Method for identifying and detecting adulteration components of fake botulinum toxin beauty product

Abstract

The invention belongs to the technical field of component identification of beauty products, and particularly relates to an identification and detection method for adulteration components of fake botulinum toxin beauty products. According to the method, the sample to be detected is prepared into the test solution, the test solution is detected according to the high performance liquid chromatography-mass spectrometry, and the red scorpion toxin, the acetyl hexapeptide-1 and the snake venom-like tripeptide are taken as key indexes for judging fake and adulterated components of the botulinum toxin product, so that the safety and the effectiveness of the botulinum toxin product are ensured. According to the method, whether the sample is adulterated or not can be accurately judged only by using a standard reference substance of adulterated components when the method is established for the first time without using a reference substance in the subsequent conventional detection process, and the method for quantitatively analyzing the red scorpion toxin by using the liquid chromatography-tandem mass spectrometry is established for the first time. The method is easy and convenient to operate, good in repeatability, high in sensitivity, high in separation speed, high in detection flux and reliable in result, and the safety and effectiveness of botulinum toxin products are effectively guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetic product ingredient identification, and in particular relates to an identification and detection method for adulterated ingredients in counterfeit botulinum toxin cosmetic products. Background Art

[0002] Botulinum toxin beauty products are also called face-slimming injections and wrinkle-removing injections. Botulinum toxin is a neurotoxin protein secreted by Clostridium botulinum during its reproduction process. Type A botulinum toxin can act on peripheral motor nerve endings, inhibit its release from the presynaptic membrane by binding to receptors related to acetylcholine release, and cause muscle relaxation and paralysis, thereby effectively removing dynamic wrinkles on the face and achieving the effect of facial rejuvenation. In addition, high-dose injections can also have the effects of face slimming, shoulder slimming, calf slimming, and scar prevention. Type A botulinum toxin works quickly, and the beauty effect can usually be observed 1-2 days after injection. However, after a period of injection, the inactivated nerve endings will produce nerve axon sprouts, re-stimulate the nerves, and cause the beauty effect to fail. Therefore, once the product is used for beauty, it needs to be injected periodically. Botulinum toxin is a biological product with extremely high biosafety risks in production, and there is a huge social demand in the field of medical beauty. Since the use of this beauty product is mostly in beauty salons rather than medical institutions, the supply chain and drug use management supervision are relatively loose, which makes it easy for criminals to take advantage of the opportunity to counterfeit and sell fakes with huge case value. Due to the rapid effect and high repurchase rate, counterfeit products without active ingredients are easily discovered and have limited sales. Therefore, many counterfeit botulinum toxin products have to add low-cost chemically synthesized botulinum toxin short peptide products. Red scorpion toxin and acetyl hexapeptide-1, snake venom tripeptide are often added to legal beauty and skin care products, and have certain beauty and wrinkle removal effects. These small molecule active peptides (red scorpion toxin and acetyl hexapeptide-1, snake venom tripeptide) are usually chemical products, which have not been produced and tested in accordance with drugs or raw materials. There is also a lack of sufficient clinical research on the adverse reactions that may be caused by injection. They are only suitable for external use in beauty, but not for injection. After injection, they may cause adverse reactions such as skin redness and swelling, and even endanger life safety. At present, there is no report on the inspection method of red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide in botulinum toxin products. The public security, procuratorial and judicial organs are unable to identify the adulterated ingredients, and it is difficult to obtain evidence to reasonably sentence and convict the counterfeiters according to the crime of manufacturing and selling counterfeit products, the crime of manufacturing and selling counterfeit drugs, or the crime of manufacturing and selling toxic substances. In order to better ensure the safety and effectiveness of botulinum toxin products, maintain market order, ensure the safety of people's lives and health, and enable judicial organs to fairly punish suspects of counterfeiting and selling counterfeit goods, it is necessary to find a detection method for identifying and controlling the adulterated ingredients of botulinum toxin products made with red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide. Summary of the invention

[0003] In response to the above needs, the present invention provides a method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin beauty products. The present invention can identify the counterfeit and adulterated ingredients of botulinum toxin beauty products by detecting red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide in suspected botulinum toxin beauty products, and provide a basis for sentencing in subsequent judicial procedures. This method only requires the use of standard reference substances of red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide when the method is initially established, and in subsequent routine detection processes, it is possible to accurately determine whether the sample contains the above-mentioned polypeptide components without the use of reference substances. In addition, the present invention has established for the first time a method for quantitative analysis of red scorpion toxin using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method has the characteristics of simple operation, good repeatability, high sensitivity, and reliable results, and can effectively control product quality and ensure product safety and effectiveness.

[0004] The present invention is achieved through the following technical solutions:

[0005] Preparation of reference solution: Accurately weigh appropriate amounts of red scorpion toxin, acetyl hexapeptide-1, and snake venom-like tripeptide, dissolve them in 0.1% BSA and dilute them step by step to prepare a series of standard working solutions. The concentrations of the red scorpion toxin calibration curve are 1, 2, 5, 10, 50, and 100 μg / L, respectively; the concentrations of the acetyl hexapeptide-1 calibration curve are 0.2, 0.4, 1, 2, 10, and 20 μg / L, respectively; the concentrations of the snake venom-like tripeptide calibration curve are 0.1, 0.2, 0.5, 1, 5, and 10 μg / L, respectively.

[0006] Preparation of test solution: Take the sample to be tested, dilute the liquid product directly 20 times with 0.1% BSA, centrifuge at 12000rpm for 10min, and take the supernatant as the test solution; dissolve the freeze-dried product in 1ml water and dilute it 20 times with 0.1% BSA, centrifuge at 12000rpm for 10min, and take the supernatant as the test solution.

[0007] After the test solution is prepared, a high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) instrument is used for analysis and detection. The high performance liquid chromatography-tandem mass spectrometry is used for analysis and detection, and the presence of red scorpion toxin, acetyl hexapeptide-1, or snake venom tripeptide is detected in the test results to preliminarily determine whether these polypeptide components exist in the sample to be tested. If a characteristic ion pair is detected, further verify whether its relative ion abundance is within the preset acceptable error range to ultimately determine whether the analyzed sample contains red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide.

[0008] Among them, the qualitative basis for the presence of red scorpion toxin in the sample to be tested is: 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 in the range of 80% to 120% and the relative ion abundance ratio of 594.75 / 129.15 to 594.75 / 70.30 is in the range of 10% to 60%; when conditions a) and b) are met at the same time, it indicates that the sample contains red scorpion toxin.

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

[0010] The qualitative basis for determining that the sample to be tested contains a snake venom-like tripeptide is as follows: 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%. When conditions a) and b) are met at the same time, it indicates that the sample contains a snake venom-like tripeptide.

[0011] The mass spectrometry conditions described in the present invention are: the ion source is in ESI+ mode; the temperature of the desolvation tube is 250°C; the temperature of the heating module is 250°C; the nebulizing gas is nitrogen, and the flow rate is 3.0L / min; the interface temperature is 350°C; the drying gas is nitrogen, and the flow rate is 5.0L / min; the heating gas is air, and the flow rate is 15.0L / min; the detection mode of the mass spectrometer detector is multiple ion selective monitoring (MRM), and the MRM parameter table is shown in Table 1.

[0012] Table 1. MRM parameters

[0013]

[0014] * is the quantitative ion

[0015] The present invention also provides a method for quantitatively identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products, comprising the following steps:

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

[0017] (2) Sample testing: Take the sample to be tested, add BSA to dilute it, centrifuge it, take the supernatant, and analyze it using high performance liquid chromatography tandem mass spectrometry. The content of the adulterated component is obtained based on the response peak area of ​​the adulterated component and the standard working curve.

[0018] The high performance liquid chromatography conditions of the present invention are: C18 chromatographic column; column temperature of 50°C; mobile phase A is 0.2% formic acid aqueous solution; mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution, initial elution concentration of mobile phase B is 5%, mobile phase A: 10-95%, mobile phase B: 5-90%; flow rate is 0.25mL / min; injection volume is 5.0μL.

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

[0020] (1) The method of the present invention can be used to screen whether red scorpion toxin, acetyl hexapeptide-1 or snake venom tripeptide is present in the sample to be tested by ion pair qualitative analysis without a reference substance. If the three ion pair channels of each polypeptide are all peaked at the same time and the ion abundance ratio is within the specified range, it is considered that the polypeptide is present in the sample. The qualitative result can be used as a basis for distinguishing the counterfeiting of botulinum toxin products and ensuring the safety and effectiveness of botulinum toxin products.

[0021] (2) The present invention is the first to establish a method for quantitative analysis of red scorpion toxin 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, and can detect 1 μg / L of red scorpion toxin, 0.2 μg / L of acetyl hexapeptide-1 and 0.1 μg / L of snake venom-like tripeptide respectively; it can also achieve a detection speed of completing one sample in 10 minutes, ensuring an efficient workflow and rapid result feedback. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 : Specificity experiment: Chromatogram of botulinum toxin beauty product solution;

[0025] Figure 3 : Specificity experiment: Chromatogram of beauty products adulterated with botulinum toxin from red scorpion toxin;

[0026] Figure 4 : Specificity experiment: Chromatogram of beauty products adulterated with botulinum toxin by snake venom tripeptide;

[0027] Figure 5 : Specificity experiment: Chromatogram of beauty products adulterated with botulinum toxin by acetyl hexapeptide-1;

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

[0029] Figure 7 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin-1μg / L, snake venom tripeptide-0.1μg / L, acetyl hexapeptide-1-0.2μg / L);

[0030] Figure 8 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin-2μg / L, snake venom tripeptide-0.2μg / L, acetyl hexapeptide-1-0.4μg / L);

[0031] Fig. 9 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin-5μg / L, snake venom tripeptide-0.5μg / L, acetyl hexapeptide-1-1μg / L);

[0032] Fig.10 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin: 10μg / L, snake venom tripeptide-1μg / L, acetyl hexapeptide-1-2μg / L);

[0033] Fig.11 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin: 50μg / L, snake venom tripeptide-5μg / L, acetyl hexapeptide-1-10μg / L);

[0034] Fig.12 :Linear relationship investigation: Chromatogram of reference standard mixed working solution (red scorpion toxin-100μg / L, snake venom tripeptide-10μg / L, acetyl hexapeptide-1-20μg / L);

[0035] Fig.13 : Repeatability experiment: Chromatogram of reference standard mixed working solution (red scorpion toxin-2μg / L, snake venom tripeptide-0.2μg / L, acetyl hexapeptide-1-0.4μg / L) - the first of six consecutive injections;

[0036] Fig.14: Chromatogram of the mixed standard working solution of repeatability test reference substance (red scorpion toxin - 2μg / L, snake venom tripeptide - 0.2μg / L, acetyl hexapeptide - 1-0.4μg / L) - the second of six consecutive injections;

[0037] Fig.15 : Chromatogram of the mixed standard working solution of repeatability experiment reference substance (red scorpion toxin-2μg / L, snake venom tripeptide-0.2μg / L, acetyl hexapeptide-1-0.4μg / L) - the third of six consecutive injections;

[0038] Fig.16 : Chromatogram of the mixed standard working solution of repeatability test reference substance (red scorpion toxin - 2μg / L, snake venom tripeptide - 0.2μg / L, acetyl hexapeptide - 1-0.4μg / L) - the fourth of six consecutive needles;

[0039] Fig.17 : Chromatogram of the mixed standard working solution of repeatability experiment reference substance (red scorpion toxin-2μg / L, snake venom tripeptide-0.2μg / L, acetyl hexapeptide-1-0.4μg / L) - the fifth of six consecutive needles;

[0040] Fig.18 : Chromatogram of the mixed standard working solution of the repeatability experiment reference substance (red scorpion toxin-2μg / L, snake venom tripeptide-0.2μg / L, acetyl hexapeptide-1-0.4μg / L) - the sixth of six consecutive needles;

[0041] Fig.19 : Recovery experiment: low concentration spike-three parallel first spikes (spiking concentrations: red scorpion toxin-1μg / L, snake venom tripeptide-0.1μg / L, acetyl hexapeptide-1-0.2μg / L);

[0042] Fig. 20 : Recovery experiment: low concentration spike - three parallel second spikes (spiking concentrations: red scorpion toxin - 1 μg / L, snake venom tripeptide - 0.1 μg / L, acetyl hexapeptide - 1-0.2 μg / L);

[0043] Fig.21 : Recovery experiment: low concentration spike - three parallel third spike (spiking concentration: red scorpion toxin - 1μg / L, snake venom tripeptide - 0.1μg / L, acetyl hexapeptide - 1-0.2μg / L);

[0044] Fig. 22 : Recovery experiment: medium concentration spike - three parallel first spike (spiking concentration: red scorpion toxin - 5μg / L, snake venom tripeptide - 0.5μg / L, acetyl hexapeptide - 1μg / L);

[0045] Fig.23: Recovery experiment: medium concentration spike - three parallel second spikes (spiking concentrations: red scorpion toxin - 5μg / L, snake venom tripeptide - 0.5μg / L, acetyl hexapeptide - 1μg / L);

[0046] Fig.24 : Recovery experiment: medium concentration spike - three parallel third spikes (spiking concentrations: red scorpion toxin - 5 μg / L, snake venom tripeptide - 0.5 μg / L, acetyl hexapeptide - 1 μg / L);

[0047] Fig.25 : Recovery experiment: high concentration spike-three parallel first spikes (spiking concentrations: red scorpion toxin-100μg / L, snake venom tripeptide-10μg / L, acetyl hexapeptide-20μg / L);

[0048] Fig.26 : Recovery experiment: high concentration spike - three parallel second spikes (spiking concentrations: red scorpion toxin - 100 μg / L, snake venom tripeptide - 10 μg / L, acetyl hexapeptide - 20 μg / L);

[0049] Fig. 27 : Recovery experiment: high concentration spike - three parallel third spike (spiking concentration: red scorpion toxin - 100μg / L, snake venom tripeptide - 10μg / L, acetyl hexapeptide - 20μg / L);

[0050] Fig.28 :Effectiveness investigation: Chromatogram of the sample containing red scorpion toxin - Shim-pack GIST C18 (100mm x2.1mm, 2μm);

[0051] Fig.29 :Effectiveness investigation: Chromatogram of the sample containing red scorpion toxin - Shim-pack GISS C18 (100mm x2.1mm, 1.9μm);

[0052] Fig.30 :Effectiveness study: Chromatogram of the sample containing red scorpion toxin - Hypersil GOLD Vanquish (100x 2.1mm, 1.9μm);

[0053] Fig.31 :Effectiveness investigation: Chromatogram of the sample containing snake venom tripeptide-Shim-pack GIST C18 (100mmx 2.1mm, 2μm);

[0054] Fig.32 :Effectiveness investigation: Chromatogram of the sample containing snake venom tripeptide-Shim-pack GISS C18 (100mmx 2.1mm, 1.9μm);

[0055] Fig.33 :Effectiveness study: Chromatogram of the sample containing snake venom tripeptide-Hypersil GOLD Vanquish (100x 2.1mm, 1.9μm);

[0056] Fig.34 :Effectiveness investigation: Chromatogram of the sample containing acetyl hexapeptide-1 - Shim-pack GIST C18 (100mm x2.1mm, 2μm);

[0057] Fig.35 :Effectiveness investigation: Chromatogram of the sample containing acetyl hexapeptide-1 - Shim-pack GISS C18 (100mm x2.1mm, 1.9μm);

[0058] Fig.36 :Effectiveness investigation: Chromatogram of the test sample containing acetyl hexapeptide-1 - Hypersil GOLD Vanquish (100x2.1mm, 1.9μm). DETAILED DESCRIPTION

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

[0060] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0061] Example 1

[0062] 1. Experimental instruments and equipment:

[0063] High pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

[0064] 2. Experimental reagents:

[0065] Acetonitrile and formic acid were of mass spectrometry grade; water was Wahaha purified water; reference substances included red scorpion toxin (CAS No. 936616-33-0), acetyl hexapeptide-1 (CAS No. 448944-47-6), and snake venom tripeptide (CAS No. 823202-99-9).

[0066] 3. Test conditions:

[0067] Chromatographic column: C18 column

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

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

[0070] Mass spectrometry conditions:

[0071] Ion source: ESI+ mode; mass spectrometer: triple quadrupole liquid-mass spectrometer; desolvation tube temperature is 250°C; heating module temperature is 250°C; nebulizing gas is nitrogen with a flow rate of 3.0 L / min; interface temperature is 350°C; drying gas is nitrogen with a flow rate of 5.0 L / min; heating gas is air with a flow rate of 15.0 L / min; mass spectrometer detection mode is multiple ion selective monitoring (MRM), and mass spectrometer detection parameters are shown in Table 1.

[0072] 4. Standard curve drawing:

[0073] 1) Preparation of standard stock solution: accurately weigh appropriate amounts of reference substances red scorpion toxin, acetyl hexapeptide-1, and snake venom tripeptide, dissolve them with 0.1% BSA to form original stock solutions with a concentration of 2 mg / ml, and then dilute the original stock solutions with 0.1% BSA to 1000 μg / L as standard stock solutions for standby use;

[0074] 2) Preparation of standard working solution: The standard stock solution was diluted stepwise with 0.1% BSA to prepare a series of standard working solutions. The concentrations of the calibration curve of red scorpion toxin were 1, 2, 5, 10, 50, and 100 μg / L, respectively; the concentrations of the calibration curve of acetyl hexapeptide-1 were 0.2, 0.4, 1, 2, 10, and 20 μg / L, respectively; the concentrations of the calibration curve of snake venom-like tripeptide were 0.1, 0.2, 0.5, 1, 5, and 10 μg / L, respectively.

[0075] 3) Drawing a standard working curve: The above standard working solution is analyzed by high performance liquid chromatography tandem mass spectrometry to obtain chromatograms of red scorpion toxin, acetyl hexapeptide-1, and snake venom-like tripeptide, and a standard working curve is obtained based on the relationship between the solution concentration and the corresponding chromatographic peak area.

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

[0077] 6. Qualitative testing basis

[0078] The qualitative basis for determining that the sample to be tested contains red scorpion toxin is as follows: 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 in the range of 80% to 120%, and the relative ion abundance ratio of 594.75 / 129.15 to 594.75 / 70.30 is in the range of 10% to 60%. When conditions a) and b) are met at the same time, it indicates 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 groups of ion pairs are detected in the sample at the same time: 435.85 / 110.15, 435.85 / 322.90, 435.85 / 86.20, b) the relative ion abundance ratio of 435.85 / 322.90 to 435.85 / 110.15 is in the range of 40% to 80%, and the relative ion abundance ratio of 435.85 / 86.20 to 435.85 / 110.15 is in the range of 35% to 75%. When conditions a) and b) are met at the same time, it indicates that the sample contains acetyl hexapeptide-1.

[0080] The qualitative basis for determining that the sample to be tested contains a snake venom-like tripeptide is as follows: 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%. When conditions a) and b) are met at the same time, it indicates that the sample contains a snake venom-like tripeptide.

[0081] 7. Specificity experiment. Red scorpion toxin (5 μg / L), acetyl hexapeptide-1 (1 μg / L), snake venom tripeptide (0.5 μg / L) reference standard solution, botulinum toxin beauty products, adulterated botulinum toxin beauty products containing red scorpion toxin, adulterated botulinum toxin beauty products containing acetyl hexapeptide-1, adulterated botulinum toxin beauty products containing snake venom tripeptide, and blank solution were injected into LC-MSMS. Figures 1 to 6 It can be seen that in the chromatogram of the mixed standard reference substance, red scorpion toxin, snake venom tripeptide, acetyl hexapeptide-1, peak at 2.95, 3.38, and 4.02, respectively, botulinum toxin cosmetic products and blank solution have no peaks, and adulterated botulinum toxin cosmetic products only peak in the channel of their adulterated compounds. The results show that this method has a high degree of specificity.

[0082] 8. Perform linear regression on the standard working curve and investigate the sensitivity of the method. The correlation coefficients of the obtained calibration curves are all greater than 0.9973, and the accuracy of each calibration point is between 95.3% and 111.3%. Labsolutions software automatically calculates the detection limit (S / N=3) and the quantitative limit (S / N=10) based on the signal-to-noise ratio. Linear equation (see chromatogram) Figure 7-12 ), detection limit and quantification limit results are shown in Table 2. The results show that each reference substance has a good linear relationship within its own 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, detection limit and quantification limit

[0084]

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

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

[0087]

[0088] 10. Recovery rate investigation: blank samples were used for spiked experiments at low, medium and high concentrations. Each concentration was tested in triplicate and the recovery rate was calculated. At different spiked concentrations, the spiked recovery rates of each small molecule peptide were between 85.2% and 114.4%, and the relative standard deviation was no higher than 3.64%. The results are shown in Table 4 and Figure 19-27 .

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

[0090]

[0091]

[0092] 11. Effectiveness evaluation: Three types of chromatographic columns were used to quantitatively determine the samples containing red scorpion toxin, snake venom tripeptide, and acetyl hexapeptide-1, as shown in Table 5. The results shown in Table 6 were obtained, and the RSD of the positive sample detection value was less than 4.62%.

[0093] Table 5. Information of three chromatographic columns

[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 make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products, characterized in that: The sample to be tested is prepared into a test solution, and the test solution is tested by high-resolution mass spectrometry in positive ion mode using high-performance liquid chromatography-tandem mass spectrometry. The presence of adulterated ingredients in the sample to be tested is determined by detecting whether the characteristic ion pairs of red scorpion toxin, acetyl hexapeptide-1, or snake venom-like tripeptide are detected in the test results and verifying whether the relative ion abundance of the characteristic ion pairs is within the preset acceptable error range.

2. The method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that: The mass spectrometry conditions are as follows: the ion source is in ESI+ mode; the temperature of the desolvation tube is 250°C; the temperature of the heating module is 250°C; the nebulizing gas is nitrogen with a flow rate of 3.0 L / min; the interface temperature is 350°C; the drying gas is nitrogen with a flow rate of 5.0 L / min; The heating gas was air with a flow rate of 15.0 L / min; the detection mode of the mass spectrometer was multiple ion selective monitoring.

3. The method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that: The HPLC conditions are as follows: C18 chromatographic column; column temperature of 50°C; mobile phase A of 0.2% formic acid aqueous solution; mobile phase B of 0.1% formic acid acetonitrile solution; gradient elution, initial elution concentration of mobile phase B of 5%, mobile phase A: 10-95%, mobile phase B: 5-90%; flow rate of 0.25 mL / min; injection volume of 5.0 μL.

4. The method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that: The qualitative basis for the sample to be tested to contain red scorpion toxin is to meet conditions a) and b) at the same time: a) The following three ion pairs were detected in the sample at the same time: 594.75 / 70.30, 594.75 / 84.05, 594.75 / 129.15; b) The relative ion abundance ratio of 594.75 / 84.05 to 594.75 / 70.30 is in the range of 80% to 120% and the relative ion abundance ratio of 594.75 / 129.15 to 594.75 / 70.30 is in the range of 10% to 60%.

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

6. The method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that: The qualitative basis for the sample to be tested to contain snake venom tripeptide is to meet conditions a) and b) at the same time: a) The following three ion pairs 376.20 / 70.15, 376.20 / 269.25, 376.20 / 359.25 were 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%.

7. The method for identifying and detecting adulterated ingredients in counterfeit botulinum toxin cosmetic products according to claim 1, characterized in that: The following steps are also included: (1) Preparation of standard working curve: Accurately weigh the adulterated component reference substance and prepare a series of standard working solutions. The standard working solutions are analyzed by high performance liquid chromatography-tandem mass spectrometry to obtain a chromatogram of the reference substance. The standard working curve is obtained based on the relationship between the concentration of the reference substance solution and the corresponding chromatographic peak area. (2) Sample testing: Take the sample to be tested, add BSA to dilute it, centrifuge it, take the supernatant, and analyze it using high performance liquid chromatography tandem mass spectrometry. The content of the adulterated component is obtained based on the response peak area of ​​the adulterated component and the standard working curve.

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