, which further verifies that the spatial structure of natural arginine / lysine polypeptides is thermodynamically unstable.

[0006] In the process of industrial synthesis of arginine / lysine peptides, the thiol groups of the six cysteines are usually protected with the same protecting group. After the linear sequence synthesis is completed, the protecting group of cysteine ​​is removed, and three pairs of disulfide bonds are synthesized by air oxidation, that is, the peptide chain is naturally oxidized in the air to form disulfide bonds. It is speculated that in the traditional mature solid phase synthesis process, arginine / lysine peptides can theoretically maintain the accuracy of their linear structure, but in the process of folding the spatial structure, it is difficult to fold into a non-thermodynamically stable configuration like natural arginine / lysine peptides. In addition, there may be a polymer form that forms intermolecular disulfide bonds through the free thiol groups of arginine / lysine peptide monomers.

[0007] In view of the various complex structural forms of arginine / lysine polypeptides, current detection methods cannot accurately detect arginine / lysine polypeptides in different forms such as the presence of various complex structures. Summary of the invention

[0008] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a method for detecting arginine / lysine polypeptides in cosmetics and its application.

[0009] To achieve the above purpose, the technical solution adopted by this application is:

[0010] The present application provides a method for detecting arginine / lysine polypeptides in cosmetics, comprising the following steps:

[0011] S1. synthesizing a linear peptide of an arginine / lysine polypeptide by a solid phase method to obtain an arginine / lysine polypeptide standard product; preparing the arginine / lysine polypeptide standard product into arginine / lysine polypeptide standard solutions of different concentrations;

[0012] S2, adding a sample extraction solvent to the cosmetic to be tested, extracting under water bath ultrasonic conditions, and then centrifuging to obtain a supernatant to obtain a sample test solution;

[0013] S3, pre-treating the arginine / lysine polypeptide standard solutions of different concentrations in step S1 and the sample solution to be tested in step S2 in the following manner;

[0014] S31, taking the arginine / lysine polypeptide standard solution and the sample solution to be tested, adding water and formic acid (quantitatively by detecting monomeric arginine / lysine polypeptide);

[0015] S32, taking the arginine / lysine polypeptide standard solution and the sample solution to be tested, adding ammonium bicarbonate buffer, dithiothreitol solution, iodoacetamide solution, trypsin solution and formic acid (quantitatively by detecting the total arginine / lysine polypeptide specific enzymatic hydrolysis polypeptide);

[0016] Respectively obtaining a pretreated standard solution and a sample solution to be tested;

[0017] S4, subjecting the pretreated standard solution to liquid chromatography tandem mass spectrometry detection, and screening out quantitative ions according to the liquid chromatography tandem mass spectrometry detection results, and then drawing a standard curve according to the concentration of the arginine / lysine polypeptide standard solution and the peak area corresponding to the quantitative ions;

[0018] The pretreated sample liquid is subjected to liquid chromatography tandem mass spectrometry detection using the method of step S3. According to the detection result of the sample liquid, whether it is an active monomer arginine / lysine polypeptide is determined by comparing the peak time; the concentration of arginine / lysine polypeptide in the sample liquid is obtained by aligning with the standard curve, thereby realizing the quantitative detection of arginine / lysine polypeptide in cosmetics.

[0019] In view of the fact that arginine / lysine polypeptides have a variety of complex structural forms, the present application defines the linear structure consistent with the theoretical sequence (pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-amide) as the total arginine / lysine polypeptide, which includes monomeric arginine / lysine polypeptides containing one to three sets of intramolecular disulfide bonds, and polymeric arginine / lysine polypeptides connected by intermolecular disulfide bonds. Monomeric arginine / lysine polypeptides can be further subdivided into active arginine / lysine polypeptides that are exactly the same as the natural structure, and inactive arginine / lysine polypeptides formed due to different disulfide bond connection positions.

[0020] Based on this, the present application further develops quantitative detection methods for different forms of arginine / lysine polypeptides. These methods can accurately quantitatively analyze total arginine / lysine polypeptides and monomeric arginine / lysine polypeptides, as well as qualitatively analyze inactive arginine / lysine polypeptides, providing important technical support for the in-depth research and application of arginine / lysine polypeptides.

[0021] The method of the present application optimizes the high performance liquid chromatography-tandem mass spectrometry method, and the method is easy to operate, has accurate quantification, stable recovery rate, high sensitivity, high precision, strong specificity and high-throughput detection advantages, and is suitable for the determination of arginine / lysine polypeptides in cosmetics with different matrices, and can detect the true situation of the product; the present application makes up for the shortcomings of the existing arginine / lysine polypeptide detection methods, and can provide a quality control basis for the application of arginine / lysine polypeptides in cosmetics.

[0022] In the technical solution of the present application, formic acid is added in both steps S31 and S32, so that the arginine / lysine polypeptide extraction process is fully protected, the accuracy of the test is improved, and it is beneficial to the quantitative analysis of arginine / lysine polypeptides.

[0023] Step S32, dithiothreitol is added. After the arginine / lysine polypeptide is reduced by dithiothreitol, its free thiol can react with iodoacetamide to form a carbamidomethyl derivative, thereby converting it into a unified linear structure. Therefore, in theory, the concentration of total arginine / lysine polypeptides can be evaluated by quantitatively detecting the linear structure.

[0024] In order to improve the detection sensitivity, the present application uses trypsin to enzymatically hydrolyze the reduced total arginine / lysine polypeptide. The polypeptide produced by enzymatic hydrolysis mainly presents the ionized form of [M+2H]2+, which is mainly because the polypeptide structure contains two amino groups on the N-terminus and the side chain of the enzymatic hydrolysis site, which together serve as the charge center in the ionization process. This method makes the ionized form more concentrated and can significantly improve the sensitivity of detection. There are four enzymatic hydrolysis sites in the amino acid sequence of arginine / lysine polypeptides, which can theoretically produce five enzymatic hydrolysis fragments of different lengths, and the number of amino acid residues in these fragments ranges from 2 to 7. Among them, the pGlu1-Gly2-Cys3-Cys4-Asn5-Gly6-Pro7-Lys8 sequence has a moderate length, which can have a high degree of specificity and avoid the problem of reduced sensitivity that may be caused by too long sequences. Therefore, it is selected as a specific peptide segment for the detection of actual samples.

[0025] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, in step S1, the concentration of arginine / lysine polypeptides in the arginine / lysine polypeptide standard solution is 5 μg / L to 100 μg / L.

[0026] The concentrations of the arginine / lysine polypeptide in the arginine / lysine polypeptide standard solution are specifically: 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L and 100.0 μg / L.

[0027] Preferably, in the step S1, in the process of synthesizing the linear peptide of arginine / lysine polypeptide by solid phase method, the resin used is Fmoc-protected amino resin, and the linear peptide of arginine / lysine polypeptide is synthesized from C-terminus to N-terminus. Among the six cysteines (C1-C6) constituting the disulfide bond, the thiol groups of C1 and C4 are protected by MMT (4-methylbenzyl), the thiol groups of C2 and C5 are protected by TRT (toluenesulfonyl), and the thiol groups of C3 and C6 are protected by ACM (acetyl).

[0028] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, in step S2, the sample extraction solvent includes a formic acid acetonitrile solution with a mass concentration of 0.01 to 0.3%;

[0029] The water bath ultrasonic conditions are as follows: ultrasonic power is 100-400W, water bath temperature is 10-40°C, and ultrasonic extraction time is 10-50min.

[0030] Preferably, in step S2, the centrifugation conditions are: centrifugation temperature is 4°C, centrifugation speed is 12000 r / min, and centrifugation is performed for 10 min. The supernatant is filtered through a 0.22 um filter membrane.

[0031] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, in step S2, the obtained sample test solution is tested within 4 to 8 hours.

[0032] This application adopts step S2 to pre-treat the cosmetics to be tested, which can better extract arginine / lysine polypeptides and improve the accuracy of arginine / lysine polypeptide detection. Experiments show that only under the processing conditions of the samples of this application can the target detection object be effectively extracted and detected. However, the sample extraction solvent is methanol or methanol / ethanol, and the lotion and cream sample extracts are turbid and difficult to filter. The target detection object is not effectively extracted, and the detection value is low.

[0033] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, in the step S32, ammonium bicarbonate buffer with a mass concentration of 50 to 150 mmol / L and dithiothreitol with a mass concentration of 40 to 140 mmol / L are added to the sample test solution, incubated in a water bath, and then iodoacetamide with a mass concentration of 260 to 350 mmol / L is added, incubated in the dark, and then ammonium bicarbonate solution and trypsin are added, incubated in a water bath, and finally formic acid is added to obtain the pretreated sample test solution.

[0034] More preferably, in step S3, the conditions for water bath incubation are: water bath temperature is 75° C., and incubation is for 30 min.

[0035] More preferably, the incubation time in the dark is 30 min.

[0036] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, the liquid chromatography adopts a Pro Chrom 300 chromatographic column (2.1 mm x 100 mm, 3 um, ), mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, flow rate was 0.2-0.4 mL / min, injection volume was 4-6 μL;

[0037] Gradient elution was adopted: 0-2 min, 5%-40% acetonitrile; 2-2.1 min, 40-100% acetonitrile; 2.1-2.5 min, 100% acetonitrile; 2.5-2.6 min, 100%-5% acetonitrile; 2.6-4.5 min, 5% acetonitrile.

[0038] As a preferred embodiment of the method for detecting arginine / lysine polypeptides in cosmetics described in the present application, the mass spectrometry detection conditions are as follows:

[0039] Electrospray ion source ESI, positive ion scanning mode multiple reaction monitoring MRM mode, capillary voltage 3.0 kV, ion source temperature 150 ° C, desolvation temperature 500 ° C, desolvation gas flow rate 800 L / h, cone hole backflush gas flow rate 160 L / h.

[0040] The present application also provides the application of the above method for detecting arginine / lysine polypeptides in cosmetics in cosmetics testing.

[0041] Compared with the prior art, this application has the following beneficial effects:

[0042] The present application provides a method for detecting arginine / lysine polypeptides in cosmetics and its application. The method of the present application optimizes the high performance liquid chromatography-tandem mass spectrometry method. The method is easy to operate, accurate in quantification, stable in recovery, high in sensitivity, high in precision, strong in specificity and high in throughput. It is suitable for the determination of arginine / lysine polypeptides in cosmetics with different matrices, and can detect the true situation of the product; the present application further develops a quantitative detection method for arginine / lysine polypeptides in different forms. These methods can accurately quantitatively analyze total arginine / lysine polypeptides and monomeric arginine / lysine polypeptides, as well as qualitatively analyze inactive arginine / lysine polypeptides, providing important technical support for the in-depth research and application of arginine / lysine polypeptides. The present application makes up for the shortcomings of existing arginine / lysine polypeptide detection methods, and can provide a quality control basis for the application of arginine / lysine polypeptides in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the chromatogram of the single arginine / lysine peptide standard;

[0044] Figure 2 is the total reduced arginine / lysine peptide chromatogram;

[0045] Figure 3 The chromatogram is of the total arginine / lysine peptide-specific peptide standard. DETAILED DESCRIPTION

[0046] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0047] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0048] Example 1: A method for detecting arginine / lysine polypeptides in cosmetics

[0049] This embodiment provides a method for detecting arginine / lysine polypeptides in cosmetics, comprising the following steps:

[0050] 1. Preparation of detection standards: The linear peptide of arginine / lysine polypeptide (pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-amide, with disulfide bonds located at Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was synthesized by solid phase method. The resin was Fmoc-protected amino resin and was synthesized from C-terminus to N-terminus. Among the six cysteines (C1-C6) that constitute the disulfide bond, the thiol groups of C1 (Cys1) and C4 (Cys4) are protected by MMT (4-methylbenzyl), the thiol groups of C2 (Cys2) and C5 (Cys5) are protected by TRT (toluenesulfonyl), and the thiol groups of C3 (Cys3) and C6 (Cys6) are protected by ACM (acetyl). After the linear polypeptide chain is synthesized, the protecting groups on each cysteine ​​residue are gradually removed in the order of MMT, TRT and ACM to promote the correct formation of disulfide bonds and finally obtain an arginine / lysine polypeptide standard with a spatial structure consistent with that of the natural μ-conotoxin peptide. The arginine / lysine polypeptide standard is finally purified by preparative chromatography and freeze-dried. The total protein of the synthesized product is detected by the Bradford method to complete the purity calibration of the reference product, and its purity is determined to be 98%.

[0051] 2. Preparation of standard solution: Prepare the arginine / lysine peptide standard (including single arginine / lysine peptide, total reduced arginine / lysine peptide and total arginine / lysine peptide specific peptide standard chromatograms as shown in Figure 1-Figure 3 Arginine / lysine peptide standard solutions with concentrations of 5.0, 10.0, 20.0, 50.0 and 100.0 μg / L were prepared with deionized water.

[0052] 3. Pretreatment of cosmetic samples: weigh about 0.2 g of sample (accurate to 0.0001 g), place in a 20 mL plastic centrifuge tube with a stopper, add 0.1% formic acid acetonitrile solution (sample extraction solvent) to make up to 10 mL, vortex for 10 min, and extract in a water bath with ultrasonic power of 250 W and temperature of 25 ° C for 30 min. Let it stand to room temperature, centrifuge at 4 ° C 12000 r / min for 10 min, take the supernatant and filter it through a 0.22 um filter membrane to obtain the sample solution to be tested, and complete the test within 6 hours.

[0053] 4. Arginine / lysine peptide standard solutions and sample solutions of different concentrations are pretreated by 1) or 2):

[0054] 1) Pretreatment of monomer arginine / lysine peptide detection: take 100uL of sample solution, add 890uL of water and 10uL of formic acid to obtain the pretreated standard solution and sample solution for injection.

[0055] 2) Pretreatment for the detection of total arginine / lysine peptide-specific enzymatic peptide (sequence: pGlu1-Gly2-Cys3-Cys4-Asn5-Gly6-Pro7-Lys8): Take 100 μL of sample, add 660 μL of ammonium bicarbonate buffer (100 mmol / L aqueous solution) and 10 μL of dithiothreitol (100 mmol / L aqueous solution), and incubate in a 75°C water bath for 30 min. Add 10 μL of iodoacetamide (300 mmol / L aqueous solution) and incubate in the dark at room temperature for 30 min. Add 200 μL of ammonium bicarbonate solution and 10 μL of alkaline trypsin (enzyme activity 10000 U / mL), and incubate in a 37°C water bath overnight. Add 10 μL of formic acid to obtain the pretreated standard solution and sample solution for injection.

[0056] 5. Test conditions, chromatographic conditions: Chromatographic column is Pro Chrom 300 (2.1mmx100 mm, 3um, ), mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, flow rate was 0.3 mL / min, injection volume was 5 μL.

[0057] Gradient elution was adopted: 0-2 min, 5%-40% acetonitrile; 2-2.1 min, 40-100% acetonitrile; 2.1-2.5 min, 100% acetonitrile; 2.5-2.6 min, 100%-5% acetonitrile; 2.6-4.5 min, 5% acetonitrile.

[0058] Mass spectrometry conditions: electrospray ion source (ESI), positive ion scan mode multiple reaction monitoring (MRM) mode, capillary voltage 3.0 kV, ion source temperature 150 °C, desolvation temperature 500 °C, desolvation gas flow rate 800 L / h, cone backflush gas flow rate 160 L / h.

[0059] 6. The above-mentioned pretreated standard solution and sample solution were tested according to step 5, with sensitive and stable ion pairs as the quantitative detection channel, peak area y as the ordinate, mass concentration x as the abscissa, and a standard curve was drawn and fitted with a linear equation, and the detection limit and quantitative limit of the method were calculated with 3 times and 10 times the signal-to-noise ratio, respectively;

[0060] 7. The content of arginine / lysine polypeptide in 9 commercially available cosmetics containing arginine / lysine (including 3 samples of skin care water, skin care lotion and skin care cream) was calculated according to the standard curve equation.

[0061] Example 2

[0062] Similar to Example 1, the only difference of Example 2 is that the sample pretreatment condition in step 3 is that the sample extraction solvent is 0.3% formic acid ethanol solution, and the water bath ultrasonic extraction is carried out for 10 minutes at an ultrasonic power of 100 W and a temperature of 40° C., and the other steps are consistent with Example 1.

[0063] Example 3

[0064] Similar to Example 1, the difference of Example 3 is that the sample pretreatment condition in step 3 is that the sample extraction solvent is 0.01% formic acid ethanol solution, and the water bath ultrasonic extraction is carried out for 50 minutes at an ultrasonic power of 400W and a temperature of 10°C. The other steps are consistent with Example 1.

[0065] Example 4

[0066] Similar to Example 1, the difference of Example 4 is that the sample pretreatment condition in step 3 is that the sample extraction solvent is 0.2% formic acid ethanol solution, and the water bath ultrasonic extraction is carried out for 40 minutes at an ultrasonic power of 200 W and a temperature of 30° C., and the other steps are consistent with Example 1.

[0067] Example 5

[0068] Similar to Example 1, the difference of Example 5 is that the sample pretreatment condition in step 3 is that the sample extraction solvent is 0.05% formic acid ethanol solution, and the water bath ultrasonic extraction is carried out for 20 minutes at an ultrasonic power of 300W and a temperature of 20°C. The other steps are consistent with Example 1.

[0069] Example 6

[0070] Similar to Example 1, the only difference of Example 6 is that in step 3, after pretreatment, the sample liquid to be tested is obtained and the test is completed within 4 hours. The other steps are consistent with Example 1.

[0071] Example 7

[0072] Similar to Example 1, the only difference of Example 7 is that in step 3, after pretreatment, the sample liquid to be tested is obtained and the test is completed within 8 hours. The other steps are consistent with Example 1.

[0073] Example 8

[0074] Similar to Example 1, the difference of Example 8 is that in step 3, after pretreatment, the sample liquid to be tested is obtained and the test is completed within 5 hours. The other steps are consistent with Example 1.

[0075] Example 9

[0076] Similar to Example 1, the difference in Example 9 is that in step 4, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 50 mmol / L, 40 mmol / L, and 260 mmol / L, respectively, and the other steps are consistent with Example 1.

[0077] Example 10

[0078] Similar to Example 1, the difference in Example 10 is that in step 4, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 150 mmol / L, 140 mmol / L, and 350 mmol / L, respectively, and the other steps are consistent with Example 1.

[0079] Embodiment 11

[0080] Similar to Example 1, the difference in Example 11 is that in step 4, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 90 mmol / L, 110 mmol / L, and 310 mmol / L, respectively, and the other steps are consistent with Example 1.

[0081] Comparative Example 1

[0082] Similar to Example 1, the difference of Comparative Example 1 is that the sample extraction solvent in step 3 is 0.1% formic acid methanol solution, and the other steps are consistent with Example 1.

[0083] Comparative Example 2

[0084] Similar to Example 1, the difference of Comparative Example 2 is that the sample extraction solvent in step 3 is 0.1% methanol and ethanol (1:1, V:V), and the other steps are consistent with Example 1.

[0085] Comparative Example 3

[0086] Similar to Example 1, the difference of Comparative Example 3 is that the sample extraction solvent in step 3 is acetonitrile solution, and the other steps are consistent with Example 1.

[0087] Comparative Example 4

[0088] Similar to Example 1, the difference of Comparative Example 4 is that the ultrasonic pretreatment conditions of the sample in step 3 are water bath ultrasonic extraction for 55 minutes at an ultrasonic power of 50 W and a temperature of 45° C., and the other steps are consistent with Example 1.

[0089] Comparative Example 5

[0090] Similar to Example 1, the difference of Comparative Example 5 is that the ultrasonic pretreatment conditions of the sample in step 3 are water bath ultrasonic extraction for 5 minutes at an ultrasonic power of 450 W and a temperature of 5° C., and the other steps are consistent with Example 1.

[0091] Comparative Example 6

[0092] Similar to Example 1, the difference of Comparative Example 6 is that the sample solution obtained in step 3 is tested within 9 hours, and the other steps are consistent with Example 1.

[0093] Comparative Example 7

[0094] Similar to Example 1, the difference of Comparative Example 7 is that step 4 is different, and 2) in step 4 is removed;

[0095] Step 4 is:

[0096] Arginine / lysine polypeptide standard solutions of different concentrations and sample test solutions were pretreated, 100uL of sample was taken, 890μL of water and 10μL of formic acid were added, and the sample was ready for injection. The other steps were consistent with Example 1.

[0097] Comparative Example 8

[0098] Similar to Example 1, the difference of Comparative Example 8 is that 2) in step 4 is different;

[0099] In step 4, 2) is:

[0100] Standard and sample test solution pretreatment, take 100 μL sample, add 870 μL ammonium bicarbonate buffer (100 mmol / L) and 10 uL dithiothreitol (100 mmol / L), incubate in a 75°C water bath for 30 min. Add 10 uL iodoacetamide (300 mmol / L) and incubate in the dark at room temperature for 30 min. Add 10 μL formic acid and wait for injection. The other steps are consistent with Example 1.

[0101] Comparative Example 9

[0102] Similar to Example 1, the difference of Comparative Example 9 is that step 4 is different, and 1) in step 4 is removed; step 4 is:

[0103] Pre-treat the sample solution with different concentrations of arginine / lysine peptide standard solution and sample solution, take 100 μL sample, add 660 μL ammonium bicarbonate buffer (100 mmol / L) and 10 μL dithiothreitol (100 mmol / L), and incubate in a 75°C water bath for 30 min. Add 10 μL iodoacetamide (300 mmol / L) and incubate in the dark at room temperature for 30 min. Add 200 μL ammonium bicarbonate solution and 10 μL trypsin, and incubate in a 37°C water bath overnight. Add 10 μL formic acid and wait for injection. The other steps are consistent with Example 1.

[0104] Comparative Example 10

[0105] Similar to Example 1, the difference of Comparative Example 10 is that step 4 is different, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 160 mmol / L, 30 mmol / L, and 360 mmol / L, respectively, and the other steps are consistent with Example 1.

[0106] Comparative Example 11

[0107] Similar to Example 1, the difference of Comparative Example 11 is that step 4 is different, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 40 mmol / L, 150 mmol / L, and 250 mmol / L, respectively, and the other steps are consistent with Example 1.

[0108] Comparative Example 12

[0109] Similar to Example 1, the difference of Comparative Example 12 is that step 4 is different, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 160 mmol / L, 150 mmol / L, and 360 mmol / L, respectively, and the other steps are consistent with Example 1.

[0110] Comparative Example 13

[0111] Similar to Example 1, the difference of Comparative Example 13 is that step 4 is different, 2) during the total arginine / lysine polypeptide-specific enzymatic polypeptide treatment, the mass concentrations of ammonium bicarbonate buffer, dithiothreitol, and iodoacetamide are 40 mmol / L, 30 mmol / L, and 250 mmol / L, respectively, and the other steps are consistent with Example 1.

[0112] Comparative Example 14

[0113] Similar to Example 1, the difference of Comparative Example 14 is that step 4 is different, 10 μL of formic acid is not added in 1) and 2), and the other steps are consistent with Example 1.

[0114] Comparative Example 15

[0115] Similar to Example 1, the difference of Comparative Example 15 is that the chromatographic column of step 5 is Shimadzu Shim-pack GIST-HP C18 (100×2.1 mm, 3 um), and the other steps are consistent with Example 1.

[0116] Comparative Example 16

[0117] Similar to Example 1, the difference of Comparative Example 16 is that in step 5, mobile phase A is water, and the other steps are consistent with Example 1.

[0118] Comparative Example 17

[0119] Similar to Example 1, the difference of Comparative Example 17 is that in step 5, the mobile phase A is 0.09% formic acid aqueous solution, and the other steps are consistent with Example 1.

[0120] Comparative Example 18

[0121] Similar to Example 1, the difference of Comparative Example 18 is that in step 5, the mobile phase A is 0.11% formic acid aqueous solution, and the other steps are consistent with Example 1.

[0122] Comparative Example 19

[0123] Similar to Example 1, the difference of Comparative Example 19 is that in step 5, mobile phase B is methanol, and the other steps are consistent with Example 1.

[0124] Comparative Example 20

[0125] Similar to Example 1, the difference of Comparative Example 20 is that in step 5, the flow rate is 0.4 mL / min, and the other steps are consistent with Example 1.

[0126] Comparative Example 21

[0127] Similar to Example 1, the difference of Comparative Example 21 is that in step 5, the flow rate is 0.2 mL / min, and the other steps are consistent with Example 1.

[0128] Comparative Example 22

[0129] Similar to Example 1, the difference of Comparative Example 22 is that in step 5, the injection volume is 4 μL, and the other steps are consistent with Example 1.

[0130] Comparative Example 23

[0131] Similar to Example 1, the difference of Comparative Example 23 is that in step 5, the injection volume is 6 μL, and the other steps are consistent with Example 1.

[0132] Experimental Example 1: Standard curves, detection limits and quantification limits of monomeric arginine / lysine peptides and total arginine / lysine peptides

[0133] The standard curves, detection limits and quantification limits of the monomeric arginine / lysine polypeptides of Examples 1, 9-11 and Comparative Examples 7-21, and the total arginine / lysine polypeptides are shown in Table 1 below:

[0134] Table 1

[0135]

[0136]

[0137]

[0138]

[0139] As shown in Table 1, the monoisotopic molecular weight of the monomeric arginine / lysine polypeptide in Example 1 is 2373.9 Da, and its [M+H] + The mass-to-charge ratio of the precursor ion was beyond the detection range of the triple quadrupole mass spectrometer and was not considered. 2+ 、[M+3H]3+、[M+4H] 4+ 、[M+5H] 5+ The theoretical mass-to-charge ratios of the parent ions were 1187.9, 792.3, 594.5 and 475.8, respectively. The arginine / lysine peptide standard was scanned in the range of m / z 300-1500 using the MS-Scan mode, and it was found that the arginine / lysine peptide was mainly [M+3H] 3+ 、[M+4H] 4+ 、[M+5H] 5+Three multi-charge ionization modes; daughter scan mode was used at the same time to perform daughter ion scanning analysis under different collision energy conditions, and sensitivity and signal stability were comprehensively considered. Finally, the ion channel of 594.9>590.4 was selected as the quantitative detection channel for monomeric arginine / lysine peptides. At the same time, the retention time with active arginine / lysine peptides was used to determine whether it was an active arginine / lysine peptide detection method.

[0140] Regardless of whether the arginine / lysine peptide exists in monomeric or polymeric form, after dithiothreitol reduction, its free thiol can react with iodoacetamide to form carbamidomethyl derivatives, thereby converting into a unified linear structure. Therefore, in theory, the concentration of total arginine / lysine peptides can be evaluated by quantitatively detecting linear structures. However, given that the multiple multi-charge forms of total arginine / lysine peptides may reduce the ionization efficiency of the parent ion in a single charge state, thereby affecting the detection sensitivity. Only detecting the total reduced arginine / lysine peptide content may result in the detection being inconsistent with the actual or being too low.

[0141] In order to improve the detection sensitivity, the present application uses alkaline trypsin to enzymatically hydrolyze the reduced total arginine / lysine peptides. The peptides produced by enzymatic hydrolysis mainly present [M+2H] 2+ The ionized form is mainly because the peptide structure contains two amino groups on the N-terminus and the side chain of the enzymatic site, which together serve as the charge center in the ionization process. This method makes the ionized form more concentrated and can significantly improve the sensitivity of detection. There are four enzymatic sites in the amino acid sequence of arginine / lysine peptides, which can theoretically produce five enzymatic fragments of different lengths, and the number of amino acid residues in these fragments ranges from 2 to 7. Among them, the pGlu1-Gly2-Cys3-Cys4-Asn5-Gly6-Pro7-Lys8 sequence has a moderate length, which can have a high degree of specificity and avoid the problem of reduced sensitivity that may be caused by too long sequences. Therefore, it was selected as a specific peptide for the detection of actual samples, and its quantitative ion is 452.2>329.1.

[0142] It can be concluded from Examples 1, 9-11 and Comparative Examples 7-23 that, under the conditions of this application, arginine / lysine polypeptides can obtain a better standard curve and can better quantify and quantify samples. Comparative Examples 7-9 have only one standard curve, because arginine / lysine polypeptides may have a variety of structures and various charge forms, and one standard curve cannot accurately reflect the true situation of the product; Comparative Examples 10-14, changing the concentration of the pretreatment reagent solution in step 4 1) and 2) will affect the response of the polypeptide in the instrument, and no effective standard curve can be obtained; Comparative Examples 15-23 show that, under the conditions of the instrument of this application, a better standard curve cannot be obtained or the obtained standard curve has a poor correlation coefficient, a high detection limit, etc., and cannot better quantify and quantify samples.

[0143] Test Example 2: Detection results of monomeric arginine / lysine peptides and total arginine / lysine peptides in lotion samples

[0144] The detection results of monomeric arginine / lysine polypeptides and total arginine / lysine polypeptides of the lotion samples in Examples 1-8 and Comparative Examples 1-6 are shown in Table 2.

[0145] Table 2

[0146]

[0147]

[0148]

[0149] From the retention time, we can see that the retention time of the monomeric arginine / lysine peptides in skin lotion 1 and skin lotion 3 has shifted compared with that of the standard, while the total arginine / lysine peptides have not shifted, indicating that the arginine / lysine peptides they contain are inactive structures (active arginine / lysine peptides have specific three disulfide bonds (Cys3-Cys15, Cys4-Cys21, Cys10-Cys22)). The reason may be that the monomeric arginine / lysine peptides artificially synthesized by air oxidation cannot obtain the same spatial configuration as the natural active arginine / lysine peptides (disulfide bonds (Cys3-Cys15, Cys4-Cys21, Cys10-Cys22)), resulting in different exposure degrees of polar / non-polar groups on the molecular surface, which in turn leads to significant changes in chromatographic retention time. The characteristic enzymatic hydrolyzed peptides of the total arginine / lysine peptides cannot reflect their original structural diversity in retention time due to reduction and enzymatic hydrolysis. It can be seen from Examples 1-5 and Comparative Examples 1-5 that the samples can be better extracted under the pretreatment conditions of the present application, which is conducive to detection. It can be seen from Examples 6-8 and Comparative Example 6 that the samples are detected within 8 hours of the present application, and the detection values ​​are more accurate.

[0150] The test results of total arginine / lysine peptides in cosmetic samples are shown in Table 3.

[0151] Table 3

[0152]

[0153]

[0154] It can be concluded from Examples 1-8 and Comparative Examples 1-6 that, in Comparative Example 1-2, because the sample extraction solvent is methanol or methanol / ethanol, the lotion and cream sample extracts are turbid and difficult to filter, the target detection object is not effectively extracted, and the detection value is low; in Comparative Example 3, because formic acid is not added, the arginine / lysine extraction process is not fully protected, and the test result is low; Comparative Examples 4-6 and Examples 1-8 can be compared to show that only under the sample processing conditions of the present application, the target detection object can be effectively extracted and detected. Comparative Example 8 tests the total reduced arginine / lysine polypeptide, because its various multi-charge forms may cause the ionization efficiency of the parent ion in a single charge state to be reduced, affecting the detection sensitivity, and low-content samples will not be detected.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for detecting arginine / lysine polypeptides in cosmetics, characterized in that: The following steps are involved: S1. synthesizing a linear peptide of an arginine / lysine polypeptide by a solid phase method to obtain an arginine / lysine polypeptide standard product; preparing the arginine / lysine polypeptide standard product into arginine / lysine polypeptide standard solutions of different concentrations; S2, adding a sample extraction solvent to the cosmetic to be tested, extracting under water bath ultrasonic conditions, and then centrifuging to obtain a supernatant to obtain a sample test solution; S3, pre-treating the arginine / lysine polypeptide standard solutions of different concentrations in step S1 and the sample solution to be tested in step S2 in the following manner; S31, taking the arginine / lysine polypeptide standard solution and the sample solution, adding water and formic acid; S32, taking the arginine / lysine polypeptide standard solution and the sample solution, adding ammonium bicarbonate buffer, dithiothreitol solution, iodoacetamide solution, trypsin solution and formic acid; Respectively obtaining a pretreated standard solution and a sample solution to be tested; S4, subjecting the pretreated standard solution to liquid chromatography tandem mass spectrometry detection, and screening out quantitative ions according to the liquid chromatography tandem mass spectrometry detection results, and then drawing a standard curve according to the concentration of the arginine / lysine polypeptide standard solution and the peak area corresponding to the quantitative ions; The pretreated sample liquid is subjected to liquid chromatography tandem mass spectrometry detection using the method of step S3. According to the detection result of the sample liquid, whether it is an active monomer arginine / lysine polypeptide is determined by comparing the peak time; the concentration of arginine / lysine polypeptide in the sample liquid is obtained by aligning with the standard curve, thereby realizing the quantitative detection of arginine / lysine polypeptide in cosmetics.

2. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: In the step S1, the concentration of the arginine / lysine polypeptide in the arginine / lysine polypeptide standard solution is 5 μg / L to 100 μg / L.

3. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: In the step S2, the sample extraction solvent includes a formic acid acetonitrile solution with a mass concentration of 0.01 to 0.3%.

4. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: In step S2, the water bath ultrasonic conditions are: ultrasonic power of 100-400W, water bath temperature of 10-40°C, and ultrasonic extraction time of 10-50min.

5. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: In step S2, the obtained sample solution is tested within 4 to 8 hours.

6. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: In the step S32, ammonium bicarbonate buffer with a mass concentration of 50 to 150 mmol / L and dithiothreitol with a mass concentration of 40 to 140 mmol / L are added to the sample solution, and the solution is incubated in a water bath. Then, iodoacetamide with a mass concentration of 260 to 350 mmol / L is added, and the solution is incubated in the dark. Then, ammonium bicarbonate solution and trypsin are added, and the solution is incubated in a water bath. Finally, formic acid is added to obtain the pretreated sample solution.

7. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: Liquid chromatography used a Pro Chrom 300 column, mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, flow rate was 0.2-0.4 mL / min, injection volume was 4-6 μL; Gradient elution was adopted: 0-2 min, 5%-40% acetonitrile; 2-2.1 min, 40-100% acetonitrile; 2.1-2.5 min, 100% acetonitrile; 2.5-2.6 min, 100%-5% acetonitrile; 2.6-4.5 min, 5% acetonitrile.

8. The method for detecting arginine / lysine polypeptide in cosmetics according to claim 1, characterized in that: The mass spectrometry detection conditions were as follows: Electrospray ion source ESI, positive ion scanning mode multiple reaction monitoring MRM mode, capillary voltage 3.0 kV, ion source temperature 150 ° C, desolvation temperature 500 ° C, desolvation gas flow rate 800 L / h, cone hole backflush gas flow rate 160 L / h.

9. Use of the method for detecting arginine / lysine polypeptide in cosmetics as claimed in claims 1 to 8 in cosmetics detection.

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