Method for simultaneously determining 55 polypeptides in cosmetics based on liquid chromatography-high resolution mass spectrometry

Abstract

The invention discloses a method for simultaneously determining polypeptide 55 in cosmetics based on liquid chromatography-high resolution mass spectrometry, and the method comprises the following steps: S1, extraction: weighing a to-be-determined cosmetic sample, uniformly mixing the to-be-determined cosmetic sample with an extraction solvent, carrying out ultrasonic extraction and centrifugal treatment, standing, and taking a supernatant and a filter membrane to obtain a to-be-determined solution; s2, detection: adopting a liquid chromatography-high resolution mass spectrometer to detect and analyze the to-be-detected solution obtained in the step S1, and the method realizes simultaneous detection of the 55 polypeptide in cosmetics with different matrixes, and has the advantages of good separation degree, high sensitivity, high accuracy and high precision.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic testing technology, and specifically relates to a method for simultaneously determining 55 peptides in cosmetics based on liquid chromatography-high resolution mass spectrometry. Background Technology

[0002] In recent years, peptides have gained widespread attention and application in the cosmetics, pharmaceutical, and food industries due to their unique biological activities. In the cosmetics field, peptides are widely used as functional ingredients in products for anti-aging, wrinkle reduction, whitening, acne treatment, and skin repair. Examples include acetyl hexapeptide-8 for wrinkle reduction, palmitoyl tetrapeptide-7 for anti-aging, and acetyl octapeptide-3 for improving expression lines. The addition of peptides not only enhances product efficacy but also improves safety and gentleness, reducing the risk of skin allergies. In the pharmaceutical field, peptide drugs are developing rapidly. As of the end of 2023, 37 new peptide drugs had been approved for marketing by the National Medical Products Administration in my country. In the food industry, peptides can be used as nutritional supplements (such as wheat oligopeptides and corn oligopeptide powder) and as functional additives, such as the dipeptide derivative aspartame, a widely used sugar substitute.

[0003] However, in the early stages of peptide application, China mainly relied on imported raw materials, and products were mostly in solution form of varying concentrations. Quality standards were relatively simple, typically only covering routine physicochemical indicators such as pH, color, and odor, as well as limits for microorganisms and heavy metals. There was a lack of precise assessment of peptide purity, structure, and active ingredients, making it difficult to control actual quality. With advancements in chemical synthesis technology, my country can now independently synthesize various functional peptides with high purity, providing a source of assurance for improving the efficacy and quality control of skincare products. Especially in the pharmaceutical field, the qualitative and quantitative analysis of peptide impurities is crucial, and high-purity samples are fundamental for accurate impurity resolution. To achieve accurate detection of peptide components, various analytical techniques have been applied to the qualitative and quantitative analysis of peptides in cosmetics, mainly including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS). For example, Chirita et al. (2009) used LC-MS / MS combined with internal standard method (pal-GHK) to achieve accurate quantification of palmitoylated pentapeptide (pal-KTTKS); Olejnik et al. used MALDI-TOF MS technology to directly detect hexapeptide-3 (ALA-ASP-LEU-LYS-PRO-THR) in cosmetic formulations without complicated pretreatment, demonstrating the convenience and specificity of the method.

[0005] However, due to the strong hydrophilicity of peptides, they exhibit weak retention on traditional reversed-phase chromatography columns, making separation difficult. Therefore, researchers have developed various improved strategies. Papagianni's team used ion-pair reversed-phase liquid chromatography with heptafluorobutyric acid as the ion-pairing reagent to achieve quantitative analysis of oligopeptide-20 on a cyanopropyl column; Giannakou et al. utilized... - A HILIC hydrophilic interaction column combined with electrospray ionization mass spectrometry (ESI-MS) was used to simultaneously determine oligopeptide-20 and oligopeptide-24 in face cream under isocratic elution conditions. This method is rapid, sensitive, and highly selective. For acetyl octapeptide-3, which has a large molecular weight, Ji et al. optimized the mobile phase conditions and constructed its mass spectrometry dataset using liquid chromatography-triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. This dataset was successfully applied to the quantitative analysis of this component in microneedle patches. Chen Yiguang's research team systematically studied the chromatographic behavior, extraction efficiency, and matrix effects of five representative palmitoyl peptides (palmitoyl pentapeptide-3, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, palmitoyl tripeptide-5, and palmitoyl hexapeptide-12), providing important technical references for the quality control of peptide cosmetics.

[0006] Although the above methods have achieved some success, due to the complexity of cosmetic matrices, conventional chromatography or low-resolution mass spectrometry techniques still have problems such as insufficient separation, low sensitivity, and easy false positives in the qualitative and quantitative analysis of peptides. Moreover, current conventional analytical methods cannot detect multiple peptides simultaneously.

[0007] Therefore, obtaining a method that can simultaneously determine peptides in cosmetics is one of the research directions in the field of cosmetic testing. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for the simultaneous determination of 55 peptides in cosmetics based on liquid chromatography-high resolution mass spectrometry. This method can simultaneously determine 55 peptides in cosmetics, and it features high separation, good recovery, and high precision.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] A method for simultaneous determination of 55 peptides in cosmetics based on liquid chromatography-high resolution mass spectrometry, the method comprising the following steps:

[0011] S1. Extraction: Weigh the cosmetic sample to be tested and mix it evenly with the extraction solvent. Perform ultrasonic extraction, centrifuge after extraction, let it stand, take the supernatant, and filter to obtain the test solution.

[0012] S2. Detection: The test solution obtained in step S1 was detected and analyzed using liquid chromatography-high resolution mass spectrometry.

[0013] This invention proposes for the first time a method for the simultaneous detection of 55 polypeptides in cosmetics with different bases. The samples are extracted with an extraction solvent and ultrasonically, and then centrifuged and filtered before being analyzed. Statistical results of this invention show that the detection limit of this method is 0.30-3.00 mg / kg, the quantitation limit is 1.00-20.0 mg / kg, the average recovery rate is 73.07%-116.74%, and the relative standard deviation is between 1.39%-12.48%.

[0014] The method provided by this invention has high separation, good recovery rate, and high precision. It has a certain detection capability for common cosmetics and can identify and quantify polypeptide active ingredients in cosmetics.

[0015] Preferably, in step S1, the mass ratio of the cosmetic sample to the volume ratio of the extraction solvent is 0.5 g: 10 mL.

[0016] Preferably, in step S1, the ultrasonic extraction time is 15 min, the ultrasonic power is 480 W, and the ultrasonic frequency is 40 kHz.

[0017] Preferably, in step S1, the centrifugation speed is 4000-6000 r / min and the centrifugation time is 5-10 min.

[0018] Preferably, the filtration method in step S1 is filtration through a filter membrane, wherein the filter membrane is a 0.45μm organic filter membrane.

[0019] Preferably, the 55 polypeptides are dipeptide-1, dipeptide-2, glutathione (reduced state), carnosine, nonapeptide-1, hexapeptide-1, hexapeptide-11, hexapeptide-3, hexapeptide-9, heptapeptide-6, myristoyl pentapeptide-4, tripeptide-1, tripeptide-1 copper, tripeptide-10 citrulline, tripeptide-2, tripeptide-3, decacapeptide-1, decacapeptide-4, tetrapeptide-1, tetrapeptide-3, tetrapeptide-4, sh-pentapeptide-1, pentapeptide-3, pentapeptide-34 trifluoroacetate, acetyl hexapeptide-1, acetyl hexapeptide-8, acetyl heptacapeptide-4, acetyl tripeptide-1, acetyl tetrapeptide-11, and acetyl hexapeptide-8. Acyl tetrapeptide-2, Acetyl tetrapeptide-3, Acetyl tetrapeptide-5, Acetyl tetrapeptide-9, Palmitoyl dipeptide-7, Palmitoyl hexapeptide-12, Palmitoyl tripeptide-1, Palmitoyl tripeptide-5, Palmitoyl tripeptide-8, Palmitoyl tetrapeptide-10, Palmitoyl tetrapeptide-7, Palmitoyl pentapeptide-4, Hexapeptide-5, Myristoyl hexapeptide-5, Trifluoroacetyl tripeptide-2, Tripeptide-32; Acetyl octapeptide-3, Acetyl hexapeptide-7, Palmitoyl hexapeptide-14, Palmitoyl hexapeptide-15, Palmitoyl pentapeptide-5, Snake venom-like peptide diacetate, Dipeptide-15, Oligopeptide-2, Oligopeptide-3, Oligopeptide-6.

[0020] Preferably, in order to meet the extraction requirements of 55 polypeptides in different base cosmetics, the extraction solvent is a 60% (v / v) methanol aqueous solution, which contains 0.15% formic acid.

[0021] Preferably, the chromatographic conditions for the liquid chromatography are as follows: column: Agilent AdvanceBio PeptidePlus (150 mm × 2.1 mm, 2.7 μm); mobile phase A: acetonitrile, mobile phase B: 0.2% formic acid-water solution; elution mode: gradient elution, flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature: 40 °C.

[0022] Preferably, the gradient elution program is as follows: 0-10 min, 90-10% (volume fraction, the same below) A; 10-15 min, 10%-0% A; 15-17 min, 0% A; 17-17.2 min, 0%-90% A; 17.2-20 min, 90% A.

[0023] Preferably, the mass spectrometry conditions are as follows: positive ion scan with electrospray ionization (ESI); monitoring mode: TOF-IDA / MS; electrospray voltage: 5000V; ion source temperature: 500℃; curtain gas pressure: 35psi; nebulizer gas pressure: 50psi; heating auxiliary gas pressure: 50psi; declustering voltage: 90V; collision energy: 35±15eV.

[0024] Compared with the prior art, the present invention has achieved beneficial technical effects:

[0025] This invention presents, for the first time, a method for the simultaneous detection of 55 peptides in cosmetics with different matrices. Samples are extracted using an extraction solvent and ultrasonically, then centrifuged and filtered before being analyzed. The method provided by this invention exhibits good separation of the 55 peptides, high sensitivity, accuracy, and precision, and the pretreatment method is simple and rapid. This method can be used for risk assessment and screening monitoring of 55 peptides in cosmetics, and has broad application prospects. Attached Figure Description

[0026] Figures 1-5 Graphs showing the separation effects of different chromatographic columns on 55 peptides ( Figure 1 For AdvanceBio Peptide Plus chromatographic columns; Figure 2 for BEH C 18 Chromatographic column; Figure 3 ZORBAX Bonus-RP column; Figure 4 For ZORBAX SB-Aq; Figure 5 for shield RP 18Chromatographic column);

[0027] Figure 6 The separation effect of 55 peptides when the organic phase of the mobile phase is methanol;

[0028] Figure 7 The diagram shows the separation effect of 55 peptides with acetonitrile as the organic phase of the mobile phase.

[0029] Figure 8 The effect of different extraction solvents on the recovery rate of four peptides in different matrices;

[0030] Figure 9 Extraction effects at different methanol concentrations;

[0031] Figure 10 Extraction effects at different formic acid concentrations;

[0032] Figure 11 The extraction effects of different extraction methods;

[0033] Figure 12 The extraction effect at different extraction times. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. However, the scope of protection of this invention is not limited to the specific embodiments described below. The reagents or instruments used in the detection method of this invention can all be purchased from the market.

[0035] Example 1

[0036] This embodiment provides a method for the simultaneous determination of 55 peptides in cosmetics (in this embodiment, the cosmetic is a toner) based on liquid chromatography-high resolution mass spectrometry. The method includes the following steps:

[0037] S1. Extraction: Weigh 0.50 g (accurate to 0.001 g) of sample into a 50 mL centrifuge tube, add 60% methanol (containing 0.15% formic acid) aqueous solution, shake for 2 min to fully mix the sample with the extraction solvent, extract by sonication for 15 min, centrifuge at 4000 r / min for 5 min, take all supernatant and filter through a 0.45 μm organic filter membrane to obtain the test solution.

[0038] S3. Detection: The test solution obtained in step S1 was detected and analyzed using liquid chromatography-high resolution mass spectrometry.

[0039] This embodiment uses a SCIEX 5600+ liquid chromatography-high resolution mass spectrometry system. The chromatographic conditions are as follows: column: Agilent AdvanceBio Peptide Plus (150mm × 2.1mm, 2.7μm); mobile phase A: acetonitrile; mobile phase B: 0.2% formic acid-water solution; elution mode: gradient elution; flow rate: 0.3mL / min; injection volume: 5μL; column temperature: 40℃. The gradient elution program is as follows: 0-10min, 90-10% (volume fraction, the same below) A; 10-15min, 10%-0% A; 15-17min, 0% A; 17-17.2min, 0%-90% A; 17.2-20min, 90% A.

[0040] Mass spectrometry conditions were as follows: Ion source: electrospray ionization (ESI); Scan mode: positive ion mode full scan, TOF scan mass range (m / z): 50-2000; Product ion scan mass range (m / z): 50-2000. Calibration solution flow rate was 0.35 mL / min, and automatic calibration was performed every 5 samples to ensure the system's accurate and stable mass numbers within the batch; Monitoring mode: TOF-IDA / MS; Electrospray voltage: 5000 V; Ion source temperature: 500 °C; Curtain gas pressure: 35 psi; Nebulizer gas pressure: 50 psi; Heating auxiliary gas pressure: 50 psi; Declustering voltage: 90 V; Collision energy: 35 ± 15 eV.

[0041] Examples 2-4

[0042] Referring to Example 1, the difference is that the cosmetics in Examples 2-4 are, in order, face cream, eye cream, and face mask.

[0043] The following examples examine the effects of different factors on the detection of 55 peptides in cosmetics with different bases.

[0044] The preparation of the 55 polypeptide standard working solutions is as follows:

[0045] (1) Preparation of standard stock solution: Accurately weigh 0.020 g of 55 polypeptide standards and place them in 10 mL volumetric flasks. Dissolve them by sonication with 60% methanol-water solution (containing 0.1% formic acid) and dilute to the mark. Shake well to prepare 55 standard stock solutions with a mass concentration of 2000 mg / L. Store in a refrigerator at 0-4℃ away from light.

[0046] (2) Preparation of mixed standard intermediate solution: Take 150 μL of each of the 55 standard stock solutions and place them in a 10 mL volumetric flask. Dilute with 60% methanol-water solution (containing 0.1% formic acid) and make up to the mark. Shake well to prepare a 30 mg / L mixed standard intermediate solution. Store in a refrigerator at 0-4℃ away from light.

[0047] (3) Preparation of matrix standard series solutions: Before use, dilute the mixed standard intermediate solution to an appropriate concentration with blank matrix solution (the liquid after the cosmetic sample without peptides has been treated by S1 in Example 1) as needed, and use it immediately.

[0048] Example 5: Optimization of liquid chromatography and mass spectrometry determination conditions

[0049] 1. Optimization of liquid phase conditions

[0050] 1.1 Selection of chromatographic column

[0051] This embodiment compares... BEH C18 (4.6mm×100mm, 2.5μm), The separation effects of five chromatographic columns—Shield RP18 (4.6mm×50mm, 3.5μm), ZORBAX SB-Aq (4.6mm×100mm, 3.5μm), ZORBAX Bonus-RP (4.6mm×100mm, 3.5μm), and AdvanceBio Peptide Plus (2.1mm×150mm, 2.7μm)—on 55 peptide components were investigated. The injection volume was 5μL, the column temperature was 40℃, and the mobile phases were acetonitrile (A) and 0.2% formic acid aqueous solution (B). The results are shown in the figure. Figure 1-5 .

[0052] BEH C 18 Columns and shield RP 18 All columns are nonpolar reversed-phase columns, exhibiting good stability under low pH conditions. Figure 5 and Figure 2 As can be seen, both columns exhibit good separation for peptides of different molecular weights, but there are instances of poor peak shape and no peak observed. Both the ZORBAX SB-Aq column and the ZORBAX Bonus-RP column are hydrophilic chromatographic columns, capable of effectively separating polar compounds and exhibiting excellent retention of hydrophilic compounds. Figure 4 and Figure 3As can be seen, both columns share the characteristic of good response to larger peptide molecules such as oligopeptide-3 and oligopeptide-6. However, most non-palmitoyl peptide analytes, after separation by the ZORBAX SB-Aq column, exhibit generally late retention times, peak tailing, and asymmetry, while the response to small peptide molecules is poor. In contrast, the ZORBAX Bonus-RP column shows better resolution, with only oligopeptide-3 exhibiting peak tailing, and a relatively higher response. The AdvanceBio Peptide Plus column is a non-polar column suitable for polar mobile phases containing formic acid, providing high sensitivity and efficiency for rapid identification of peptides and target proteins. Figure 1 As can be seen, the peak shapes of all 55 peptides were normal, and there were no issues such as tailing or poor response. Given that it exhibited the best overall separation performance among the five chromatographic columns, the AdvanceBio Peptide Plus column was selected for this invention.

[0053] 2.2 Selection of Mobile Phase

[0054] The chromatographic column was an AdvanceBio Peptide Plus (2.1 mm × 150 mm, 2.7 μm), and the injection volume was 5 μL. Under a column temperature of 40 °C, the effects of two different organic phases (methanol and acetonitrile) on the response and resolution of 55 polypeptide components were first investigated. The aqueous phase was 0.1% formic acid solution. Chromatographic analysis was performed, and the results are shown in Figures 6 and 7. When methanol was used as the organic phase, the response and resolution of the 55 polypeptide components were slightly better than in the acetonitrile system. However, when methanol was used as the organic phase, the peaks of acetyl tetrapeptide-11, acetyl tripeptide-1, acetyl tetrapeptide-9, acetyl octapeptide-3, oligopeptide-3, and oligopeptide-6 were broadened and exhibited spikes, which was detrimental to analysis. Considering that acetonitrile has a stronger elution ability than methanol, acetonitrile was selected as the organic phase in the method of this invention.

[0055] Then, using acetonitrile as the organic phase, the effects of different aqueous phases (0.1% formic acid water, 0.2% formic acid water, and 0.3% formic acid water) on the response and resolution of 55 polypeptide components were analyzed by chromatography, and parameters such as peak area (see Table 1 below) and retention time (see Table 2 below) of the analytes were recorded.

[0056] Table 1. Effects of different aqueous phases (different formic acid concentrations) on the peak area of ​​55 peptide components by acetonitrile systems.

[0057]

[0058]

[0059]

[0060] As shown in Table 2, the pH of the acetonitrile-0.1% formic acid aqueous mobile phase is higher than the other two systems, resulting in almost no peaks for glutathione and larger molecular weight peptides (such as nonapeptide-1, oligopeptide-3, and oligopeptide-6). With increasing acid concentration, the overall peak shape and peak area of ​​the acetonitrile-0.2% formic acid aqueous mobile phase improved. Further increasing the acid concentration slightly increased the peak area of ​​small molecular weight peptides, but the peak areas of palmitoyl peptides and larger molecular weight peptides generally showed a decreasing trend. This is likely because increasing formic acid concentration may increase the ionic strength in the mobile phase, triggering an ion inhibition effect and affecting peptide stability. Small molecular weight peptides experience less ion inhibition, resulting in a slight increase in peak area, but palmitoyl peptides and larger molecular weight peptides have weaker polarity, and their ionization may be more strongly inhibited, leading to a decrease in peak area. Therefore, this invention uses a 0.2% formic acid aqueous solution as the aqueous phase.

[0061] This invention employs a nonpolar reversed-phase chromatographic column (AdvanceBio Peptide Plus column) and a polar mobile phase. The more nonpolar the analyte, the later the retention time. Figure 1 And as shown in Table 2 below.

[0062] Table 2 Retention times of 55 peptide components using AdvanceBio Peptide Plus columns

[0063]

[0064]

[0065]

[0066] Table 2 shows that small molecule peptides retained relatively early on this column, while non-derivative peptides with molecular weights greater than 600 (such as hexapeptide-11 and oligopeptide-3) had moderate retention times. Acetyl peptides generally retained earlier, while palmitoyl peptides generally retained later. Small molecule peptides and acetyl peptides, due to their shorter carbon chains and the presence of polar groups such as terminal carboxyl groups (—COOH), amino groups (—NH2), guanidinyl groups (—NH—C(=NH)—NH—), amide groups (—CONH2), acetyl groups (—COCH3), and imidazole rings), possess hydrophilicity and enhance molecular polarity, resulting in retention times generally around 1 min. However, acetyl tetrapeptide-11 is an exception, with a retention time of 4.27 min. Based on its molecular structure, it contains two tetrahydropyrrole rings. Even with the presence of carboxyl and acetyl groups, its overall hydrophilicity is significantly reduced, hence the relatively late retention time of acetyl tetrapeptide-11.

[0067] Larger polypeptide molecules, due to their longer carbon chains and the presence of nonpolar groups such as methylthio (-SCH3), n-propyl, isobutyl, and benzene rings, exhibit significantly reduced molecular polarity. In contrast, palmitoyl (-COC) groups... 15 H 31 Because these are long carbon-chain nonpolar groups, larger molecular weight peptides and palmitoyl peptides generally have later retention times. Palmitoyl pentapeptide-4, with its larger molecular weight, has a relatively earlier retention time. This is likely because its molecular structure lacks nonpolar groups such as tetrahydropyrrole rings and benzene rings, instead containing three hydroxyl groups (—OH) and two amino groups, resulting in relatively higher hydrophilicity. Although oligopeptide-3 and oligopeptide-6 have long carbon chains, the number of hydrophilic groups is correspondingly increased, and they lack nonpolar group modifications; therefore, their retention times are similar to those of hexapeptide-11, heptapeptide-6, and nonapeptide-1.

[0068] 2. Optimization of mass spectrometry conditions

[0069] The choice of ionization method directly affects the detection sensitivity and fragmentation pattern of the analyte. As shown in Table 3 below, this experiment used an electrospray ionization source to screen out the optimal ionization methods for 55 peptide components, mainly including four forms: +H, +2H, +3H, and +4H.

[0070] The +H ionization method is suitable for small molecule peptides, such as glutathione, dipeptide-10, and copper tripeptide-1. Due to their small molecular weight, single-charge ionization can provide sufficient detection sensitivity. For larger molecular weight peptides and derivatives, such as nonapeptide-1, acetyl heptapeptide-4, and palmitoyl pentapeptide-4, +2H and +3H ionization methods are required. Oligopeptide-6 has a more complex molecular structure and the largest molecular weight, therefore, a +4H ionization method is required to provide sufficient detection sensitivity.

[0071] The precise mass fraction of the parent ion and its isotopic abundance ratio can be used for qualitative analysis; therefore, the parent ion is used for both qualitative and quantitative analysis, while secondary fragments with higher abundance serve as auxiliary references for qualitative analysis. The m / z values ​​of the daughter ions can be used to resolve the structure of polypeptide molecules. Specific mass spectrometry parameters are shown in Table 3.

[0072] Table 3. Parameters of 55 peptides

[0073]

[0074]

[0075] As shown in Table 3, this invention collected the molecular ion peaks of each polypeptide component and identified the corresponding parent ion and the three secondary fragments with the highest peaks based on the corresponding ionization methods. For polypeptides, peptide bonds have relatively weak bond energies in the overall molecular structure, making them most susceptible to breakage when subjected to calibration transmission system (CDS) collisions. The mass spectrometry fragmentation analysis of glutathione and acetyl hexapeptide-8 is as follows: Glutathione has a molecular weight of 307.32 g / mol and its amino acid sequence is γ-Glu-Cys-Gly, composed of glutamic acid, cysteine, and glycine. After removing the glycine fragment (75.07 g / mol), the remaining mass is approximately 232.25 g / mol, which is very close to the molecular ion peak of 231.0431. Therefore, this fragment is likely the part of the glutathione molecule after the glycine fragment has fallen off.

[0076] Acetyl hexapeptide-8 has a molecular weight of 888.97 g / mol and its amino acid sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH2, which consists of two glutamic acid molecules, methionine, glutamic acid, two arginine molecules, and alanine. Structural analysis reveals that the value of the secondary fragment 572.3096 is extremely close to that of the Met-Gln-Arg-Arg-Ala-NH2 fragment (641.81 - 18 × 4 = 569.81 g / mol), possibly related to the ionization mode of the +2H group.

[0077] Example 6: Effect of different extraction solvents and solvent concentrations on peptide recovery rate in cosmetics

[0078] 1. Effects of different extraction solvents on the recovery rate of peptides in cosmetics

[0079] A mixed solution of 50 μL of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides at a concentration of 100 mg / L was added to a blank cosmetic sample (the cosmetic sample did not contain peptide molecules). The sample was treated according to step S1 in Example 1. The difference from Example 1 was that four different extraction reagents (methanol, methanol-0.20% formic acid, acetonitrile, and acetonitrile-0.20% formic acid from Table 5) were used to prepare the test solution. The solution was then analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the recoveries of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides were calculated. The results are shown below. Figure 8 As shown. From Figure 8The results show that, compared to acetonitrile, the recovery rates of small molecule peptides, acetyl peptides, and palmitoyl peptides are higher when using an extraction reagent containing methanol. Therefore, the extraction reagent needs to contain methanol. Compared to pure solvents, using 0.20% formic acid to increase the acid concentration for extraction resulted in recovery rates of small molecule peptides, acetyl peptides, and palmitoyl peptides all around 100%. However, the recovery rate of medium-molecular-weight oligopeptides was the lowest (<80%), with a maximum of only 41.62%. This indicates that using organic solvents alone, without an acidic system, cannot adequately disperse larger molecule peptide components and cannot achieve good extraction results. Therefore, the extraction reagent in this method needs to contain formic acid.

[0080] Table 4 Different extraction reagents

[0081]

[0082] 2. Effects of methanol and formic acid concentrations on the recovery rate of peptide molecules in cosmetics

[0083] A mixed solution of 50 μL of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides at a concentration of 100 mg / L was added to a blank cosmetic sample (the cosmetic sample did not contain peptide molecules). The sample was treated according to step S1 in Example 1, except that different extraction reagents were selected (all extraction reagents listed in Table 5 except for methanol, methanol-0.20% formic acid, acetonitrile, and acetonitrile-0.20% formic acid). The resulting test solutions were then analyzed using liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the recoveries of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides were calculated. The results are shown below. Figure 9 , 10 As shown, thus Figure 9 It was found that the recovery rates of small molecule peptides and acetyl peptides remained stable above 90% in methanol solutions of five different concentrations. With increasing methanol concentration, the recovery rate of palmitoyl peptides also increased, rising from a minimum of 27.31% to 107.35%. Medium molecule oligopeptides showed good recovery rates only in 50% and 60% methanol aqueous solutions; therefore, this invention selected a 60% methanol aqueous solution. Figure 10 As can be seen, the overall recovery rate of palmitoyl peptides was significantly improved under the influence of formic acid, remaining stable at 80%–100%. However, the recovery rate of medium-molecular-weight oligopeptides was low at a formic acid concentration of 0.20%. When the formic acid concentration increased to 0.25%, the recovery rate of acetyl peptides decreased accordingly, possibly due to the presence of H+ in the solution. +As the concentration increases, some acetyl groups undergo hydrolysis, causing them to detach from the polypeptide, altering its structure and properties, and consequently affecting extraction and detection. Therefore, considering the above factors, this invention selects a 60% methanol-water solution (containing 0.15% formic acid) as the extraction solvent for 55 polypeptide components.

[0084] Example 9: Effect of different extraction methods on peptide recovery rate in cosmetics

[0085] A mixed solution of 50 μL of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides at a concentration of 100 mg / L was added to a blank cosmetic sample (the cosmetic sample did not contain peptide molecules). The sample was processed according to step S1 in Example 1, using ultrasonic extraction, vortex extraction, and horizontal shaking extraction to obtain the test solution. The solution was then analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the recoveries of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides were calculated. The results are shown below. Figure 11 As shown, ultrasonic extraction yielded good extraction results for different types of peptides. Vortex extraction showed slightly better extraction results for small molecule peptides, acetyl peptides, and palmitoyl peptides than ultrasonic extraction. However, for medium-molecular-weight oligopeptides, vortex extraction actually reduced the recovery rate. This may be because oligopeptides have larger molecular weights and more complex structures, making them more likely to bind firmly to the sample matrix. The gentle shear force of the vortex is insufficient for effective dissociation, while the high energy of ultrasound can more effectively dissociate larger molecule oligopeptides from the matrix, thus improving the recovery rate. While oscillation extraction is similar to ultrasonic extraction, the recovery rate of acetyl peptides showed a decreasing trend. Therefore, this invention chose to use ultrasonic extraction.

[0086] Example 7: Effect of different extraction times on peptide recovery rate in cosmetics

[0087] A mixed standard solution of 50 μL containing 100 mg / L small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides was added to a blank cosmetic sample (the cosmetic sample did not contain peptide molecules). The sample was processed according to step S1 in Example 1, with different extraction times (see table below) to obtain the test solution. The sample was then analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the recoveries of small molecule peptides, acetyl peptides, palmitoyl peptides, and medium molecule oligopeptides were calculated. The results are shown below. Figure 12 As shown, from Figure 12It was observed that within the extraction time range of 5–15 min, the recovery rate increased with increasing extraction time. However, after 20 min, the recovery rates of small molecule peptides, acetyl peptides, and palmitoyl peptides decreased, while the recovery rate of medium-molecule oligopeptides exceeded 120%. This is presumably because prolonged ultrasound exposure can cause a thermal effect, potentially leading to peptide bond breakage in the oligopeptides and affecting their integrity. The resulting small molecule fragments exhibit higher response signals, thus causing an inflated calculated recovery rate. Therefore, this invention selected an extraction time of 15 min.

[0088] Table 5 Different extraction times

[0089]

[0090] Example 8: Effect of matrix effect on peptide recovery rate in cosmetics

[0091] This embodiment investigates the influence of matrix effect. Blank samples of toner and face cream were selected and processed according to step S1 of Example 1 to obtain blank matrix solutions. Standard solutions were prepared using the blank matrix solutions and 60% methanol-water (containing 0.1% formic acid), and standard curves were plotted to obtain the slope k of the standard curves prepared by these two matrices and 60% methanol-water (containing 0.1% formic acid). The specific process was as follows: An appropriate amount of 30 mg / L standard solution was accurately measured and diluted with the blank matrix solution and 60% methanol-water (containing 0.1% formic acid) to prepare a series of matrix standard working solutions, which were prepared and used immediately. The standard working solutions were analyzed using a liquid chromatography-high resolution mass spectrometer under the above-mentioned determination conditions, with the standard working solution concentrations increasing sequentially. Chromatographic data were obtained, and the chromatographic data obtained using TraceFinder 3.2.368.22 data processing software were plotted with the peak area of ​​the quantitative ion chromatographic peaks of 55 peptides as the ordinate and the corresponding standard solution concentration as the abscissa to create a standard working curve. The matrix effect ME was calculated as follows:

[0092]

[0093] In this invention, blank samples without toners or creams were selected to investigate the matrix effect. A series of working solutions of varying concentrations were obtained by diluting the standard intermediate solution with blank matrix solution and 60% methanol-water (containing 0.1% formic acid). These solutions were then analyzed to obtain blank matrix curves and solvent curves. The slope k of the matrix curve was used as the criterion. 基质 The slope k of the solvent curve 溶剂 Substitute into the K value calculation formula: (k 基质 / k 溶剂 Calculate K by multiplying K by 100% (where K) 溶剂The slope of the standard curve for a non-matrix-prepared 60% methanol-water (containing 0.1% formic acid) solvent is used to derive the matrix effect (ME), i.e., the K value, which is then used as the evaluation index.

[0094] If K is between -10% and 10%, it indicates that the matrix effect is weak and not obvious;

[0095] If K < -10%, a strong inhibitory effect is observed.

[0096] If K > 10%, a strong enhancement effect will be observed.

[0097] The K-values ​​for cosmetics with different bases are shown in Table 6 below.

[0098] Table 655 matrix effects of peptide components in different cosmetics

[0099]

[0100]

[0101]

[0102] Table 6 shows that 55 peptide components exhibit significant matrix effects. Small molecule peptides and acetyl peptides mainly show inhibitory effects, while palmitoyl peptides mainly show enhancing effects. Experimental results indicate that toners and creams significantly interfere with the determination of the target compounds. Among them, the matrix effect of the cream sample has the most significant interference with the detection of the target compounds, while the matrix effect of the toner is relatively weak. Therefore, this invention aims to eliminate the interference of matrix effects by preparing a standard curve using a blank matrix solution.

[0103] Examples 9 and 10 are used to verify the accuracy, sensitivity and repeatability of the detection method of the present invention.

[0104] Example 9

[0105] The mixed standard solution was diluted to different concentration gradients using blank matrix solution, and analyzed by injection according to the optimized instrument method. A standard curve was plotted with the quantitative ion peak area as the ordinate (y) and the corresponding concentration as the abscissa (x), and the corresponding linear equation was fitted, as shown in Table 7 below. The linear range was determined according to the detection sensitivity of each peptide as follows: the linear range of most small molecule peptides and acetyl peptides is 50-2000 μg / L; the linear range of 15 peptides, including myristoyl pentapeptide-4, is 25-1000 μg / L; the linear range of 5 peptides, including tripeptide-1, is 250-10000 μg / L; and the linear range of 2 oligopeptides is 500-20000 μg / L.

[0106] Table 755 shows the linear parameters, limits of detection, and limits of quantitation for each polypeptide component.

[0107]

[0108]

[0109]

[0110] As shown in Table 7, the 55 polypeptides exhibited good linearity, with a correlation coefficient (R0). 2 All values ​​were greater than 0.99, the limit of detection (LOD) was 0.30-3.00 mg / kg, and the limit of quantitation (LOQ) was 1.00-20.0 mg / kg.

[0111] Example 9

[0112] Two types of cosmetic blank matrices (toner and cream) were used, and 55 peptides were spiked at three concentration levels (2.0 mg / kg, 10.0 mg / kg, and 20.0 mg / kg). Six parallel experiments were conducted simultaneously. The recovery rate (%) and relative standard deviation (RSD) of the three spiked concentration levels and the test results are shown in Table 8.

[0113] Table 8. Recovery rates and relative standard deviations of 855 polypeptide components in two cosmetic matrices (n=6)

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] As shown in Table 8, the average recoveries of the 55 peptides ranged from 3.07% to 116.74%, with relative standard deviations (RSDs) ranging from 1.39% to 11.75%. The experimental results indicate that this method has good accuracy and precision.

[0120] Application Examples

[0121] Using the detection methods of Examples 1-4, 40 batches of peptide cosmetic samples from well-known market brands were tested. These cosmetics included 14 batches of essence water, 15 batches of face cream, 9 batches of eye cream, and 2 batches of face mask. The results are recorded in Table 9.

[0122] Table 9. Detection content of peptide components in cosmetic samples

[0123]

[0124]

[0125] Table 9 shows that 14 out of 40 batches of peptide-based cosmetic samples from well-known brands on the market tested positive for peptide components, including carnosine, acetyl hexapeptide-8, and palmitoyl tripeptide-1. Among these, carnosine, palmitoyl tetrapeptide-7, and palmitoyl tetrapeptide-7 have relatively high usage rates in peptide-based cosmetics. The remaining 26 batches did not test positive for the above 20 peptide components. The reasons for this could be that the peptide content in some products may be below the detection limit of this method, resulting in undetectable data; alternatively, there may be discrepancies between the label information and the actual ingredients, which could be due to regulatory loopholes, cost pressures, and market competition. In summary, the experimental method developed in this invention has a certain detection capability for common types of cosmetics on the market and can identify and quantify the effective peptide components in cosmetics.

[0126] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the invention.

Claims

1. A method for the simultaneous determination of 55 peptides in cosmetics based on liquid chromatography-high resolution mass spectrometry, characterized in that, The method includes the following steps: S1. Extraction: Weigh the cosmetic sample to be tested and mix it evenly with the extraction solvent. Perform ultrasonic extraction, centrifuge after extraction, let it stand, take the supernatant, and filter to obtain the test solution. S2. Detection: The test solution obtained in step S1 was detected and analyzed using liquid chromatography-high resolution mass spectrometry.

2. The method according to claim 1, characterized in that, The ultrasonic extraction time in S1 is 15 min.

3. The method according to claim 1, characterized in that, In S1, the centrifugation speed is 4000-6000 r / min, and the centrifugation time is 5-10 min.

4. The method according to claim 1, characterized in that, The filtration method in step S1 is filtration through a filter membrane, which is a 0.45μm organic filter membrane.

5. The method according to claim 1, characterized in that, The 55 polypeptides are dipeptide-1, dipeptide-2, glutathione (reduced state), carnosine, nonapeptide-1, hexapeptide-1, hexapeptide-11, hexapeptide-3, hexapeptide-9, heptapeptide-6, myristoyl pentapeptide-4, tripeptide-1, tripeptide-1 copper, tripeptide-10 citrulline, tripeptide-2, tripeptide-3, decacapeptide-1, decacapeptide-4, tetrapeptide-1, tetrapeptide-3, tetrapeptide-4, sh-pentapeptide-1, pentapeptide-3, pentapeptide-34 trifluoroacetate, acetyl hexapeptide-1, acetyl hexapeptide-8, acetyl heptacapeptide-4, acetyl tripeptide-1, acetyl tetrapeptide-11, and acetyl... Tetrapeptide-2, Acetyl Tetrapeptide-3, Acetyl Tetrapeptide-5, Acetyl Tetrapeptide-9, Palmitoyl Dipeptide-7, Palmitoyl Hexapeptide-12, Palmitoyl Tripeptide-1, Palmitoyl Tripeptide-5, Palmitoyl Tripeptide-8, Palmitoyl Tetrapeptide-10, Palmitoyl Tetrapeptide-7, Palmitoyl Pentapeptide-4, Hexapeptide-5, Myristoyl Hexapeptide-5, Trifluoroacetyl Tripeptide-2, Tripeptide-32; Acetyl Octapeptide-3, Acetyl Hexapeptide-7, Palmitoyl Hexapeptide-14, Palmitoyl Hexapeptide-15, Palmitoyl Pentapeptide-5, Snake Venom Peptide Diacetate, Dipeptide-15, Oligopeptide-2, Oligopeptide-3, Oligopeptide-6.

6. The method according to claim 1, characterized in that, The extraction solvent is a 60% (v / v) methanol aqueous solution, which contains 0.15% formic acid.

7. The method according to claim 1, characterized in that, The chromatographic conditions for the liquid chromatography were as follows: column: Agilent Advance Bio Peptide Plus (150 mm × 2.1 mm, 2.7 μm); mobile phase A: acetonitrile; mobile phase B: 0.2% formic acid-water solution; elution mode: gradient elution; flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature: 40 °C.

8. The method according to claim 7, characterized in that, Gradient elution program: 0-10 min, 90-10% (volume fraction, the same below) A; 10-15 min, 10%-0% A; 15-17 min, 0% A; 17-17.2 min, 0%-90% A; 17.2-20 min, 90% A.

9. The method according to claim 1, characterized in that, The mass spectrometry conditions were as follows: positive ion scan with electrospray ionization (ESI); monitoring mode: TOF-IDA / MS; electrospray voltage: 5000V; ion source temperature: 500℃; curtain gas pressure: 35psi; nebulizer gas pressure: 50psi; heating auxiliary gas pressure: 50psi. Declustering voltage: 90V; Collision energy: 35±15eV.

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