Method for detecting 18 local anesthetics in cosmetics

Abstract

The invention provides a method for analyzing 18 local anesthetics in cosmetics. According to the method, an ultra-high performance liquid chromatography-tandem mass spectrometry method is adopted for qualitative screening and quantitative analysis. Chromatographic conditions are as follows: a chromatographic column is a C8 chromatographic column; mobile phases: A-0.1% ammonia water solution and B-0.1% ammonia water acetonitrile; the flow velocity is 0.2 to 0.4 mL / min; the column temperature is 30-40 DEG C; the sample size is 0.1 to 5 [mu] L; the elution procedure is as follows: 0-1.0 min, 10% B; in 1.0-2.5 min, 10%-40% of B is added; when the time is 2.5-6.0 min, 40% of B is reached; when the time is 6.0-14.0 min, 40-50% of B is used; in 14.0-15.0 min, 50%-90% of B is used; when the time is 15.0-17.0 min, 90% of B is reached; when the time is 17.0-17.2 min, 90-10% of B is used; and in 17.2-18.5 min, 10% of B is used. The method disclosed by the invention can be used for exclusively, sensitively and quickly screening and quantifying 18 local anesthetics such as tetracaine impurity 2 and the like in cosmetics, and is mainly suitable for analyzing cosmetic dosage forms such as cream, cream, emulsion, water aqua and the like.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics detection, and particularly to a method for detecting 18 local anesthetics in cosmetics. Background Art

[0002] Caine local anesthetics are a class of drugs that can block the conduction of nerve impulses locally and are commonly used in the medical field. Adding these substances to cosmetics can play roles such as repair, soothing, anti-wrinkle, and anti-aging, but they can also cause adverse reactions such as nerve or cardiovascular toxicity. Therefore, the "Technical Specifications for Cosmetics Safety" (2015 Edition) in China lists such components as prohibited components and stipulates the detection methods for 7 local anesthetics such as procainamide. However, some illegal merchants, in order to evade supervision, add local anesthetics outside the legal detection standards, or modify the structures of such drugs, or use by-products in the drug production process as raw materials, making them difficult to be identified in cosmetics detection. In recent years, our laboratory has first detected tetracaine impurity 2, tetracaine impurity 3, and tetracaine impurity 6 in cosmetics claimed to have a soothing effect, but there has been no report on the detection methods for the above substances. Given that tetracaine impurity 2, benzocaine impurity 23, and benzocaine impurity 30 are isomers of each other, and tetracaine impurity 6, tetracaine impurity 17, and tetracaine impurity 37 are isomers of each other, the structures of isomers are similar, which is likely to lead to misjudgment in the actual detection process. The legal standard adopts HPLC-DAD combined with QQQ-MS method, which has a long inspection time and is difficult to effectively distinguish the above isomers. The present invention adopts UPLC-QTRAP-MS through the MRM-IDA-EPI mode, which can obtain the secondary mass spectrometry information of target compounds while performing routine mass spectrometry qualitative and quantitative analysis, thereby more effectively distinguishing isomers, and its analysis time is shorter, the sensitivity is higher, and the number of compounds that can be screened simultaneously in a single analysis is more. Therefore, on the basis of the 7 local anesthetics stipulated in the legal detection standard, the present invention integrates clinically commonly used local anesthetics outside the standard and substances such as tetracaine impurity 2 that have not been reported in the literature, and establishes a separation and analysis method for 18 caine local anesthetics such as tetracaine impurity 2 for qualitative screening and content determination. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method that can detect the contents of 18 local anesthetics, and at the same time, the method can be simple to operate, specific and sensitive, and avoid misjudgment caused by similar structures.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] A method for detecting 18 local anesthetics in cosmetics, comprising the following steps:

[0006] (1) Preparation of reference solution: Weigh accurately tetracaine impurity 2, tetracaine impurity 3, tetracaine impurity 6, tetracaine impurity 17, tetracaine impurity 37, benzocaine impurity 30, benzocaine impurity 23, procainamide, lidocaine, procaine, tetracaine, chloroprocaine, bupivacaine, ropivacaine, cinchocaine, oxybuprocaine, benzocaine, prilocaine, dissolve and dilute them to prepare a reference solution for use;

[0007] (2) Preparation of test solution;

[0008] (3) Qualitative screening by ultra-high performance liquid chromatography-tandem mass spectrometry: Take blank solution, test solution and reference solution respectively, inject them into the liquid chromatography-mass spectrometry instrument, and detect the above 18 local anesthetics.

[0009] Preferably, for the preparation of the reference solution: Weigh accurately 2 mg each of tetracaine impurity 2, tetracaine impurity 3, tetracaine impurity 6, tetracaine impurity 17, tetracaine impurity 37, benzocaine impurity 30, benzocaine impurity 23, procainamide, lidocaine, procaine, tetracaine, chloroprocaine, bupivacaine, ropivacaine, cinchocaine, oxybuprocaine, benzocaine, prilocaine, place them in a 10 mL volumetric flask, dissolve with methanol and make up to the mark to obtain a single standard stock solution; then transfer 0.1 mL to the same volumetric flask and gradually dilute it 10,000 times with the negative sample extract to prepare a reference solution for use with a concentration of 20 ng / mL;

[0010] Preferably, for the preparation of the test solution: Weigh accurately 0.2 g of the test sample, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to 10 mL, centrifuge at 13,000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the test solution;

[0011] Preferably, qualitative screening is carried out by ultra-high performance liquid chromatography-tandem mass spectrometry: Chromatographic conditions:

[0012] Chromatographic column: C 8 chromatographic column, 100 mm × 2.1 mm, 1.7 μm / 1.8 μm;

[0013] Mobile phase: A - 0.1% ammonia water solution, B - 0.1% ammonia water acetonitrile;

[0014] Flow rate: 0.2 - 0.4 mL / min;

[0015] Column temperature: 30 - 40 °C;

[0016] Injection volume: 0.1 - 5 μL;

[0017] Elution program: 0 - 1.0 min, 10% B; 1.0 - 2.5 min, 10 - 40% B; 2.5 - 6.0 min, 40% B; 6.0 - 14.0 min, 40 - 50% B; 14.0 - 15.0 min, 50 - 90% B; 15.0 - 17.0 min, 90% B; 17.0 - 17.2 min, 90 - 10% B; 17.2 - 18.5 min, 10% B;

[0018] Mass spectrometry conditions:

[0019] Electrospray ionization positive ion mode (ESI + );

[0020] Ion source spray voltage (IS): +5500 V / -4500 V;

[0021] Ion source temperature (TEM): 450 - 550 °C;

[0022] Heating gas (GS1): 40 - 55 psi;

[0023] Auxiliary heating gas (GS2): 40 - 60 psi;

[0024] Curtain gas (CUR): 15 - 35 psi;

[0025] Collision gas (CAD): Medium;

[0026] Acquisition mode: Multiple reaction monitoring triggered enhanced product ion scan (MRM-IDA-EPI);

[0027] Preferably, ultra-high performance liquid chromatography-tandem mass spectrometry is used for qualitative screening: Blank solution, test solution and reference solution are respectively taken and injected into the liquid chromatography-mass spectrometry instrument, and 18 local anesthetics are detected according to the above chromatographic and mass spectrometry conditions.

[0028] Table 1. Mass spectrometry parameters of compounds

[0029] Serial number Name CAS No. Molecular formula <![CDATA[[M+H] + > Product ion CE / V DP / V 1 Tetracaine impurity 2 4740-24-3 <![CDATA[C 11 H 15 NO 2 > 194.1 94.2、138.0* 26、18 40 2 Tetracaine impurity 3 71839-12-8 <![CDATA[C 12 H 17 NO 2 > 208.1 120.0、152.0* 25、19 40 3 Tetracaine impurity 6 94-32-6 <![CDATA[C 13 H 19 NO 2 > 222.1 138.1、166.1* 23、19 50 4 Tetracaine impurity 17 1056627-94-1 <![CDATA[C 13 H 19 NO 2 > 222.1 138.1、166.1* 28、16 30 5 Tetracaine impurity 37 512175-79-0 <![CDATA[C 13 H 19 NO 2 > 222.1 138.1、166.1* 24、17 40 6 Benzocaine impurity 23 94-25-7 <![CDATA[C 11 H 15 NO 2 > 194.1 120.0、138.0* 28、13 5 7 Benzocaine impurity 30 10287-53-3 <![CDATA[C 11 H 15 NO 2 > 194.1 151.1*、166.1 32、22 60 8 Procainamide hydrochloride 614-39-1 <![CDATA[C 13 H 22 ClN 3 O]]> 236.1 120.0、163.1* 39、21 40 9 Lidocaine 137-58-6 <![CDATA[C 14 H 22 N 2 O]]> 235.1 58.0、86.0* 52、21 40 10 Procaine hydrochloride 51-05-8 <![CDATA[C 13 H 21 ClN 2 O 2 > 237.1 100.0*、120.0 20、32 40 11 Tetracaine 94-24-6 <![CDATA[C 15 H 24 N 2 O 2 > 265.2 72.0、176.1* 20、20 40 12 Chloroprocaine hydrochloride 3858-89-7 <![CDATA[C 13 H 20 Cl 2 N 2 O 2 > 271.1 100.0*、154.0 21、32 40 13 Bupivacaine hydrochloride 73360-54-0 <![CDATA[C 18 H 31 ClN 2 O 2 > 289.2 84.0、140.1* 55、27 60 14 Ropivacaine mesylate 854056-07-8 <![CDATA[C 18 H 30 N 2 O 4 S]]> 275.1 84.1、126.1* 56、26 60 15 Cinchocaine hydrochloride 61-12-1 <![CDATA[C 20 H 30 ClN 3 O 2 > 344.2 215.1、271.2* 40、28 80 16 Oxybuprocaine hydrochloride 5987-82-6 <![CDATA[C 17 H 29 ClN 2 O 3 > 309.2 100.1、192.1* 23、24 60 17 Benzocaine 94-09-7 <![CDATA[C 9 H 11 NO 2 > 166.0 120.0、138.0* 25、14 30 18 Prilocaine 721-50-6 <![CDATA[C 13 H 20 N 2 O]]> 221.1 86.1*、136.0 17、25 30

[0030] Note: *Quantitative ion pair

[0031] Preferably, the test sample in step (2) is a cream, lotion, emulsion or aqueous cosmetic.

[0032] Preferably, the chromatographic column in step (3) is a C8 chromatographic column, 100 mm × 2.1 mm, 1.7 μm.

[0033] Preferably, the column temperature in step (3) is 35 °C.

[0034] Preferably, the flow rate in step (3) is 0.3 mL / min.

[0035] Preferably, the ionization voltage in step (3) is 5500 V.

[0036] Preferably, the ion source temperature in step (3) is 500 °C;

[0037] Preferably, the heating gas in step (3) is 55 psi;

[0038] Preferably, the auxiliary heating gas in step (3) is 55 psi;

[0039] Preferably, the curtain gas in step (3) is 25 psi;

[0040] Preferably, the sample injection volume in step (3) is 1 μL.

[0041] In the present invention, if there is a conflict between the Chinese name and the structural formula of the compound, the structural formula shall prevail.

[0042] The principle of this method is as follows: The test sample is extracted with methanol, separated by a C 8 chromatographic column, eluted with 0.1% ammonia aqueous solution and 0.1% ammonia acetonitrile solution, ionized in the positive ion mode of the electrospray ionization source (ESI + ), detected by the multiple reaction monitoring-triggered enhanced product ion scan (MRM-IDA-EPI) mode, preliminarily determined as a suspected positive sample according to the chromatographic retention time and relative ion abundance ratio, and further confirmed by comparing the secondary mass spectra of the suspected positive sample and the reference substance, and quantified by the external standard method.

[0043] Result judgment:

[0044] If the test sample shows qualitative / quantitative ion pair chromatographic peaks with the same retention time as those of the 18 local anesthetic reference substances, and the relative ion abundance ratio (k) does not exceed the maximum allowable deviation (k > 50%, the maximum allowable deviation is ±20%; 50% ≥ k > 20%, the maximum allowable deviation is ±25%; 20% ≥ k > 10%, the maximum allowable deviation is ±30%; k ≤ 10%, the maximum allowable deviation is ±50%), it is determined as a suspected positive sample. By further comparing the secondary mass spectra of the suspected positive sample and the reference substance, if they are consistent, it is determined that the local anesthetic component in this experiment is added, and the component is quantified by the external standard method.

[0045] If the test sample shows ion pair chromatographic peaks with the same retention time as those of the 18 local anesthetic reference substances, and if the relative ion abundance ratio (k) exceeds the maximum allowable deviation and is inconsistent with the secondary mass spectrum of the reference substance, it is determined that these 18 local anesthetics are not added to the sample, and the detected concentration is calculated.

[0046] If no chromatographic peaks of ion pairs consistent with the retention times of the 18 local anesthetic reference substances appear in the test sample, it is determined that these 18 local anesthetics are not added to the sample, and the detected concentration is calculated.

[0047] The beneficial effects of the present invention are as follows:

[0048] 1. The method of the present invention can specifically, sensitively and rapidly conduct qualitative screening and quantitative analysis on 18 local anesthetics such as tetracaine impurity 2 in cosmetics, avoiding misjudgment caused by similar compound structures.

[0049] 2. The method of the present invention is mainly applicable to the inspection of 18 local anesthetics such as tetracaine impurity 2 in creams, emulsions and aqueous solutions.

[0050] 3. Among the 18 local anesthetics such as tetracaine impurity 2 detected in this experiment, there are no detection means for tetracaine impurity 2, tetracaine impurity 3, tetracaine impurity 6, tetracaine impurity 17, tetracaine impurity 37, benzocaine impurity 23 and benzocaine impurity 30. Through this method, accurate screening and confirmation can be carried out, providing strong technical support for supervision. Description of the Drawings

[0051] Figure 1 Extracted ion chromatogram (EIC) of 18 local anesthetics such as tetracaine impurity 2 (1: tetracaine impurity 2, 2: procainamide, 3: benzocaine, 4: procaine, 5: chloroprocaine, 6: prilocaine, 7: benzocaine impurity 23, 8: lidocaine, 9: benzocaine impurity 30, 10: tetracaine, 11: ropivacaine, 12: tetracaine impurity 3, 13: oxybuprocaine, 14: tetracaine impurity 17, 15: bupivacaine, 16: tetracaine impurity 37, 17: tetracaine impurity 6, 18: cinchocaine);

[0052] Figure 2 Secondary mass spectrum of the tetracaine impurity 2 reference substance solution;

[0053] Figure 3 Secondary mass spectrum of the prilocaine reference substance solution;

[0054] Figure 4 Secondary mass spectrum of the lidocaine reference substance solution;

[0055] Figure 5 Secondary mass spectrum of the tetracaine reference substance solution;

[0056] Figure 6 Secondary mass spectrum of the tetracaine impurity 3 reference substance solution;

[0057] Figure 7 Secondary mass spectrum of the tetracaine impurity 6 reference substance solution;

[0058] Figure 8 EIC chromatogram of the test solution in Example 2 (1: Tetracaine impurity 2, 2: Prilocaine, 3: Lidocaine, 4: Tetracaine);

[0059] Figure 9 EIC chromatogram of the test solution in Example 2 (5: Tetracaine impurity 3, 6: Tetracaine impurity 6);

[0060] Figure 10 EIC chromatogram of the test solution in Example 3 (1: Tetracaine impurity 2, 2: Prilocaine, 3: Lidocaine, 4: Tetracaine);

[0061] Figure 11 EIC chromatogram of the test solution in Example 3 (5: Tetracaine impurity 3, 6: Tetracaine impurity 6);

[0062] Figure 12 EIC chromatogram of the test solution in Example 4 (1: Tetracaine impurity 2, 2: Prilocaine, 3: Lidocaine, 4: Tetracaine);

[0063] Figure 13 EIC chromatogram of the test solution in Example 4 (5: Tetracaine impurity 3). Detailed implementation mode

[0064] The present invention will be described below with reference to examples, but the present invention is not limited thereto. In the art, simple substitutions or improvements made by those skilled in the art to the present invention all fall within the scope of the technical solutions protected by the present invention.

[0065] Example 1:

[0066] Screening of chromatographic, mass spectrometric and sample treatment conditions

[0067] 1. Reagents and materials

[0068] Ultra-pure water; methanol and acetonitrile are mass spectrometry-grade alcohols; ammonia water is of analytical grade; reference substances of Tetracaine impurity 2, Tetracaine impurity 3, Tetracaine impurity 6, Benzocaine impurity 23, Benzocaine impurity 30 (source: STD PHARM), Tetracaine impurity 17, Tetracaine impurity 37 (source: CATO), Procainamide hydrochloride, Procaine hydrochloride, Chloroprocaine hydrochloride, Benzocaine, Lidocaine, Tetracaine, Cinchocaine hydrochloride, Prilocaine reference substances (source: BePure), Bupivacaine hydrochloride, Ropivacaine mesylate, Oxybuprocaine hydrochloride reference substances (source: NIFDC).

[0069] 2. Instruments

[0070] Sciex Q-trap 6500+ triple quadrupole-linear ion trap hybrid mass spectrometer (Shanghai AB SCIEX Trading Co., Ltd.), KQ-300DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), Metler-Toledo XP56 electronic balance (accuracy 0.001 mg, Mettler-Toledo Instruments (Shanghai) Co., Ltd.), Milli-Q pure water system (Millipore).

[0071] 3. Chromatographic conditions

[0072] Chromatographic column: C8 chromatographic column, ACQUITY UPLC BEH C8, 1.7 μm, 2.1×100 mm;

[0073] Mobile phase: A - 0.1% ammonia aqueous solution, B - 0.1% ammonia acetonitrile;

[0074] Flow rate: 0.3 mL / min;

[0075] Column temperature: 35 °C;

[0076] Injection volume: 1 μL;

[0077] Elution program: 0 - 1.0 min, 10% B; 1.0 - 2.5 min, 10 - 40% B; 2.5 - 6.0 min, 40% B; 6.0 - 14.0 min, 40 - 50% B; 14.0 - 15.0 min, 50 - 90% B; 15.0 - 17.0 min, 90% B; 17.0 - 17.2 min, 90 - 10% B; 17.2 - 18.5 min, 10% B;

[0078] 4. Mass spectrometry conditions

[0079] Electrospray ionization positive ion mode (ESI + );

[0080] Ion source spray voltage (IS): 5500 V;

[0081] Ion source temperature (TEM): 500 °C;

[0082] Heating gas (GS1): 55 psi;

[0083] Auxiliary heating gas (GS2): 55 psi;

[0084] Curtain gas (CUR): 25 psi;

[0085] Collision gas (CAD): Medium;

[0086] Sampling mode: Multiple reaction monitoring-triggered enhanced product ion scan (MRM-IDA-EPI).

[0087] 5. Preparation of analytes

[0088] (1) Preparation of reference solutions: Accurately weigh 2 mg each of tetracaine impurity 2, tetracaine impurity 3, tetracaine impurity 6, tetracaine impurity 17, tetracaine impurity 37, benzocaine impurity 30, benzocaine impurity 23, procainamide, lidocaine, procaine, tetracaine, chloroprocaine, bupivacaine, ropivacaine, cinchocaine, oxybuprocaine, benzocaine, and prilocaine, and place them in separate 10 mL volumetric flasks. Dissolve with methanol and make up to the mark to obtain single-standard stock solutions. Then transfer 0.1 mL to the same volumetric flask and gradually dilute 10,000-fold with negative sample extract to prepare reference working solutions with a concentration of 20 ng / mL each.

[0089] (2) Sample preparation: Accurately weigh 0.2 g of the test sample, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to 10 mL, centrifuge at 13,000 r / min for 5 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the test sample solution.

[0090] (3) Qualitative screening by ultra-high performance liquid chromatography-tandem mass spectrometry: Take blank solution, test sample solution, and reference solution respectively, and inject them into the liquid chromatography-mass spectrometry instrument for detection.

[0091] Table 2. Selection of mobile phase

[0092]

[0093] Table 3. Selection of ammonia concentration in mobile phase

[0094] Method number Ammonia water concentration (%) Result 1 0.02 The tailing of cinchocaine is serious 2 0.05 The peak shapes of each compound have a slight tailing, and the response of the compound is good 3 0.10 The peak shapes of each compound are good, and the response of the compound is good 4 0.15 The peak shapes of each compound are good, and the response of the compound is slightly low

[0095] Table 4. Selection of stationary phase

[0096]

[0097] Table 5. Selection of column temperature

[0098] Method number Column temperature Result 1 30℃ The resolution of each compound is good, but the system pressure is too high 2 35℃ The resolution of each compound is good 3 40℃ The resolution of each compound is poor

[0099] Table 6. Selection of extraction solvent

[0100]

[0101] Methodology investigation

[0102] Linear range:

[0103] Appropriately transfer an appropriate amount of the reference substance stock solutions of 18 components such as tetracaine impurity 2, and successively dilute with the negative sample extract to prepare a series of control solutions with final concentrations of 1, 2, 5, 10, 20, and 50 ng / mL respectively. Perform linear fitting with the peak area (Y) against the mass concentration (X), and calculate the linear equation and correlation coefficient (see Table 7). The results show that within the range of 1 - 50 ng / mL, each compound has a good linear relationship (r > 0.999).

[0104] Table 7. Linear ranges, linear equations, and correlation coefficients of 18 local anesthetics

[0105]

[0106]

[0107] Detection limit and quantification limit:

[0108] Appropriately transfer an appropriate amount of the reference substance stock solutions of 18 components such as tetracaine impurity 2. Using the negative sample extract of creams as the solvent, take the concentrations of the respective standard solutions corresponding to 3 and 10 times the signal-to-noise ratio as the detection limit and quantification limit respectively. The results are shown in Table 8.

[0109] Table 8. Detection limits and quantification limits of 18 local anesthetics

[0110]

[0111]

[0112] Recovery and precision:

[0113] Using the negative aqueous solution and cream samples in the detection results as the matrix, conduct the determination of the recovery rate of added standards. Weigh 0.2 g of the negative sample and place it in a 15 mL graduated centrifuge tube. Add 40 μL of the mixed reference substance solution of 18 local anesthetics with a mass concentration of 500 ng / mL, in parallel for 6 portions. Prepare the sample according to the sample preparation in step (2), inject the sample, and measure. Prepare the blank matrix extract spiked solution and measure it concomitantly. Calculate the ratio of the mass spectrometric response of the analyte in the sample solution to that of the reference substance solution at the same concentration to evaluate the recovery rate and precision of this method (Table 9).

[0114] Table 9. Recovery rates and precisions of 18 local anesthetics (n = 6)

[0115]

[0116] Matrix effect:

[0117] The standard curves were prepared using the extracts of aqueous and cream negative samples as blank matrices, and the matrix effect (ME) was calculated as the ratio of the slopes of the matrix standard curve to the solvent standard curve. The results are shown in Table 10. The results showed that the matrix effects of the 18 compounds were between 0.44 and 1.16, indicating that different matrix types had a certain degree of influence on the determination of the target compounds. Therefore, the matrix-matched blank standard curve was used in this method.

[0118] Table 10. Matrix effects of 18 local anesthetics

[0119]

[0120] Stability

[0121] A mixed reference solution with a concentration of 10 ng / mL was placed in the autosampler and injected at 1 h, 6 h, 24 h, 48 h, and 72 h after preparation. The mass spectrometry responses of each component were calculated, and the results showed good stability.

[0122] Example 2:

[0123] Accurately weigh 0.2 g of a certain batch of soothing lotion, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to 10 mL, centrifuge at 13000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and inject it into the liquid chromatography-mass spectrometry instrument for detection.

[0124] Result analysis:

[0125] The results showed that chromatographic peaks appeared in the sample solution at retention times of 3.11, 6.58, 9.39, 10.60, 11.98, and 14.86 min, which were consistent with the retention times of the reference substances of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, tetracaine impurity 3, and tetracaine impurity 6 (see Appendix Figure 1 , 8, 9), the relative ion abundance ratio (k) did not exceed the maximum allowable deviation, and the corresponding secondary mass spectrometry Figure 1 was consistent. The contents of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, tetracaine impurity 3, and tetracaine impurity 6 were measured to be 2775, 9206, 9621, 13757, 40, and 100 mg / kg, respectively.

[0126] Example 3:

[0127] Accurately weigh 0.2 g of a certain batch of soothing lotion, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to 10 mL, centrifuge at 13000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and inject it into the liquid chromatography-mass spectrometry instrument for detection.

[0128] Result analysis:

[0129] The results showed that chromatographic peaks appeared in the sample solution at retention times of 3.11, 6.59, 9.41, 10.61, 11.98, and 14.86 min, which were consistent with the retention times of the reference substances of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, tetracaine impurity 3, and tetracaine impurity 6, respectively (see Appendix Figure 1 , 10, 11). The relative ion abundance ratio (k) did not exceed the maximum allowable deviation, and the corresponding secondary mass spectra Figure 1 were consistent. The contents of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, tetracaine impurity 3, and tetracaine impurity 6 were measured to be 3849, 10315, 8229, 11221, 38, and 98 mg / kg, respectively.

[0130] Example 4:

[0131] Accurately weigh 0.2 g of a certain batch number of soothing paste, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to make up to 10 mL, centrifuge at 13000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm filter membrane, and inject it into the liquid chromatography-mass spectrometry instrument for detection.

[0132] Result analysis:

[0133] The results showed that chromatographic peaks appeared in the sample solution at retention times of 3.13, 6.58, 9.40, 10.60, and 12.00 min, which were consistent with the retention times of the reference substances of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, and tetracaine impurity 3, respectively (see Appendix Figure 1 , 12, 13). The relative ion abundance ratio (k) did not exceed the maximum allowable deviation, and the corresponding secondary mass spectra Figure 1 were consistent. The contents of tetracaine impurity 2, prilocaine, lidocaine, tetracaine, and tetracaine impurity 3 were measured to be 3140, 1171, 44244, 39945, and 145 mg / kg, respectively.

[0134] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for detecting 18 local anesthetics in cosmetics, characterized in that: The steps include: (1) Preparation of reference substance solution: accurately weigh tetracaine impurity 2, tetracaine impurity 3, tetracaine impurity 6, tetracaine impurity 17, tetracaine impurity 37, benzocaine impurity 30, benzocaine impurity 23, procainamide, lidocaine, procaine, tetracaine, chloroprocaine, bupivacaine, ropivacaine, cinchocaine, oxybuprocaine, benzocaine, and prilocaine, dissolve and dilute to prepare a reference substance solution; (2) Preparation of test solution; (3) Qualitative screening was performed using ultra-high performance liquid chromatography-tandem mass spectrometry: blank solution, test solution, and reference solution were injected into the LC-MS instrument to detect the above 18 local anesthetics.

2. The method according to claim 1, characterized in that The step (1) is specifically as follows: accurately weigh 2 mg of each of the 18 local anesthetic reference substances, place them in a 10 mL volumetric flask, dissolve them with methanol and dilute to the mark as a single standard stock solution; then transfer 0.1 mL to the same volumetric flask, gradually dilute them 10,000 times with the negative sample extract to prepare a reference substance working solution with a concentration of 20 ng / mL.

3. The method according to claim 1, characterized in that: The step (2) is specifically as follows: accurately weigh 0.2 g of the test sample, place it in a 15 mL centrifuge tube, add 8 mL of methanol, vortex for 1 min, ultrasonically extract for 30 min, then add methanol to 10 mL, centrifuge at 13000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain a test sample solution.

4. The method according to claim 1, characterized in that The conditions for qualitative screening by ultra-high performance liquid chromatography-tandem mass spectrometry in step (3) are specifically as follows: Chromatographic conditions: Chromatographic column: C8 / C 18 Chromatographic column, 100 mm × 2.1 mm, 1.7 μm / 1.8 μm; Mobile phase: A-0.1% ammonia solution, B-0.1% ammonia acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 30-40°C; Injection volume: 0.1-5μL; Elution program: 0-1.0 min, 10% B; 1.0-2.5min, 10-40%B; 2.5-6.0min, 40%B; 6.0-14.0min, 40-50%B; 14.0-15.0min, 50-90%B; 15.0-17.0min, 90%B; 17.0-17.2min, 90-10%B; 17.2-18.5min, 10%B; Mass spectrometry conditions: Electrospray ionization positive ion mode; Ion source spray voltage: +5500V / -4500V; Ion source temperature: 450-550℃; Heating gas: 40-55psi; Auxiliary heating gas: 40-60psi; Air curtain gas: 15-35psi; Collision gas: Medium; Acquisition mode: Multiple reaction monitoring triggered enhanced product ion scan.

5. The method according to claim 1, characterized in that The test sample in step (2) is an ointment, cream, lotion or aqueous cosmetic.

6. The method according to claim 1, characterized in that The chromatographic column in step (3) is an ACQUITY UPLC BEH C8 chromatographic column, 1.7 μm, 2.1×100 mm.

7. The method according to claim 1, characterized in that In step (3), the column temperature is 35° C. and the flow rate is 0.3 mL / min.

8. The method according to claim 1, characterized in that: In the step (3), the ionization voltage is 5500 V and the ion source temperature is 500° C.

9. The method according to claim 1, characterized in that: In step (3), the heating gas is 55 psi and the auxiliary heating gas is 55 psi.

10. The method according to claim 1, characterized in that The curtain gas in step (3) is 25 psi, and the injection volume in S3 is 1 μL.

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