Method for identification and detection of adulterating ingredients for counterfeit botulinum toxin cosmetic products

Abstract

The present application belongs to the technical field of cosmetic product ingredient identification, and particularly relates to a method for identifying and detecting counterfeit ingredients of counterfeit botulinum toxin cosmetic products. The present application prepares a test sample into a test solution, detects it according to high performance liquid chromatography mass spectrometry, takes telencephalin, acetyl hexapeptide-1 and snake venom-like tripeptide as key indicators for distinguishing counterfeit ingredients of counterfeit botulinum toxin products, and ensures the safety and effectiveness of the botulinum toxin products. The present application only needs to use standard control samples of counterfeit ingredients when the method is established for the first time, and does not need to use control samples in the subsequent routine detection process to accurately determine whether the sample is counterfeit. Moreover, the present application first establishes a method for quantitatively analyzing telencephalin by using liquid chromatography-tandem mass spectrometry. The present application is simple in operation, good in repeatability, high in sensitivity, fast in separation speed, high in detection flux, reliable in results, and effectively ensures the safety and effectiveness of the botulinum toxin products.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic product ingredient identification technology, specifically relating to a method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products. Background Technology

[0002] Botox, also known as facial slimming injections or wrinkle-reducing injections, is a neurotoxin protein secreted by Clostridium botulinum during its reproduction. Type A botulinum toxin acts on peripheral motor nerve endings by binding to receptors associated with acetylcholine release, inhibiting its release from the presynaptic membrane, causing muscle relaxation and paralysis. This effectively removes dynamic wrinkles on the face, achieving a facial rejuvenation effect. High-dose injections can also slim the face, shoulders, and calves, and prevent scarring. Type A botulinum toxin is fast-acting, with visible results typically within 1-2 days after injection. However, after a period of time, the inactivated nerve endings can undergo axonal sprouting, reactivating the nerves and causing the cosmetic effect to fail. Therefore, once this product is used, periodic injections are necessary. Botox is a biological product with extremely high biosafety risks during production, yet there is a huge social demand in the medical aesthetics field. Because this cosmetic product is mostly used in beauty salons rather than medical institutions, the supply chain and drug use are subject to more relaxed supervision, making it easy for unscrupulous individuals to exploit the situation, resulting in the production and sale of counterfeit products with substantial sums involved. Because of their rapid effectiveness and high repurchase rate, counterfeit products without added active ingredients are easily detected, leading to limited sales. Therefore, many counterfeit botulinum toxin products add low-cost, chemically synthesizable botulinum toxin-like short peptides. Scorpion venom and acetyl hexapeptide-1, snake venom-like tripeptides, are often added to legitimate cosmetic skincare products, offering some anti-wrinkle effects. These small-molecule active peptides (scorpion venom and acetyl hexapeptide-1, snake venom-like tripeptides) are usually chemical products, not manufactured or tested as pharmaceuticals or raw materials. There is a lack of sufficient clinical research on potential adverse reactions from injection. They are only suitable for topical cosmetic use, not injection. Injection may cause adverse reactions such as skin redness and swelling, and even endanger life. Currently, there are no reported testing methods for scorpion venom, acetyl hexapeptide-1, and snake venom-like tripeptides in botulinum toxin products. Public security organs, procuratorates, and courts cannot identify adulterated ingredients and find it difficult to obtain evidence to reasonably sentence and convict those involved in counterfeiting under the crimes of manufacturing and selling counterfeit and substandard products, manufacturing and selling counterfeit and substandard drugs, or manufacturing and selling toxic substances. To better ensure the safety and efficacy of botulinum toxin products, maintain market order, safeguard people's lives and health, and enable judicial organs to fairly punish suspects involved in the manufacture and sale of counterfeit products, it is necessary to find a detection method for identifying and controlling adulterated components in counterfeit botulinum toxin products made from scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide. Summary of the Invention

[0003] In view of the above requirements, the application provides a method for identifying and detecting counterfeit components of counterfeit botulinum toxin cosmetic products. By detecting the red scorpion toxin, acetyl hexapeptide-1 and snake venom-like tripeptide in suspected botulinum toxin cosmetic products, the identification of counterfeit components of botulinum toxin cosmetic products can be realized, and the basis for subsequent judicial procedure sentencing can be provided. The method only needs to use red scorpion toxin, acetyl hexapeptide-1 and snake venom-like tripeptide standard controls in the initial method establishment, and does not need to use controls in the subsequent regular detection process to accurately determine whether the polypeptide components are contained in the sample. In addition, the application first establishes a method for quantitatively analyzing the red scorpion toxin by using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method has the characteristics of simple operation, good repeatability, high sensitivity and reliable results, and can effectively control the product quality and ensure the safety and effectiveness of the product.

[0004] The application is realized by the following technical solutions:

[0005] Preparation of the control solution: a certain amount of red scorpion toxin, acetyl hexapeptide-1 and snake venom-like tripeptide are precisely weighed, dissolved with 0.1% BSA and diluted step by step to prepare a series of standard working solutions. The calibration curve concentrations of the red scorpion toxin are 1, 2, 5, 10, 50 and 100 μg / L respectively; the calibration curve concentrations of the acetyl hexapeptide-1 are 0.2, 0.4, 1, 2, 10 and 20 μg / L respectively; and the calibration curve concentrations of the snake venom-like tripeptide are 0.1, 0.2, 0.5, 1, 5 and 10 μg / L respectively.

[0006] Preparation of the test sample solution: the test sample is taken, the liquid product is directly diluted 20 times with 0.1% BSA, and the supernatant obtained by centrifugation at 12000 rpm for 10 min is the test sample solution; the freeze-dried product is dissolved with 1 ml of water, then diluted 20 times with 0.1% BSA, and the supernatant obtained by centrifugation at 12000 rpm for 10 min is the test sample solution.

[0007] After the preparation of the test sample solution, high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for analysis and detection. The analysis and detection are performed by using high performance liquid chromatography-tandem mass spectrometry, and whether the characteristic ion pairs of the red scorpion toxin or the acetyl hexapeptide-1 or the snake venom-like tripeptide are detected in the detection results is used to preliminarily judge whether these polypeptide components exist in the test sample. If the characteristic ion pairs are detected, it is further verified whether the relative ion abundance is within the preset acceptable error range, so as to finally determine whether the red scorpion toxin, the acetyl hexapeptide-1 and the snake venom-like tripeptide are contained in the analysis sample.

[0008] The qualitative basis that the red scorpion toxin is contained in the sample to be detected is that: a) the following three groups of ion pairs 594.75 / 70.30, 594.75 / 84.05 and 594.75 / 129.15 are detected in the sample at the same time; b) the relative ion abundance ratio of 594.75 / 84.05 and 594.75 / 70.30 is within the range of 80% to 120%, and the relative ion abundance ratio of 594.75 / 129.15 and 594.75 / 70.30 is within the range of 10% to 60%; when conditions a) and b) are met at the same time, it is indicated that the sample contains red scorpion toxin.

[0009] The qualitative basis that acetyl hexapeptide-1 is contained in the sample to be detected is that: a) the following three groups of ion pairs 435.85 / 110.15, 435.85 / 322.90 and 435.85 / 86.20 are detected in the sample at the same time; b) the relative ion abundance ratio of 435.85 / 322.90 and 435.85 / 110.15 is within the range of 40% to 80%, and the relative ion abundance ratio of 435.85 / 86.20 and 435.85 / 110.15 is within the range of 35% to 75%; when conditions a) and b) are met at the same time, it is indicated that the sample contains acetyl hexapeptide-1.

[0010] The qualitative basis that the snake venom-like tripeptide is contained in the sample to be detected is that: a) the following three groups of ion pairs 376.20 / 70.15, 376.20 / 269.25 and 376.20 / 359.25 are detected in the sample at the same time; b) the relative ion abundance ratio of 376.20 / 269.25 and 376.20 / 70.15 is within the range of 25% to 75%, and the relative ion abundance ratio of 376.20 / 359.25 and 376.20 / 70.15 is within the range of 15% to 65%; when conditions a) and b) are met at the same time, it is indicated that the sample contains the snake venom-like tripeptide.

[0011] The mass spectrometry condition in the application is that: the ion source is ESI+ mode; the desolvation tube temperature is 250 DEG C; the heating module temperature is 250 DEG C; the atomization gas is nitrogen, and the flow rate is 3.0 L / min; the interface temperature is 350 DEG C; the drying gas is nitrogen, and the flow rate is 5.0 L / min; the heating gas is air, and the flow rate is 15.0 L / min; the mass spectrometer detector detection mode is multi-ion selection monitoring (MRM), and the MRM parameter table is shown in Table 1.

[0012] Table 1. MRM parameter table

[0013]

[0014] * is a quantitative ion

[0015] The application also provides a quantitative identification and detection method for the adulterated components of the fake botulinum toxin cosmetic product, which comprises the following steps:

[0016] (1) Preparation of standard working curve: Accurately weigh the adulterated reference standard and prepare a series of standard working solutions: Analyze the standard working solutions by high performance liquid chromatography-tandem mass spectrometry to obtain the chromatogram of the reference standard. The standard working curve is obtained based on the relationship between the concentration of the reference standard solution and the corresponding chromatographic peak area.

[0017] (2) Sample detection: Take the sample to be tested, add BSA to dilute, centrifuge, take the supernatant, and analyze and detect it by high performance liquid chromatography-tandem mass spectrometry. Based on the response peak area of ​​the adulterant and the standard working curve, the content of the adulterant is obtained.

[0018] The high-performance liquid chromatography (HPLC) conditions described in this invention are as follows: C18 column; column temperature 50℃; mobile phase A is 0.2% formic acid aqueous solution; mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution, with an initial elution concentration of 5% for mobile phase B, mobile phase A: 10-95%, mobile phase B: 5-90%; flow rate 0.25 mL / min; injection volume 5.0 μL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The method of this invention can screen for the presence of scorpion toxin, acetyl hexapeptide-1, or snake venom-like tripeptide in a sample by ion pair qualitative analysis in the absence of a reference standard. If all three ion pair channels of each polypeptide emit peaks at the same time and the ion abundance ratio is within the specified range, the polypeptide is considered to be present in the sample. This qualitative result can serve as a basis for identifying counterfeit botulinum toxin products, ensuring the safety and efficacy of botulinum toxin products.

[0021] (2) This invention establishes for the first time a method for quantitative analysis of red scorpion venom using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0022] (3) The present invention has the characteristics of fast separation speed, high detection throughput and high detection sensitivity. It can detect 1 μg / L of red scorpion venom, 0.2 μg / L of acetyl hexapeptide-1 and 0.1 μg / L of snake venom tripeptide respectively. It can also complete the detection of one sample in 10 minutes, ensuring an efficient workflow and rapid result feedback. Attached Figure Description

[0023] Figure 1 Specificity test: Chromatogram of the mixed standard working solution (red scorpion toxin - 5 μg / L, snake venom-like tripeptide - 0.5 μg / L, acetyl hexapeptide - 1-1 μg / L);

[0024] Figure 2 Specificity test: Chromatogram of botulinum toxin cosmetic product solution;

[0025] Figure 3 Specificity experiment: chromatogram of red scorpion toxin adulterated botulinum toxin cosmetic product;

[0026] Figure 4 Specificity experiment: chromatogram of botulinum toxin cosmetic product adulterated with snake venom-like tripeptide;

[0027] Figure 5 Specificity experiment: chromatogram of botulinum toxin cosmetic product adulterated with acetyl hexapeptide-1;

[0028] Figure 6 Specificity experiment: chromatogram of blank solution;

[0029] Figure 7 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 1 μg / L, snake venom-like tripeptide - 0.1 μg / L, acetyl hexapeptide-1 - 0.2 μg / L);

[0030] Figure 8 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 2 μg / L, snake venom-like tripeptide - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L);

[0031] Figure 9 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 5 μg / L, snake venom-like tripeptide - 0.5 μg / L, acetyl hexapeptide-1 - 1 μg / L);

[0032] Figure 10 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 10 μg / L, snake venom-like tripeptide - 1 μg / L, acetyl hexapeptide-1 - 2 μg / L);

[0033] Figure 11 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 50 μg / L, snake venom-like tripeptide - 5 μg / L, acetyl hexapeptide-1 - 10 μg / L);

[0034] Figure 12 Linearity test: chromatogram of control mixed standard working solution (red scorpion toxin - 100 μg / L, snake venom-like tripeptide - 10 μg / L, acetyl hexapeptide-1 - 20 μg / L);

[0035] Figure 13 Repeatability test: chromatogram of control mixed standard working solution (red scorpion toxin - 2 μg / L, snake venom-like tripeptide - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, first injection;

[0036] Figure 14Chromatogram of the repeatability experiment of the control mix working solution (inotadine - 2 μg / L, eptadote - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, first injection;

[0037] Figure 15 Chromatogram of the repeatability experiment of the control mix working solution (inotadine - 2 μg / L, eptadote - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, third injection;

[0038] Figure 16 Chromatogram of the repeatability experiment of the control mix working solution (inotadine - 2 μg / L, eptadote - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, fourth injection;

[0039] Figure 17 Chromatogram of the repeatability experiment of the control mix working solution (inotadine - 2 μg / L, eptadote - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, fifth injection;

[0040] Figure 18 Chromatogram of the repeatability experiment of the control mix working solution (inotadine - 2 μg / L, eptadote - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) - consecutive six injections, sixth injection;

[0041] Figure 19 Recovery experiment: low concentration spike - first spike in triplicate (spike concentration: inotadine - 1 μg / L, eptadote - 0.1 μg / L, acetyl hexapeptide-1 - 0.2 μg / L);

[0042] Figure 20 Recovery experiment: low concentration spike - second spike in triplicate (spike concentration: inotadine - 1 μg / L, eptadote - 0.1 μg / L, acetyl hexapeptide-1 - 0.2 μg / L);

[0043] Figure 21 Recovery experiment: low concentration spike - third spike in triplicate (spike concentration: inotadine - 1 μg / L, eptadote - 0.1 μg / L, acetyl hexapeptide-1 - 0.2 μg / L);

[0044] Figure 22 Recovery experiment: medium concentration spike - first spike in triplicate (spike concentration: inotadine - 5 μg / L, eptadote - 0.5 μg / L, acetyl hexapeptide-1 - 1 μg / L);

[0045] Figure 23Recovery experiment: medium concentration spike - 3 replicates 2nd spike (spike concentration: red scorpion toxin - 5 pg / L, snake venom-like tripeptide - 0.5 pg / L, acetyl hexapeptide - 1 pg / L);

[0046] Figure 24 Recovery experiment: medium concentration spike - 3 replicates 3rd spike (spike concentration: red scorpion toxin - 5 pg / L, snake venom-like tripeptide - 0.5 pg / L, acetyl hexapeptide - 1 pg / L);

[0047] Figure 25 Recovery experiment: high concentration spike - 3 replicates 1st spike (spike concentration: red scorpion toxin - 100 pg / L, snake venom-like tripeptide - 10 pg / L, acetyl hexapeptide - 20 pg / L);

[0048] Figure 26 Recovery experiment: high concentration spike - 3 replicates 2nd spike (spike concentration: red scorpion toxin - 100 pg / L, snake venom-like tripeptide - 10 pg / L, acetyl hexapeptide - 20 pg / L);

[0049] Figure 27 Recovery experiment: high concentration spike - 3 replicates 3rd spike (spike concentration: red scorpion toxin - 100 pg / L, snake venom-like tripeptide - 10 pg / L, acetyl hexapeptide - 20 pg / L);

[0050] Figure 28 Effectiveness investigation: chromatogram of sample containing red scorpion toxin to be tested - Shim-pack GIST C18 (100 mm x 2.1 mm, 2 pm);

[0051] Figure 29 Effectiveness investigation: chromatogram of sample containing red scorpion toxin to be tested - Shim-pack GISS C18 (100 mm x 2.1 mm, 1.9 pm);

[0052] Figure 30 Effectiveness investigation: chromatogram of sample containing red scorpion toxin to be tested - Hypersil GOLD Vanquish (100 x 2.1 mm, 1.9 pm);

[0053] Figure 31 Effectiveness investigation: chromatogram of sample containing snake venom-like tripeptide to be tested - Shim-pack GIST C18 (100 mm x 2.1 mm, 2 pm);

[0054] Figure 32 Effectiveness investigation: chromatogram of sample containing snake venom-like tripeptide to be tested - Shim-pack GISS C18 (100 mm x 2.1 mm, 1.9 pm);

[0055] Figure 33 : Effectiveness investigation: chromatogram of test sample containing acetyl hexapeptide-1 - Shim-pack GISS C18 (100mm x 2.1mm, 1.9μm) ;

[0056] Figure 34 : Effectiveness investigation: chromatogram of test sample containing acetyl hexapeptide-1 - Shim-pack GISS C18 (100mm x 2.1mm, 1.9μm) ;

[0057] Figure 35 : Effectiveness investigation: chromatogram of test sample containing acetyl hexapeptide-1 - Shim-pack GISS C18 (100mm x 2.1mm, 1.9μm) ;

[0058] Figure 36 : Effectiveness investigation: chromatogram of test sample containing acetyl hexapeptide-1 - Shim-pack GISS C18 (100mm x 2.1mm, 1.9μm). DETAILED DESCRIPTION

[0059] The application will be further described in conjunction with the following examples. However, the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0060] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods, etc. already existing in the prior art.

[0061] Example 1

[0062] 1. Experimental instruments and equipment:

[0063] High-pressure binary pump, degassing machine, automatic sampler, column oven and triple quadrupole mass spectrometer.

[0064] 2. Experimental reagents:

[0065] Acetonitrile and formic acid are mass spectrometry pure; water is Wahaha purified water; reference substance scorpion venom (CAS No. 936616-33-0), acetyl hexapeptide-1 (CAS No. 448944-47-6), and scorpion-like tripeptide (CAS No. 823202-99-9).

[0066] 3. Detection conditions:

[0067] Chromatographic column: C18 chromatographic column

[0068] Mobile phase: A-formic acid: water (2:1000, v / v); B-formic acid: acetonitrile (1:1000, v / v);

[0069] Gradient: 0-5.00 min 5% B-85% B, 5.00-5.01 min 85%-90% B, 5.01-7.00 min 90% B, 7.00-7.20 min 90%-5% B, 7.20-10 min 5% B; Column temperature: 50°C; Flow rate: 0.25 mL / min; Injection volume: 5.0 μL.

[0070] Mass spectrometry conditions:

[0071] Ion source: ESI+ mode; Mass spectrometer: triple quadrupole liquid chromatograph-mass spectrometer; Desolvation tube temperature: 250°C; Heating module temperature: 250°C; Atomization gas: nitrogen, flow rate: 3.0 L / min; Interface temperature: 350°C; Dry gas: nitrogen, flow rate: 5.0 L / min; Heating gas: air, flow rate: 15.0 L / min; Mass spectrometer detector detection mode: multiple ion selection monitoring (MRM), mass spectrometer detection parameters are shown in Table 1.

[0072] 4. Standard curve drawing:

[0073] 1) Preparation of standard mother liquor: accurately weigh appropriate amount of control product scorpion toxin, acetyl hexapeptide-1 and snake venom-like tripeptide, dissolve them into original mother liquor with a concentration of 2 mg / ml using 0.1% BSA, and then dilute the original mother liquor to 1000 μg / L using 0.1% BSA as a standard mother liquor for standby;

[0074] 2) Preparation of standard working solution: dilute the standard mother liquor with 0.1% BSA step by step to prepare a series of standard working solutions, the calibration curve concentrations of scorpion toxin are 1, 2, 5, 10, 50, 100 μg / L respectively; the calibration curve concentrations of acetyl hexapeptide-1 are 0.2, 0.4, 1, 2, 10, 20 μg / L respectively; the calibration curve concentrations of snake venom-like tripeptide are 0.1, 0.2, 0.5, 1, 5, 10 μg / L respectively.

[0075] 3) Draw the standard working curve: analyze the above standard working solution by high performance liquid chromatography tandem mass spectrometry, and the chromatograms of scorpion toxin, acetyl hexapeptide-1 and snake venom-like tripeptide can be obtained, and the standard working curve can be obtained according to the relationship between the solution concentration and the corresponding chromatographic peak area.

[0076] 5. Preparation of test solution: Take the suspected botulinum toxin cosmetic product (sample to be tested), dilute the liquid product 20 times with 0.1% BSA, centrifuge at 12000 rpm for 10 min, and take the supernatant as the test solution; dissolve the freeze-dried product with 1 ml water, dilute 20 times with 0.1% BSA, centrifuge at 12000 rpm for 10 min, and take the supernatant as the test solution.

[0077] 6. Qualitative detection basis

[0078] The qualitative basis for the presence of red scorpion toxin in the sample to be tested is: a) the following three ion pairs 594.75 / 70.30, 594.75 / 84.05, and 594.75 / 129.15 are detected in the sample at the same time, b) the relative ion abundance ratio of 594.75 / 84.05 to 594.75 / 70.30 is within the range of 80% to 120%, and the relative ion abundance ratio of 594.75 / 129.15 to 594.75 / 70.30 is within the range of 10% to 60%, and conditions a) and b) are met at the same time, indicating that the sample contains red scorpion toxin.

[0079] The qualitative basis for the presence of acetyl hexapeptide-1 in the sample to be tested is: a) the following three ion pairs 435.85 / 110.15, 435.85 / 322.90, and 435.85 / 86.20 are detected in the sample at the same time, b) the relative ion abundance ratio of 435.85 / 322.90 to 435.85 / 110.15 is within the range of 40% to 80%, and the relative ion abundance ratio of 435.85 / 86.20 to 435.85 / 110.15 is within the range of 35% to 75%, and conditions a) and b) are met at the same time, indicating that the sample contains acetyl hexapeptide-1.

[0080] The qualitative basis for the presence of snake venom-like tripeptide in the sample to be tested is: a) the following three ion pairs 376.20 / 70.15, 376.20 / 269.25, and 376.20 / 359.25 are detected in the sample at the same time, b) the relative ion abundance ratio of 376.20 / 269.25 to 376.20 / 70.15 is within the range of 25% to 75%, and the relative ion abundance ratio of 376.20 / 359.25 to 376.20 / 70.15 is within the range of 15% to 65%, and conditions a) and b) are met at the same time, indicating that the sample contains snake venom-like tripeptide.

[0081] 7. Specificity Test. Mixed standard solutions of scorpion venom (5 μg / L), acetyl hexapeptide-1 (1 μg / L), and snake venom-like tripeptide (0.5 μg / L), botulinum toxin cosmetic products, cosmetic products adulterated with scorpion venom, cosmetic products adulterated with acetyl hexapeptide-1, cosmetic products adulterated with snake venom-like tripeptide, and blank solutions were injected into LC-MS. Figures 1 to 6 As can be seen in the chromatogram of the mixed standard reference, scorpion toxin, snake venom-like tripeptide, and acetyl hexapeptide-1 eluted at 2.95, 3.38, and 4.02, respectively. Botulinum toxin cosmetic products and blank solutions showed no peaks. Adulterated botulinum toxin cosmetic products eluted only in the channels of the adulterated compounds. The results indicate that this method has high specificity.

[0082] 8. Linear regression was performed on the above standard working curves, and the sensitivity of the method was investigated. The correlation coefficients of the obtained calibration curves were all greater than 0.9973, and the accuracy at each calibration point was between 95.3% and 111.3%. Labsolutions software automatically calculated the limit of detection (S / N = 3) and limit of quantitation (S / N = 10) based on the signal-to-noise ratio. The linear equation (chromatogram is shown below) Figures 7-12 The results of the limits of detection and limits of quantitation are shown in Table 2. The results indicate that each reference standard has a good linear relationship within its respective mass range, proving that the method of the present invention has high accuracy and high sensitivity within the selected linear range.

[0083] Table 2. Linear equation, limit of detection and limit of quantitation

[0084]

[0085] 9. Repeatability test: The mixed standard working solution (red scorpion toxin - 2 μg / L, snake venom-like tripeptide - 0.2 μg / L, acetyl hexapeptide-1 - 0.4 μg / L) was measured 6 times consecutively to test the precision of the instrument. The results are shown in Table 3 and [Table data missing]. Figures 13-18 .

[0086] Table 3. Retention time and peak area repeatability results (n=6)

[0087]

[0088] 10. Recovery rate assessment: Blank samples were used for spiking experiments at low, medium, and high concentrations, with three replicates for each concentration. The recovery rate was calculated. At different spiking concentrations, the recoveries of each small molecule peptide ranged from 85.2% to 114.4%, with a relative standard deviation not exceeding 3.64%. The results are shown in Table 4 and [Table data would be inserted here]. Figures 19-27 .

[0089] Table 4. Sample determination results and spiked recovery rate results (n=3)

[0090]

[0091]

[0092] 11. Effectiveness investigation: using three types of chromatographic columns, as shown in Table 5, the samples containing, respectively, red scorpion toxin, snake venom-like tripeptide, and acetyl hexapeptide-1 were quantitatively determined. The results are shown in Table 6, and the RSD of the positive sample detection value is less than 4.62%.

[0093] Table 5. Three chromatographic column information tables

[0094]

[0095] Table 6. Comparison of positive sample detection results using different chromatographic columns

[0096]

[0097] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for the identification and detection of adulterating ingredients of counterfeit botulinum toxin cosmetic products, characterized by, The sample to be detected is prepared into a test solution, the test solution is detected by high resolution mass spectrometry in a positive ion mode using high performance liquid chromatography-tandem mass spectrometry, whether the characteristic ion pairs of red scorpion toxin and acetyl hexapeptide-1 and snake venom-like tripeptide are detected in the detection result is determined, and whether the relative ion abundance of the characteristic ion pairs is verified within a preset acceptable error range, to determine whether the sample to be detected contains a counterfeit component; The mass spectrometry conditions are that the ion source is in an ESI+ mode; the desolvation tube temperature is 250 DEG C; the heating module temperature is 250 DEG C; the atomizing gas is nitrogen with a flow rate of 3.0 L / min; the interface temperature is 350 DEG C; the drying gas is nitrogen with a flow rate of 5.0 L / min; the heating gas is air with a flow rate of 15.0 L / min; and the mass spectrometry detector detection mode is multi-ion selection monitoring; The high performance liquid chromatography conditions are that the C18 chromatographic column is used; the column temperature is 50 DEG C; the mobile phase A is 0.2% formic acid aqueous solution; the mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution is used; the flow rate is 0.25 mL / min; and the injection volume is 5.0 µL; The gradient elution program is 0-5.00 min 5%B-85%B, 5.00-5.01 min 85%-90%B, 5.01-7.00 min 90%B, 7.00-7.20 min 90%-5%B, and 7.20-10 min 5%B; The qualitative basis for the sample to be detected containing red scorpion toxin is that conditions a) and b) are both satisfied: a) the following three groups of ion pairs 594.75 / 70.30, 594.75 / 84.05, and 594.75 / 129.15 are detected in the sample at the same time; b) the relative ion abundance ratio of 594.75 / 84.05 to 594.75 / 70.30 is within the range of 80% to 120%, and the relative ion abundance ratio of 594.75 / 129.15 to 594.75 / 70.30 is within the range of 10% to 60%.

2. The method for identifying and detecting adulterated ingredients of counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that, The qualitative basis for the sample to be detected containing acetyl hexapeptide-1 is that conditions a) and b) are both satisfied: a) the following three groups of ion pairs 435.85 / 110.15, 435.85 / 322.90, and 435.85 / 86.20 are detected in the sample at the same time; b) the relative ion abundance ratio of 435.85 / 322.90 to 435.85 / 110.15 is within the range of 40% to 80%, and the relative ion abundance ratio of 435.85 / 86.20 to 435.85 / 110.15 is within the range of 35% to 75%.

3. The method for identification and detection of adulterants of counterfeit botulinum toxin cosmetic product according to claim 1, wherein, The qualitative basis for the sample to be detected containing snake venom-like tripeptide is that conditions a) and b) are both satisfied: a) the following three groups of ion pairs 376.20 / 70.15, 376.20 / 269.25, and 376.20 / 359.25 are detected in the sample at the same time; b) the relative ion abundance ratio of 376.20 / 269.25 to 376.20 / 70.15 is in the range of 25% to 75% and the relative ion abundance ratio of 376.20 / 359.25 to 376.20 / 70.15 is in the range of 15% to 65%.

4. The method for identifying and detecting adulterated ingredients of counterfeit botulinum toxin cosmetic products according to claim 1, wherein, Also comprising the following steps: (1) Standard working curve preparation: precisely weigh the adulteration component reference substance, prepare a series of standard working solutions: analyze the standard working solutions by high performance liquid chromatography tandem mass spectrometry to obtain the chromatogram of the reference substance, and obtain the standard working curve according to the concentration of the reference substance solution and the corresponding chromatographic peak area; (2) Sample detection: dilute the sample to be detected with BSA, centrifuge, take the supernatant, and analyze and detect by high performance liquid chromatography tandem mass spectrometry, and obtain the content of the adulteration component according to the response peak area of the adulteration component and the standard working curve.

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