Method for detecting 21 oligopeptides in cosmetics

Abstract

The application discloses a method for detecting 21 oligopeptides in cosmetics, comprising the following steps: adding an ammonium acetate aqueous solution to the cosmetic to be detected, uniformly vortexing and dispersing, then adding methanol for ultrasonic extraction, centrifuging, filtering the supernatant, and then performing ultra-high performance liquid chromatography tandem mass spectrometry detection. In the application, the extraction solvent and extraction method of the sample pretreatment are optimized, and the chromatographic separation conditions and mass spectrometry parameters are optimized, so that the recovery of the 21 oligopeptides is improved, the 21 oligopeptides are effectively separated, a simultaneous detection method for the 21 oligopeptides in the cosmetics is established, and an effective detection means is provided for the qualitative and quantitative detection of the oligopeptides in the cosmetics. The detection method is simple in operation, accurate and reliable in determination result, effectively makes up for the defects of the existing detection methods, and provides important technical support for product research and development, product quality control and market supervision of the cosmetic industry.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and more specifically, this invention relates to a method for detecting 21 oligopeptides in cosmetics. Background Technology

[0002] Peptides are composed of specific amino acids arranged in different sequences. While their structure is relatively simple, they possess important physiological activities. Many small-molecule oligopeptides have been proven to have functions such as wrinkle reduction, regulating cell metabolism, repairing and replacing damaged and aging cells, delaying aging, and supplementing nutrition for human skin. Oligopeptides ranging from dipeptides to decapeptides are commonly used in cosmetics, such as acetyl hexapeptide-8, glutathione, L-carnosine, tripeptide-1, palmitoyl tripeptide-1, and palmitoyl pentapeptide-4. Oligopeptides are safe, highly active, and easily absorbed, playing an increasingly important role as active ingredients in cosmetics. However, oligopeptide raw materials are diverse and generally expensive. Commercially available cosmetics may contain errors or false labeling, making it impossible to guarantee the actual efficacy of the products. Therefore, there is an urgent need to establish scientific and efficient detection methods for oligopeptides in cosmetics, enabling rapid and accurate determination of oligopeptide components in products.

[0003] Currently, the main methods for detecting oligopeptides in cosmetics include electrophoresis, high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among these, LC-MS / MS is the preferred method for qualitative and quantitative detection of oligopeptides due to its high specificity and sensitivity. However, because different types of oligopeptides have vastly different properties, solubility, and chromatographic retention behaviors, the currently reported LC-MS / MS detection methods are only suitable for the simultaneous detection of a few types of oligopeptides, and these oligopeptides must all be either hydrophilic or lipophilic.

[0004] Therefore, it is essential to develop a method that can simultaneously detect multiple oligopeptides, including hydrophilic and lipophilic oligopeptides. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for detecting 21 oligopeptides in cosmetics.

[0006] The technical solutions for achieving the above-mentioned objectives include the following.

[0007] In a first aspect, the present invention provides a method for detecting 21 oligopeptides in cosmetics, comprising the following steps: adding an ammonium acetate aqueous solution to the cosmetic to be tested, vortexing to disperse evenly, then adding methanol for ultrasonic extraction, centrifuging, filtering the supernatant, and then performing ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0008] In this invention, by optimizing the extraction solvent and extraction method in sample pretreatment, and combining optimized chromatographic separation conditions and mass spectrometry parameters, the spiked recovery rate of 21 oligopeptides was improved, achieving effective separation of the 21 oligopeptides. A method for the simultaneous detection of 21 oligopeptides in cosmetics was established, providing an effective detection means for the qualitative and quantitative detection of oligopeptides in cosmetics. The detection method of this invention is simple to operate, and the measurement results are accurate and reliable, effectively making up for the deficiencies of existing testing methods. This method provides important technical support for product development, product quality control, and market supervision in the cosmetics industry. Attached Figure Description

[0009] Figure 1 The image shows the MRM chromatogram of a mixed standard solution (500 μg / L) of 21 oligopeptides in Example 1 of this invention, where 1 to 21 represent palmitoyl hexapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, dipeptide-2, tripeptide-1, palmitoyl tripeptide-1, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, acetyl tetrapeptide-2, myristoyl pentapeptide-4, acetyl tetrapeptide-9, glutathione, tetrapeptide-4, hexapeptide-9, dipeptide diaminobutyryl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-5, L-carnosine, pentapeptide-3, and tripeptide-10 citrulline, respectively.

[0010] Figure 2 The following are MRM chromatograms of 21 oligopeptides under different column conditions in Experimental Example 1 of this invention, wherein (a) is a C18 column; (b) is a cyano column; (c) is a HILIC column (Agilent); (d) is an amino column; and (e) is a HILIC column (Waters). 1 to 21 represent palmitoyl hexapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, dipeptide-2, tripeptide-1, palmitoyl tripeptide-1, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, acetyl tetrapeptide-2, myristoyl pentapeptide-4, acetyl tetrapeptide-9, glutathione, tetrapeptide-4, hexapeptide-9, dipeptide diaminobutyryl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-5, L-carnosine, pentapeptide-3, and tripeptide-10 citrulline, respectively.

[0011] Figure 3The images show the MRM chromatograms of 21 oligopeptides under different mobile phase conditions in Experimental Example 3 of this invention. In these chromatograms, (a) to (u) represent palmitoyl hexapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, dipeptide-2, tripeptide-1, palmitoyl tripeptide-1, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, acetyl tetrapeptide-2, myristoyl pentapeptide-4, acetyl tetrapeptide-9, glutathione, tetrapeptide-4, hexapeptide-9, dipeptide diaminobutyryl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-5, L-carnosine, pentapeptide-3, and tripeptide-10 (citrulline), respectively. 1 to 10 represent mobile phases of: water + methanol, water + acetonitrile, 0.1% formic acid aqueous solution + acetonitrile, 0... 0.1% formic acid aqueous solution + acetonitrile (containing 0.1% formic acid), 5 mmol / L ammonium acetate aqueous solution (containing 0.05% formic acid) + acetonitrile (containing 0.05% formic acid), 10 mmol / L ammonium acetate aqueous solution (containing 0.05% formic acid) + acetonitrile (containing 0.05% formic acid), 15 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid) + acetonitrile (containing 0.1% formic acid), 20 mmol / L ammonium acetate aqueous solution (containing 0.2% formic acid) + acetonitrile (containing 0.2% formic acid), 25 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid) + acetonitrile (containing 0.1% formic acid), 15 mmol / L ammonium acetate aqueous solution (containing 0.2% formic acid) + acetonitrile (containing 0.2% formic acid). Detailed Implementation

[0012] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0013] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0014] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Green and Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2013), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0015] In the detection method for 21 oligopeptides in cosmetics in this invention, the pretreatment method of the cosmetics to be tested was first optimized (the cosmetics were dispersed by vortexing in an aqueous solution of ammonium acetate containing formic acid, followed by ultrasonic extraction with methanol; the addition of ammonium acetate increased the ionic strength of the extraction solution, disrupting the original ionic system of the matrix and making the oligopeptides easier to release; the addition of formic acid adjusted the pH of the solution, making the hydrophilic oligopeptides more soluble in the solution; the addition of methanol for ultrasonic extraction made the extraction of lipophilic oligopeptides more complete; the synergistic effect improved the spiked recovery rate of oligopeptides). Then, the chromatographic conditions of ultra-high performance liquid chromatography were further optimized (HILIC column was selected, preferably Poroshell). A 120HILIC-Z column was used, which can effectively separate oligopeptides and effectively separate them from the matrix. The mobile phases selected were A: 10 mmol / L to 20 mmol / L ammonium acetate aqueous solution containing 0.05% to 0.2% formic acid, and B: acetonitrile containing 0.05% to 0.2% formic acid. The addition of formic acid can improve ionization efficiency and oligopeptide response. The addition of ammonium acetate, which has a competitive effect, can effectively improve the peak tailing phenomenon caused by secondary retention. The mass spectrometry conditions (including ion mode, parent ion, daughter ion, collision energy, auxiliary gas temperature, spray voltage, sheath gas and auxiliary gas flow rate) were also selected to achieve effective separation of 21 oligopeptides (including hydrophilic and lipophilic oligopeptides) in cosmetics. A method for simultaneous detection of 21 oligopeptides in cosmetics was established.

[0016] In some embodiments of the present invention, a method for detecting 21 oligopeptides in cosmetics is disclosed, comprising the following steps: adding ammonium acetate aqueous solution to the cosmetic to be tested, vortexing to disperse evenly, then adding methanol for ultrasonic extraction, centrifuging, filtering the supernatant, and then performing ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0017] In some embodiments, the concentration of ammonium acetate in the aqueous solution is 0.05 mol / L to 0.15 mol / L, preferably 0.1 mol / L.

[0018] In some embodiments, the aqueous ammonium acetate solution also contains 0.1% to 0.5% (volume concentration) of formic acid, preferably 0.25%.

[0019] In some embodiments, the volume ratio of the ammonium acetate aqueous solution to methanol is 30–60:70–40.

[0020] In some embodiments, the ultrasonic extraction time is 10 min to 20 min.

[0021] In some embodiments, the method for detecting 21 oligopeptides in the cosmetic includes the following steps: accurately weighing 0.1g to 0.5g of the cosmetic to be tested, adding 1.5mL to 3mL of a 0.05mol / L to 0.15mol / L ammonium acetate aqueous solution containing 0.1% to 0.5% formic acid, vortexing and dispersing evenly, adding methanol to 5mL, mixing and ultrasonically extracting for 10min to 20min, centrifuging, filtering the supernatant through a 0.22μm organic filter membrane, and performing ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0022] In some embodiments, the ultra-high performance liquid chromatography uses a Poroshell 120 HILIC-Z column.

[0023] In some embodiments, the mobile phase used in the ultra-high performance liquid chromatography is: A: 10 mmol / L to 20 mmol / L ammonium acetate aqueous solution containing 0.05% to 0.2% formic acid, and B: acetonitrile containing 0.05% to 0.2% formic acid.

[0024] In some embodiments, the ultra-high performance liquid chromatography employs gradient elution with the following gradient elution program: 0.0–1.0 min, 90% B; 1.0–8.0 min, 90% B–50% B; 8.0–12.0 min, 50% B; 12.1–17.0 min, 90% B.

[0025] In some embodiments, the mass spectrometry conditions include:

[0026] Electrospray ionization source in positive ion mode;

[0027] Spray voltage: 3500±500V;

[0028] Sheath gas: 40±10 Unit;

[0029] Auxiliary gas: 12±5 Unit;

[0030] Auxiliary gas temperature: 350±50℃;

[0031] Ion transfer tube temperature: 325±50℃;

[0032] The data acquisition mode is Multi-Response Monitoring (MRM).

[0033] In some embodiments, the 21 oligopeptides include palmitoyl hexapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, dipeptide-2, tripeptide-1, palmitoyl tripeptide-1, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, acetyl tetrapeptide-2, myristoyl pentapeptide-4, acetyl tetrapeptide-9, glutathione, tetrapeptide-4, hexapeptide-9, dipeptide diaminobutyryl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-5, L-carnosine, pentapeptide-3, and tripeptide-10 citrulline.

[0034] Information on the retention time, parent ion, daughter ion, collision energy, etc. of the 21 oligopeptides is shown in Table 1.

[0035] Table 121 Mass Spectrometry Parameters of Oligopeptides

[0036]

[0037]

[0038] Note: "*" indicates quantitative ions.

[0039] The instruments used in the following examples include: Ultimate 3000 ultra-high performance liquid chromatograph (UHPLC): Thermo Fisher Scientific, USA; TSQ Quantiva triple quadrupole mass spectrometer (MS / MS): Thermo Fisher Scientific, USA, equipped with an electrospray ionization (ESI) source; BSA224S-CW electronic balance: Sartorius, Germany; MS3 basic vortex mixer: IKA, Germany; KQ-250DV CNC ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd.; and Milli-Q pure water system: Millipore, USA.

[0040] The reagents used in the following examples include: 21 oligopeptide reference standards (see Table 2 for details); methanol and acetonitrile: LC-MS grade, products of Merck, Germany; formic acid: LC-MS grade, products of Sigma-Aldrich, USA; ammonium acetate: analytical grade, products of Guangzhou Chemical Reagent Factory; laboratory purified water (18.2 MΩ·cm) was prepared by a Milli-Q purified water system; organic filter membrane: nylon, 0.22 μm, products of Tianjin Jinteng Experimental Equipment Co., Ltd.; oligopeptide-containing cosmetics: commercially available products.

[0041] Table 221 Information on Oligopeptide Reference Standards

[0042]

[0043]

[0044] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1: Detection method for 21 oligopeptides in cosmetics

[0046] Includes the following steps:

[0047] 1. Preparation of standard solutions

[0048] Weigh 10.0 mg (accurate to 0.1 mg) of each of the 21 oligopeptide reference standards and place them in a 10 mL amber volumetric flask. Dilute to the mark with 60% methanol-water (containing 0.25% formic acid) and mix well to obtain an oligopeptide standard stock solution with a mass concentration of 1000 μg / mL. Accurately transfer 100 μL of each standard stock solution to a 10 mL amber volumetric flask and dilute to the mark with 60% methanol-water (containing 0.25% formic acid) to prepare mixed standard solutions with mass concentrations of 10 μg / mL. Then dilute with the same solvent to prepare standard working solutions of 5 μg / L, 10 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L.

[0049] 2. Sample pretreatment

[0050] Weigh approximately 0.25 g (accurate to 0.0001 g) of the cosmetic sample into a 10 mL plastic centrifuge tube, add 2 mL of 0.1 mol / L ammonium acetate aqueous solution (containing 0.25% formic acid), vortex to disperse evenly, add methanol to 5 mL, mix well, sonicate for 15 min, centrifuge at 5000 r / min for 10 min, filter the supernatant through a 0.22 μm organic filter membrane, and use the filtrate for instrumental analysis.

[0051] 3. UPLC-MS / MS detection

[0052] (1) Chromatographic conditions

[0053] Chromatographic column: Agilent Poroshell 120HILIC-Z column (100mm×3.0mm, 2.7μm, Agilent Technologies, USA);

[0054] Mobile phase: A: 15 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid), B: acetonitrile (containing 0.1% formic acid);

[0055] Gradient elution procedure:

[0056] 0.0–1.0 min, 90% B;

[0057] 1.0~8.0min, 90%B~50%B;

[0058] 8.0–12.0 min, 50% B;

[0059] 12.1–17.0 min, 90% B;

[0060] Flow rate: 0.3 mL / min;

[0061] Column temperature: 30℃;

[0062] Injection volume: 2 μL.

[0063] (2) Mass spectrometry conditions

[0064] Electrospray ionization source in positive ion mode (ESI+);

[0065] Spray voltage: 3500V;

[0066] Sheath gas: 40 Units;

[0067] Auxiliary gas: 12 Units;

[0068] Auxiliary gas temperature: 350℃;

[0069] Ion transfer tube temperature: 325℃;

[0070] The data acquisition mode is multiple response monitoring (MRM).

[0071] Information on the retention time, parent ion, daughter ion, collision energy, etc. of the 21 oligopeptides is shown in Table 1.

[0072] MRM chromatograms of 21 oligopeptide standard solutions are shown below. Figure 1 .

[0073] Example 2: Detection method for 21 oligopeptides in cosmetics

[0074] Includes the following steps:

[0075] 1. Preparation of standard solutions

[0076] Same as the operation steps in Example 1.

[0077] 2. Sample pretreatment

[0078] Weigh approximately 0.1 g (accurate to 0.0001 g) of the cosmetic sample (same as in Example 1) into a 10 mL plastic centrifuge tube, add 1.5 mL of 0.05 mol / L ammonium acetate aqueous solution (containing 0.1% formic acid), vortex to disperse evenly, add methanol to 5 mL, mix well, sonicate for 10 min, centrifuge at 5000 r / min for 10 min, filter the supernatant through a 0.22 μm organic filter membrane, and use the filtrate for instrumental analysis.

[0079] 3. UPLC-MS / MS detection

[0080] (1) Chromatographic conditions

[0081] Chromatographic column: Agilent Poroshell 120HILIC-Z column (100mm × 3.0mm, 2.7μm);

[0082] Mobile phase: A: 10 mmol / L ammonium acetate aqueous solution (containing 0.05% formic acid), B: acetonitrile (containing 0.05% formic acid);

[0083] Gradient elution procedure:

[0084] 0.0–1.0 min, 90% B;

[0085] 1.0~8.0min, 90%B~50%B;

[0086] 8.0–12.0 min, 50% B;

[0087] 12.1–17.0 min, 90% B;

[0088] Flow rate: 0.3 mL / min;

[0089] Column temperature: 30℃;

[0090] Injection volume: 2 μL.

[0091] (2) Mass spectrometry conditions

[0092] Electrospray ionization source in positive ion mode (ESI+);

[0093] Spray voltage: 3000V;

[0094] Sheath gas: 30 Units;

[0095] Auxiliary gas: 7 Units;

[0096] Auxiliary gas temperature: 300℃;

[0097] Ion transfer tube temperature: 275℃;

[0098] The data acquisition mode is multiple response monitoring (MRM).

[0099] Information on the retention time, parent ion, daughter ion, collision energy, etc. of the 21 oligopeptides is shown in Table 1.

[0100] Example 3: Detection method for 21 oligopeptides in cosmetics

[0101] Includes the following steps:

[0102] 1. Preparation of standard solutions

[0103] Same as the operation steps in Example 1.

[0104] 2. Sample pretreatment

[0105] Weigh approximately 0.5 g (accurate to 0.0001 g) of the cosmetic sample (same as in Example 1) into a 10 mL plastic centrifuge tube, add 3 mL of 0.15 mol / L ammonium acetate aqueous solution (containing 0.5% formic acid), vortex to disperse evenly, add methanol to 5 mL, mix well, sonicate for 20 min, centrifuge at 5000 r / min for 10 min, filter the supernatant through a 0.22 μm organic filter membrane, and use the filtrate for instrumental analysis.

[0106] 3. UPLC-MS / MS detection

[0107] (1) Chromatographic conditions

[0108] Chromatographic column: Agilent Poroshell 120HILIC-Z column (100mm × 3.0mm, 2.7μm);

[0109] Mobile phase: A: 20 mmol / L ammonium acetate aqueous solution (containing 0.2% formic acid), B: acetonitrile (containing 0.2% formic acid);

[0110] Gradient elution procedure:

[0111] 0.0–1.0 min, 90% B;

[0112] 1.0~8.0min, 90%B~50%B;

[0113] 8.0–12.0 min, 50% B;

[0114] 12.1–17.0 min, 90% B;

[0115] Flow rate: 0.3 mL / min;

[0116] Column temperature: 30℃;

[0117] Injection volume: 2 μL.

[0118] (2) Mass spectrometry conditions

[0119] Electrospray ionization source in positive ion mode (ESI+);

[0120] Spray voltage: 4000V;

[0121] Sheath gas: 50 Units;

[0122] Auxiliary gas: 17 Units;

[0123] Auxiliary gas temperature: 400℃;

[0124] Ion transfer tube temperature: 375℃;

[0125] The data acquisition mode is multiple response monitoring (MRM).

[0126] Information on the retention time, parent ion, daughter ion, collision energy, etc. of the 21 oligopeptides is shown in Table 1.

[0127] Table 3 shows the average spiked recovery and precision results of the detection methods in Examples 1-3 (n=6).

[0128]

[0129]

[0130] Table 3 shows that the detection methods in Examples 1 to 3 can all determine 21 oligopeptides in cosmetics, with an average spiked recovery rate of more than 80% and a relative standard deviation of less than 10%, which meets the actual detection requirements.

[0131] Example 4: Methodological verification of the method of the present invention

[0132] 1. Linearity, method detection limit, and quantitation limit

[0133] Appropriate amounts of standard working solutions of 21 oligopeptides were transferred and analyzed according to the method in Example 1, and a standard curve was plotted. The mass concentrations of all 21 oligopeptides showed a linear relationship with their corresponding peak areas within a certain range. The limits of detection (LOD) and quantitation (LOQ) were calculated using a signal-to-noise ratio (S / N = 3) and a signal-to-noise ratio (S / N = 10), respectively. The linear regression equation, correlation coefficient, LOD, and LOQ are detailed in Table 4. The results show that the 21 oligopeptides exhibit a wide linear range and good linearity under this method, with correlation coefficients all greater than 0.995. Furthermore, the method demonstrates high sensitivity, with LODs reaching 0.1–1 μg / g, fully meeting the qualitative and quantitative requirements for various oligopeptides in cosmetics.

[0134] Table 421 lists the linear range, regression equation, correlation coefficient, limit of detection, and limit of quantitation for each oligopeptide.

[0135]

[0136]

[0137] 2. Recovery rate and precision

[0138] Standard working solutions at low, medium, and high concentrations were added to blank samples of three different bases (aqueous, cream, and emulsion). Six parallel samples were prepared for each concentration level, and sample pretreatment and analysis were performed according to the method in Example 1. The results are shown in Table 5.

[0139] Table 5. Spike recoveries and precision of 521 oligopeptides (n=6)

[0140]

[0141]

[0142] Table 5 shows that the recoveries of the 21 oligopeptides ranged from 81.7% to 95.3% at three spiking concentration levels in three matrices, with relative standard deviations ranging from 1.44% to 8.92%. This indicates that the method of the present invention has high accuracy and good repeatability, and the recovery rate and precision can meet the actual detection requirements.

[0143] Example 5: Actual Sample Measurement

[0144] The detection method established in Example 1 of this invention was used to test 28 batches of cosmetics labeled as containing oligopeptides. The test results are shown in Table 6.

[0145] Table 6. Detection results of 21 oligopeptides in actual samples (n=2)

[0146]

[0147]

[0148] As shown in Table 6, most samples tested positive for oligopeptides that matched the label; however, five batches of samples were labeled as containing glutathione, acetyl hexapeptide-8, acetyl tetrapeptide-2, etc., but the test results showed that they were not detected, indicating that the actual components did not match the label.

[0149] Experimental Example 1: The Influence of Different Chromatographic Columns on Detection Results

[0150] Five different chromatographic columns were used, including a C18 column (Hypersil Gold C18, 50 mm × 2.1 mm, 1.9 μm), a cyano column (Waters HSS Cyano, 100 mm × 2.1 mm, 1.8 μm), an amino column (Waters BEH Amide, 100 mm × 2.1 mm, 2.5 μm), and a HILIC column (Agilent Poroshell 120HILIC-Z, 100 mm × 3.0 mm, 2.7 μm; Waters ACQUITYBEH HILIC, 100 mm × 2.1 mm, 1.7 μm). The remaining steps were the same as in Example 1.

[0151] The effects of the different chromatographic columns on the detection of a mixed standard solution of 21 oligopeptides (500 μg / L) were compared. Figure 2The results show the MRM chromatograms of 21 oligopeptides under different column conditions. The C18 and cyano columns showed good retention of fatty acyl-modified oligopeptides (such as palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, and myristoyl pentapeptide-4), but weak retention of most other oligopeptides (such as tripeptide-1, L-carnosine, acetyl tetrapeptide-5, glutathione, and tetrapeptide-4) and failed to achieve effective separation. Dipeptide diaminobutyryl benzylamide diacetate showed peak separation, and most oligopeptide peaks exhibited tailing and broadening. The cyano column showed better peak shape and stronger retention than the C18 column, but seven oligopeptides, including glutathione, were still not effectively retained. The amino column showed some retention of all oligopeptides, but the peak shape was poor, with severe peak separation, broadening, and tailing; adjusting the mobile phase did not significantly improve the situation. The HILIC column separated oligopeptides well; except for tripeptide-1, the other oligopeptides showed good peak shape and were effectively separated from the matrix. Since tripeptide-1 was not retained on C18 and cyano columns, but was strongly retained on amino and HILIC columns (though tailing was more pronounced on amino columns), adjusting the mobile phase could improve the tailing phenomenon. Therefore, considering all factors, the HILIC column was chosen as the separation column, as it could effectively separate all oligopeptides.

[0152] Further comparison of different brands of HILIC columns (Agilent Poroshell 120HILIC-Z, 100mm × 3.0mm, 2.7μm; Waters ACQUITY BEH HILIC, 100mm × 2.1mm, 1.7μm) showed that all oligopeptides could be effectively separated on the chromatographic columns, but the Agilent Poroshell 120HILIC-Z column exhibited superior peak shape. Therefore, the Agilent Poroshell 120HILIC-Z column was the preferred choice.

[0153] Experimental Example 2: The Influence of Different Sample Pretreatment Methods on Detection Results

[0154] The sample pretreatment methods were as follows: (1) methanol; (2) acetonitrile; (3) direct extraction with water / methanol (40 / 60, v / v); (4) dispersion with ammonium acetate aqueous solution followed by extraction with methanol (40 / 60, v / v); (5) dispersion with ammonium acetate aqueous solution (containing formic acid) followed by extraction with methanol (40 / 60, v / v). The remaining detection steps were the same as in Example 1.

[0155] The effects of different pretreatment methods on the spiked recovery rates of 21 oligopeptides were compared, and the results are detailed in Table 7.

[0156] Table 7. Recovery results of 21 oligopeptides spiked using different pretreatment methods.

[0157]

[0158]

[0159] Table 7 shows that when methanol and acetonitrile were used as solvents for direct ultrasonic extraction, methanol extraction was slightly better than acetonitrile extraction, but the recoveries of dipeptide-2, tripeptide-1, palmitoyl pentapeptide-4, and acetyl tetrapeptide-2 were still less than 80%. When 60% methanol-water (v / v) was used as the extraction solvent for direct ultrasonic extraction, the recoveries of palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, and acetyl tetrapeptide-2 were less than 80%, but the recoveries of hydrophilic oligopeptides such as dipeptide-2 and tripeptide-1 were significantly improved.

[0160] A sample pretreatment method that involves dispersion with aqueous solution followed by ultrasonic extraction with organic solvent is beneficial for improving the recovery rate of oligopeptides. The effects of pretreatment methods—dispersion / extraction followed by ultrasonic extraction with methanol (40 / 60, v / v) and ammonium acetate aqueous solution (containing formic acid) / methanol (40 / 60, v / v)—on the spiked recovery rate of oligopeptides were compared. Table 7 shows that dispersing with ammonium acetate aqueous solution (containing formic acid) followed by methanol extraction yields the best results.

[0161] The 21 oligopeptides to be detected ranged in Log P values ​​from -5.53 to 5.50, with some being hydrophilic compounds and others being lipophilic compounds with fatty acyl lipophilic ends. The oligopeptides exhibit significant differences in chemical properties and solubility in solvents, making extraction with a single solvent difficult to achieve satisfactory results. Furthermore, the complexity of cosmetic matrices further hinders extraction. This invention, after extensive experimentation, discovered that using an ammonium acetate aqueous solution (containing formic acid) to first disperse and extract the oligopeptide compounds increases the ionic strength of the extraction solution, disrupting the original ionic system of the matrix and making it easier for hydrophilic oligopeptides to dissolve and for the matrix to disperse better. The addition of formic acid adjusts the pH of the solution, further increasing the solubility of the oligopeptides and thus improving the spiked recovery rate of the hydrophilic oligopeptides. Then, methanol is added to break the emulsion, and ultrasonic extraction further extracts the lipophilic oligopeptides encapsulated in the matrix. The synergistic effect of dispersion extraction and ultrasonic extraction results in better extraction of the 21 oligopeptides from cosmetics, significantly improving the accuracy of the method.

[0162] The ammonium acetate aqueous solution has an ammonium acetate concentration of 0.05 mol / L to 0.15 mol / L and contains 0.1% to 0.5% formic acid (volume concentration). The volume ratio of ammonium acetate aqueous solution to methanol is 30 to 60: 70 to 40. Preferably, the solution is first dispersed in an ammonium acetate aqueous solution (ammonium acetate concentration of 0.1 mol / L, containing 0.25% formic acid), and then methanol is added for ultrasonic extraction. The volume ratio of ammonium acetate aqueous solution to methanol is 40: 60.

[0163] Experimental Example 3: The Influence of Different Flow Rates on Detection Results

[0164] Five different mobile phase systems were used: I. water + methanol, II. water + acetonitrile, III. formic acid aqueous solution + acetonitrile, IV. formic acid aqueous solution + acetonitrile (containing formic acid), and V. ammonium acetate aqueous solution (containing formic acid) + acetonitrile (containing formic acid). The remaining detection steps were the same as in Example 1.

[0165] The effects of different flow rates on the peak shape and intensity of oligopeptides were compared. Figure 3 MRM chromatograms of 21 oligopeptides under different mobile phase conditions. Figure 3 As shown, when using mobile phase systems I, II, and III, all oligopeptides exhibited tailing, particularly those with long retention times. The broadening and tailing of the tripeptide-1 peak resulted in no peak being observed. The addition of formic acid to mobile phase systems III and IV enhanced the response and retention. Using mobile phase V, the addition of ammonium acetate significantly enhanced oligopeptide retention and improved peak shape; the improvement became more pronounced with increasing concentration, but the oligopeptide response intensity was inhibited. A relative equilibrium was reached at ammonium acetate concentrations of 10–20 mmol / L, where the decrease in response intensity was not significant. Simultaneously, the addition of formic acid enhanced the response; when the formic acid content was 0.05%–0.2%, the oligopeptide peak shape was better, and the response was superior.

[0166] The above results indicate that adding formic acid can improve ionization efficiency and enhance the response of oligopeptides. Since oligopeptides possess strong hydrogen-bonding groups such as peptide bonds, amino groups, or carboxyl groups, they exhibit strong retention on HILIC columns. They can interact with silanol groups on the surface of stationary phase particles, leading to secondary retention and peak tailing. Adding ammonium acetate, which has a competitive effect, to the mobile phase can effectively improve peak tailing caused by secondary retention. Considering that higher concentrations of ammonium acetate are more prone to crystallization in high-proportion organic phases, potentially causing wear on the liquid phase plunger seal or crystallization at the mass spectrometer ion source port, a mobile phase of 10 mmol / L–20 mmol / L ammonium acetate aqueous solution (containing 0.05%–0.2% formic acid) + acetonitrile (containing 0.05%–0.2% formic acid) was selected, with 15 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid) + acetonitrile (containing 0.1% formic acid) being the preferred mobile phase.

[0167] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0168] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting 21 oligopeptides in cosmetics, characterized in that, Includes the following steps: Add 0.05 mol / L to 0.15 mol / L ammonium acetate aqueous solution to the cosmetic to be tested, vortex to disperse evenly, then add methanol for ultrasonic extraction, centrifuge, filter the supernatant, and perform ultra-high performance liquid chromatography-tandem mass spectrometry detection; the ammonium acetate aqueous solution also contains 0.1% to 0.5% formic acid; the volume ratio of the ammonium acetate aqueous solution to methanol is 30 to 60: 70 to 40; The ultra-high performance liquid chromatography used a HILIC column; the mobile phase used was: A: 10 mmol / L to 20 mmol / L ammonium acetate aqueous solution containing 0.05% to 0.2% formic acid, B: acetonitrile containing 0.05% to 0.2% formic acid; Gradient elution was employed, and the gradient elution program was as follows: 0.0–1.0 min, 90% B; 1.0–8.0 min, 90% B–50% B; 8.0~12.0min, 50%B; 12.1~17.0min, 90%B; The 21 oligopeptides include palmitoyl hexapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tripeptide-8, dipeptide-2, tripeptide-1, palmitoyl tripeptide-1, palmitoyl tetrapeptide-10, acetyl hexapeptide-1, palmitoyl pentapeptide-4, acetyl tetrapeptide-2, myristoyl pentapeptide-4, acetyl tetrapeptide-9, glutathione, tetrapeptide-4, hexapeptide-9, dipeptide diaminobutyryl benzylamide diacetate, acetyl hexapeptide-8, acetyl tetrapeptide-5, L-carnosine, pentapeptide-3, and tripeptide-10 citrulline.

2. The method for detecting 21 oligopeptides in cosmetics according to claim 1, characterized in that, The concentration of ammonium acetate in the aqueous solution is 0.1 mol / L.

3. The method for detecting 21 oligopeptides in cosmetics according to claim 1, characterized in that, The concentration of formic acid in the ammonium acetate aqueous solution is 0.25%.

4. The method for detecting 21 oligopeptides in cosmetics according to claim 1, characterized in that, The volume ratio of the ammonium acetate aqueous solution to methanol is 40:

60.

5. The method for detecting 21 oligopeptides in cosmetics according to claim 1, characterized in that, The ultrasonic extraction time is 10 min to 20 min.

6. The method for detecting 21 oligopeptides in cosmetics according to claim 1, characterized in that, Includes the following steps: Accurately weigh 0.1g to 0.5g of the cosmetic product to be tested, add 1.5mL to 3mL of 0.05mol / L to 0.15mol / L ammonium acetate aqueous solution containing 0.1% to 0.5% formic acid, vortex to disperse evenly, add methanol to 5mL, mix well, ultrasonically extract for 10min to 20min, centrifuge, filter the supernatant through a 0.22μm organic filter membrane, and perform ultra-high performance liquid chromatography-tandem mass spectrometry detection.

7. The method for detecting 21 oligopeptides in cosmetics according to any one of claims 1 to 6, characterized in that, The mass spectrometry conditions include: positive ion mode of electrospray ionization source; spray voltage: 3500±500V; sheath gas: 40±10Unit; auxiliary gas: 12±5Unit; auxiliary gas temperature: 350±50℃; ion transmission tube temperature: 325±50℃; and data acquisition mode of multiple reaction monitoring (MRM).

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