Rapid screening method for adding prohibited antibiotics in cosmetics

Abstract

The invention provides a rapid screening method for adding prohibited antibiotics in cosmetics, which comprises the following steps: carrying out ultrasonic extraction, centrifugation, cyclohexane back extraction, sample introduction and C18 chromatographic column separation on a test sample by using methanol, extracting parent ions (EIC peaks) of each compound in a database, and carrying out preliminary qualitative judgment according to the accurate mass number of the detected substance peaks. The suspected detected substance needs to be independently prepared with reference substance solutions and injected, and confirmation is completed by comparing ultraviolet absorption spectrum and secondary mass spectrum characteristics of the detected substance and the reference substance (when a plurality of matching results with the same mass number appear in qualitative results, the reference substance solutions need to be respectively prepared, and a chromatographic gradient elution procedure is properly adjusted according to needs; and baseline separation is achieved. The content of the detected substance is obtained by performing standard curve fitting calculation on the primary mass spectrum signal. According to the procedure provided by the invention, under the condition that 90% of reference substances are deficient, rapid screening of ten types and 296 antibiotics in cosmetics can be completed through one experiment.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic testing and relates to methods for detecting chemicals, specifically a rapid screening and quantitative method for the addition of prohibited antibiotics to cosmetics. Background Technology

[0002] Antibiotics mainly include quinolones, macrolides, sulfonamides, nitroimidazoles, tetracyclines, and amide alcohols. The addition of these drugs gives cosmetics certain effects such as acne treatment, anti-acne, and mite removal; however, long-term use can induce dysbiosis, easily causing contact dermatitis, allergies, and antibiotic resistance in the human body. my country's "Cosmetic Safety Technical Specifications" (2022 edition) explicitly stipulates that antibiotics are prohibited substances. In recent years, the cosmetics market has grown rapidly, and the illegal addition of various antibiotics has occurred frequently. The legally prescribed inspection methods only cover dozens of antibiotics, while hundreds of compounds can be used as cosmetic additives (see Table 1). Therefore, rapid screening for illegally added antibiotics in cosmetics is of great significance for purifying the cosmetics market environment. Although there are many antibiotics listed above, drugs within the same class share similar or identical structural cores, resulting in similar retention behaviors in liquid chromatography (LC). According to literature reports, all of these compounds can be separated using reversed-phase chromatography columns. The LC retention time of substances within the same class primarily depends on polarity, which can be measured by the logP value. This provides a possibility for establishing a unified chromatographic separation method. Therefore, by selecting a column with a wide polarity range, using a wide range of mobile phase elution programs, and leveraging the differences in logP values ​​among components, representative compounds can be selected to establish a chromatographic method. This allows for the separation of almost all commercially available antibiotics with a single injection. Similar to chromatographic methods, the structural similarity of a class of compounds determines the similarity of their mass spectrometric behaviors. Therefore, a single set of mass spectrometry parameters can be used to detect all antibiotics. In conclusion, by selecting representative components from major prohibited components and establishing chromatographic and mass spectrometric methods, rapid screening and content determination of several major categories of components in a database can be achieved. Table 1. Summary of prohibited ingredients in cosmetics listed in the standards Summary of the Invention

[0003] The technical problem to be solved: The purpose of this invention is to provide a rapid screening method for prohibited antibiotics added to cosmetics, which can simultaneously screen for common illegal additives in cosmetics such as quinolones, tetracyclines, macrolides, sulfonamides, azoles, lincosamides, amide alcohols, nitrofurans, and allylamines. It integrates the existing seven testing standards, so that it can not only meet the needs of supervision and inspection, but also lay the foundation for subsequent standard improvement, without increasing the testing cost.

[0004] Technical solution: A rapid screening method for prohibited antibiotics in cosmetics, comprising the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection data, and according to the logP values ​​of compounds in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or near-largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic conditions: Chromatographic column: Waters CORTECS UPLC HSS T3, 100mm × 2.1mm, 1.6μm or a similarly structured C18 HPLC or UPLC column capable of withstanding high proportions of aqueous phase; Mobile phase: A - methanol-acetonitrile solution of 0.1% formic acid, B - 0.1% formic acid solution; Flow rate: 0.2-1.0 mL / min; Column temperature: 25-40℃; Injection volume: 0.5-50 μL; Gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B; Spectral acquisition wavelength range: 210-400nm; Mass spectrometry conditions: Electrospray positive and negative ion modes; Capillary voltage: 2000-3500V(+) / 1500-3000V(-); Drying gas flow rate: 12.0 L / min; Nebulizer gas pressure: 40 psig; Drying gas temperature: 350℃; Breaking voltage: 50-125V; Scanning method: Full scan, first-order mass spectrometry; Acquisition mode: MS (Auto); Mass spectrometry scanning range: 50-1200. Table 2. Database of Antibiotics That May Be Added to Cosmetics Table 3. Methods for establishing a collection of antibiotic compounds Preferably, the method for obtaining the antibiotic compound set is as follows: based on the parent nucleus structure category of the antibiotic to be screened in the database, select several substances with the largest and smallest or close to the largest and smallest logP values ​​to form the set. Preferably, the test sample in step S3 includes ointments, creams, emulsions, films, and aqueous cosmetics. Preferably, the calibration deviation of the positive and negative ion mass spectrometer in the ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry method is <2ppm. Preferably, in the methanol-acetonitrile solution of 0.1% formic acid in S4, the ratio of methanol to acetonitrile is 1:1. Preferably, the column temperature in the mass spectrometry conditions is 30-40℃, more preferably 30-35℃. Preferably, the chromatographic conditions are: UPLC with an injection volume of 0.5-2 μL; or HPLC with an injection volume of 1-20 μL. Preferably, the capillary voltage in the mass spectrometry conditions is 2000V(+) / 3000V(-). Preferably, the fragmentation voltage in the mass spectrometry conditions is 100-125V. Principle: The test sample is extracted with methanol by ultrasonication, centrifuged, back-extracted with cyclohexane, injected, and separated on a C18 column. In the acquired total ion chromatogram, the precursor ions corresponding to the compounds in the positive and negative modes listed in Table 2 are extracted, and preliminary screening is completed based on the extracted ion chromatograms. For suspected detectable substances, the results are compared with literature search information, and their reference standards are obtained (if multiple matching results are found, all isomers must be obtained). The sample is then injected again, and the chromatographic behavior, UV spectral characteristics, and mass spectrometric patterns of the test sample and reference standards are compared for comprehensive confirmation. Confirmed detectable substances are quantified using the external standard method. Using the above detection methods, total ion current chromatograms of the test sample and blank solution were collected. The [M+H] ions of each compound in Table 2 were then extracted using the Extract chromatogram function. + [MH] - The primary mass spectrum was used to obtain the precise mass numbers of the peaks detected in the test sample and the fragment information of the secondary mass spectrometry. Combined with database information and literature search, background interference was eliminated to preliminarily determine the presence of added antibiotic components. Confirmation: Further confirmation of the detected components was performed by preparing a separate reference solution (concentration approximately 10 μg / mL) and sequentially injecting it along with the blank solution and the test solution. The UV spectrum and secondary mass spectrum of the detected compounds were collected, and the results were obtained by comparing the retention time, UV absorption characteristics, parent ion, daughter ion, and their relative abundance ratios from the primary extraction ion chromatogram (EIC). Content determination: Quantification of the detected components was performed using the external standard method: a series of standard solutions of the analyte (range 0.5 μg / mL-20 μg / mL) were prepared and injected. The dilution factor of the test solution was adjusted appropriately according to the primary mass spectrometry response of the compound to ensure it fell within the standard curve range. The peak area of ​​the primary parent ion of the compound was linearly fitted to its concentration, and the content of the detected component in the test sample was calculated. Limit of detection: An appropriate amount of the mixed reference solution from Table 13 was transferred and diluted to prepare mixed reference solutions with concentrations of 0.1 μg / mL. These solutions were injected, and the detection concentration was calculated as a reference value for the sensitivity of this method for detecting antibiotics. If the final comparison finds one or more substances in the database, and these substances have no isomers, then proceed with the mass spectrometry confirmation steps described above. If the final comparison finds two or more compounds in the database, and these substances contain isomers, then the chromatographic elution procedure in the separation method should be adjusted according to the detected components to achieve chromatographic separation of the detected isomers, and then the mass spectrometry confirmation procedure described above should be followed. Beneficial effects: 1. This invention covers multiple testing standards in the current national testing standards and the 2022 edition of the "Cosmetic Safety Technical Specifications" (see Table 1). It can significantly reduce the consumption of reference standards and reagents while basically meeting the testing requirements of current standards. This method can rapidly screen for 296 antibiotics across ten categories in cosmetics in a single experiment, even with 90% reference standards as a default setting. This saves the country considerable procurement costs and reduces the maintenance costs associated with laboratory management and periodic verification resulting from the consumption of large amounts of reference standards and reagents. 2. The analysis time for completing a screening in this invention is much shorter than that of the current standard (see Table 4), saving time caused by differences in sample pretreatment, chromatographic column, mobile phase, and analysis procedure in the current standard method, and effectively improving the testing efficiency. 3. Current methods for detecting antibiotics in cosmetics often use QQQ instruments in MRM mode. While this method has high sensitivity (detection concentration ≈ 1 ng / mL), the low detection levels are far below the effective dose for most antibiotics, thus limiting its practical significance. Furthermore, adulterated cosmetics typically contain high concentrations of antibiotics (ranging from approximately 1-80,000 mg / kg), easily leading to system residues and false positives (e.g., terbinafine). This invention uses a time-of-flight mass spectrometer (TOF-MS) with automatic scanning. Although its sensitivity is slightly lower than a quadrupole mass spectrometer (detection concentration ≈ 100 ng / mL), its mass accuracy is high (approximately 1000 times higher than QQQ), meeting both sensitivity requirements and minimizing system contamination. For suspected detectable substances, comparison with a reference standard using UV spectroscopy and secondary mass spectrometry effectively eliminates false positives. 4. This invention provides a method for screening quinolones, tetracyclines, macrolides, sulfonamides, azoles, lincosamides, amide alcohols, nitrofurans, allylamines, and other chemical substances with the highest possible efficiency. It also provides methods for further confirmation and quantification after the detection of these substances, which can not only meet the preliminary screening in the actual detection process, but also achieve accurate identification and content detection. 5. The database system established by this invention can facilitate the upgrading of standards for the illegal addition of antibiotic ingredients to cosmetics. For newly discovered prohibited components, it is not necessary to optimize the extraction, separation, and detection parameters of the compounds according to the current national standard model; it is only necessary to examine the applicability of the method in this invention to the newly discovered components. 6. The wide-area database established by this invention can achieve rapid screening of a large number of prohibited ingredients in the absence of reference standards, greatly increasing the cost of counterfeiting and helping to combat illegal activities. Table 4. Summary of Comparison between this Patent and Current Standards Attached Figure Description Figure 1 To establish a method for the extraction ion current (EIC) of antibiotic compound collection reference standards. + Overlay chromatogram; Figure 2 To establish a method for the extraction ion current (EIC) of antibiotic compound collection reference standards. - Overlay chromatogram; Figure 3 To validate the method, the extraction ion current (EIC) of quinolone reference standards in the concentration of antibiotic compounds was used. + Overlay chromatogram; Figure 4 To validate the method, the extraction ion current (EIC) of antibiotic compounds, specifically sulfonamide reference standards, was concentrated. + Overlay chromatogram; Figure 5 To validate the method, a concentrated extraction ion current (EIC) was used to extract tetracycline reference standards from antibiotic compounds. + Overlay chromatogram; Figure 6 To validate the method, the extraction ion current (EIC) of macrolide antibiotic reference standards was concentrated. + Overlay chromatogram; Figure 7 To validate the method, a concentrated ion exchange current (EIC) was used to extract antibiotic compounds, specifically azole reference standards. + Overlay chromatogram; Figure 8 To validate the method, the extracted ion current (EIC+) superimposed chromatograms of antibiotic compounds and nitrofuran reference standards were used. Figure 9 To validate the method, the extraction ion current (EIC) of lincosamide reference standards for antibiotic compounds was concentrated. + Overlay chromatogram; Figure 10 To validate the method, the extraction ion current (EIC) of allylamine reference standards was concentrated in the antibiotic compound. + Overlay chromatogram; Figure 11 The ion chromatogram (EIC) of sulfamethoxazole extracted from the test sample in Example 2 is shown below. + ); Figure 12The mass spectrum (CE:15V) of sulfamethoxazole in the test sample of Example 2 is shown below. Figure 13 The extractive ion chromatogram (EIC) of trimethoprim in the test sample of Example 2 is shown below. + ); Figure 14 The mass spectrum (CE:25V) of trimethoprim in the test sample of Example 2 is shown below. Figure 15 The extractive ion chromatogram (EIC) of hydroxyterbinafine and terbinafine N-oxide in the reference standard of Example 3 is shown below. + ); Figure 16 The extractive ion chromatogram (EIC) of terbinafine N-oxide in the test sample of Example 3 is shown below. + ); Figure 17 The mass spectrum (CE:15V) of terbinafine N-oxide in the test sample of Example 3 is shown. Figure 18 The ion chromatogram (EIC) of terbinafine extracted from the sample in Example 3 is shown below. + ); Figure 19 The mass spectrum (CE:15V) of terbinafine in the test sample of Example 3 is shown below. Figure 20 The extractive ion chromatogram (EIC) of miconazole impurity H in the test sample of Example 4 is shown below. + ); Figure 21 This is a secondary mass spectrum (CE:20V) of miconazole impurity H in the test sample of Example 4. Detailed Implementation The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Method establishment and verification: A rapid screening method for the addition of prohibited antibiotics to cosmetics includes the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection data, and according to the logP values ​​of compounds in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or near-largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic conditions: Column: Waters CORTECS UPLC HSS T3, 100mm × 2.1mm, 1.6μm; Mobile phase: A - 0.1% formic acid (methanol-acetonitrile), B - 0.1% formic acid solution; Flow rate: 0.3 mL / min; Column temperature: 35℃; Injection volume: 1 μL; Gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B; Mass spectrometry conditions: Electrospray positive and negative ion modes; Capillary voltage: 2000V(+) / 3000V(-); Drying gas flow rate: 12.0 L / min; Nebulizer gas pressure: 40 psig; Drying gas temperature: 350℃; Breaking voltage: 125V; Scanning method: Full scan primary and secondary mass spectrometry; Acquisition mode: MS (Auto); Mass spectrometry scanning range: 50-1200. In a preferred embodiment, the test sample in step S3 includes ointments, creams, emulsions, films, and aqueous cosmetics. In a preferred embodiment, the filter membrane in step S3 is a 0.22 μm organic filter membrane. In a preferred embodiment, the ratio of methanol to acetonitrile in A-0.1% formic acid (methanol-acetonitrile) in step S4 is 1:1. In a preferred embodiment, the calibration positive and negative ion mass deviations of the time-of-flight mass spectrometer in the ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry method are both <2ppm. Using the above detection methods, total ion current chromatograms of the test sample and blank solution were collected. The [M+H] ions of each compound in Table 1 were then extracted using the Extract chromatogram function. + [MH] - The primary mass spectrometry chromatogram, by comparing the spectra of the test sample and the reference sample, eliminates background interference and preliminarily detects the added antibiotic component. Mass spectrometry confirmation: Further confirmation of the detected components is carried out by preparing a separate reference solution (concentration of 1-50 μg / mL) and injecting it sequentially with the blank solution and the test solution. The secondary mass spectrum of the detected compound is collected, and the confirmation is completed by comparing the retention time, parent ion, daughter ion and their relative abundance ratio of the primary extraction ion chromatogram (EIC). Content determination: The external standard method was used to quantify the detected components: a series of standard solutions of the components to be tested (range 1-100 μg / mL) were prepared and injected. The dilution factor of the test solution was adjusted appropriately according to the primary mass spectrometry response of the compound to make it fall within the standard curve range. The peak area of ​​the primary precursor ion of the compound was linearly fitted with its concentration, and the content of the detected components in the test sample was calculated. Determination of detection limit: Take an appropriate amount of the mixed reference solution from Tables 3 and 13, dilute it to prepare a reference detection limit solution with a concentration of 0.1 μg / mL, inject it into the sample, and calculate its detection concentration as a reference value for the sensitivity of this method in detecting antibiotics. If the final comparison finds one or more substances in the database, and these substances have no isomers, then proceed with the mass spectrometry confirmation steps described above. If the final comparison reveals isomers of the compounds in the database, the chromatographic elution procedure in the separation method should be adjusted according to the detected components to achieve chromatographic separation of the detected isomers, and then the mass spectrometry confirmation steps described above should be followed. The screening process for chromatography, mass spectrometry, and sample processing conditions is as follows: Table 5 Selection of Organic Phase Types in the Mobile Phase Method number Organic phase types result 1 methanol High system pressure 2 Acetonitrile One-way valve of liquid phase binary high-pressure pump is prone to sticking. 3 Methanol:acetonitrile (1:1) The compounds all responded well, and the high-pressure pump check valve showed no sticking. Table 6 Selection of Stationary Phase serial number Fixed phase specifications result 1 HSS T3C18, 2.1*100mm, 1.7μm Various compounds are well preserved 3 Cortecs T3C18,2.1*100mm,1.6μm Various compounds are well preserved 4 BEH C8, 2.1*100mm, 1.7μm Strongly polar compounds are poorly retained Table 7 Selection of Flow Rate Method number Flow rate (mL / min) result 1 0.20 The chromatographic system operates at low pressure, resulting in slow elution of compound peaks. 2 0.30 The chromatographic system pressure was moderate, and the compound peaks were satisfactory. 4 0.40 The chromatographic system operates at high pressure, resulting in rapid elution of compounds. Table 8 Selection of Mass Spectrometry Spray Voltage Method number Spray voltage (V) result 1 2000 The parent ions of each compound showed high mass spectrometry response. 2 2500 The mass spectrometry response of the parent ions of each compound was low. 3 3000 The mass spectrometry response of the parent ions of each compound was low. 4 3500 The mass spectrometry response of the parent ions of each compound was low. 5 4000 The mass spectrometry response of the parent ions of each compound was low. Table 9 Selection of Mass Spectrometry Spray Voltage Method number Spray voltage (V) result 1 -1500 The mass spectrometry response of the parent ions of each compound was low. 2 -2000 The mass spectrometry response of the parent ions of each compound was low. 3 -2500 The mass spectrometry response of the parent ions of each compound was low. 4 -3000 The parent ions of each compound showed high mass spectrometry response. Table 10 Selection of Desolventizing Temperature Table 11 Selection of Mass Spectrometry Fragmentation Voltage Table 12 Selection of Impurity Removal Methods for Sample Extracts Methodological Validation 1. Verification of the universality of the analytical method Each reference standard from the antibiotic compound set (Table 13) was accurately weighed, dissolved in methanol and diluted to 100 mL to prepare a mixed reference standard working solution with a concentration of 10 μg / mL. The antibiotic compound set mixed control solution was validated using the injection method described in S4. The chromatographic retention and first-order mass spectrometry response of the compounds were examined (results are attached). Figure 3-10 ). Table 13. Collection of antibiotic compounds used for method validation 2. Limit of detection and limit of quantitation: Take an appropriate amount of mixed reference solution of various antibiotics from the antibiotic compound pool for method establishment and validation, dilute with methanol to a concentration of 0.1 μg / mL, inject and determine, extract the EIC chromatogram of each compound, calculate the signal-to-noise ratio, and use the concentration of each reference solution corresponding to a signal-to-noise ratio of 3 and 10 times as the detection concentration and quantification concentration. The calculation results are shown in Table 14. Table 14. Method establishment and validation: Limit of detection for concentrated substances in compounds Methodological validation of the chromatographic retention behavior of the antibiotic compound set (see Appendix) Figure 3-10 As can be seen from the results, the separation and analytical methods established based on the antibiotic compound set in this experiment are also suitable for method validation of each component in the antibiotic compound set. From the detection limits of each component (see Table 14), it can be seen that the sensitivities of the major component groups in this experiment are similar, with detection concentrations all below 4 μg / g. By extension, it can be considered that when the additive content is higher than 4 μg / g, the analytical methods established in this experiment are applicable to all compounds in the database in Table 2. Therefore, the following method validation items will be performed using the antibiotic compound set established by this method. 4. Linear range A stock solution of each antibiotic compound was prepared using a transfer method. A mixed control solution with a concentration of 100 μg / mL was prepared, and then serially diluted to prepare mixed control series solutions with concentrations of 20, 10, 5, 2, 1, 0.5, and 0.2 μg / mL. These solutions were injected under the conditions described in S4, and the EIC spectra of the precursor ions in Table 3 were extracted. The results were integrated, and a linear fit was performed on the peak area based on the compound concentration to calculate the correlation coefficient. The results are shown in Table 15 below. Table 15. Linear fitting of antibiotic compounds Serial Number Peak name Linear range (μg / ml) Fitting equation Correlation coefficient 1 Erythromycin succinate 0.2-5 y = 186x + 7634 0.9997 2 erythromycin 0.2-5 y = 5x + 698 0.9965 3 furazolidone 0.2-10 y = 131x + 24949 0.9993 4 Fusidic acid 0.2-10 y = 103x + 36601 0.9969 5 Sulfadosin 0.2-10 y = 243x - 8499 0.9989 6 sulfonamides 0.2-10 y = 10x + 6962 0.9973 7 sulfaquinoxaline 0.2-10 y = 70x - 5163 0.9970 8 sulfaguanidine 0.2-10 y = 163x + 67624 0.9989 9 Trimethoprim 0.2-10 y = 483x + 164515 0.9992 10 sulfanitrobenzene 0.2-10 y = 14x + 2540 0.9992 11 Lincomycin 0.2-10 y = 271x + 18754 0.9990 12 Chloramphenicol 0.5-20 y = 113x + 80646 0.9980 13 griseofulvin 0.2-10 y = 79x - 3990 0.9977 14 Terbinafine 0.2-10 y = 885x + 306909 0.9984 15 Mupiro Star 0.2-10 y = 45x - 3839 0.9980 16 tetracycline 0.2-10 y = 94x - 6690 0.9986 17 Minocycline 0.2-10 y = 25x + 2481 0.9959 18 Doxycycline 0.2-10 y = 95x - 11828 0.9982 19 Salad Starch 0.2-10 y = 115x + 11299 0.9993 20 Difloxacin 0.2-10 y = 226x + 21869 0.9996 21 Enoxastatin 0.2-10 y = 236x + 19642 0.9996 22 Pazufloxacin 0.2-10 y = 33x + 1378 0.9998 23 Piperidin 0.2-10 y = 297x + 62338 0.9995 24 Miconazole 0.2-10 y = 164x + 16876 0.9992 25 Metronidazole 0.2-10 y = 309x + 88186 0.9993 26 tinidazole 0.2-10 y = 167x + 33247 0.9993 5. Precision A mixed control solution with a concentration of 10 μg / mL was taken and injected six times according to the conditions in S4. The EIC chromatograms of the precursor ions in the table were extracted, integrated, and the relative standard deviations of retention time and peak area were calculated. Results: The retention time RSD of each compound was <1.0%, and the peak area RSD was <10%. 6. Stability A mixed control solution with a concentration of 10 μg / mL was placed in an autosampler and injected at 1 h, 2 h, 4 h, and 8 h. The EIC chromatograms of each compound were extracted, integrated, and the changes were calculated. The results showed that the peak area change was less than 10%. 7. Accuracy Two samples, a cream and a liquid formulation without added antibiotics, were used as negative samples for the recovery test. 1.0 g of each was accurately weighed, and six replicates were prepared. 1.0 mL of a mixed standard solution containing 100 μg / mL antibiotic compounds was added to each sample. The samples were processed according to step S3, injected, and the peak areas of the analytes treated with the two matrices were calculated and compared with the mixed standard solution containing the same concentration of blank solvent. The recovery rates were then calculated. The results showed that the average recovery rate of all compounds in the cream was 48.0%, and the average recovery rate of all components in the liquid formulation was 64.2%. Qualitative screening result determination: From the chromatogram of the test sample, extract the [M+H] compounds from the database in Table 2. + / [MH] - If no chromatographic peaks are found in the EIC chromatogram, it is determined that the sample does not contain any of the antibiotic components listed in Table 2. From the chromatogram of the test sample, extract the [M+H] compounds from the database in Table 2. + / [MH] - If an EIC chromatogram shows a peak, antibiotics may have been added to the sample. A reference standard for the suspected substance needs to be obtained and prepared into a solution with a concentration of 10 μg / mL. Then, the blank solution, reference solution, and test solution are injected separately, and the retention time, UV absorption spectrum, and primary and secondary ion mass spectrometry are collected to confirm the substance. From the chromatogram of the test sample, extract the [M+H] compounds from the database in Table 2. + / [MH] - If an EIC chromatogram shows a peak, antibiotics may have been added to the sample. If the suspected component has one or more isomers, reference standards for all suspected substances must be obtained. At the same time, the current gradient elution procedure should be examined and modified to ensure that the suspected substance and its isomers are separated. Then, blank solution, reference solution, and test solution are injected separately, and the retention time of the compound, UV absorption spectrum, and primary and secondary ion mass spectrometry are collected to complete the confirmation. Determination of the content of the detected substances: Accurately weigh 1 mg of the reference standard for the detected substance and prepare a series of standard curve solutions (1-100 μg / mL). Adjust the dilution factor of the test sample according to the first-order mass spectrometry response of the detected substance in the test sample so that the response of the analyte is within the standard curve range, and calculate the content of the detected component in the sample according to the external standard method. The following are specific examples: instrument: Agilent 1290 UHPLC; Agilent 6538ESI-Q-TOF / MS mass spectrometer (Agilent, America); KQ-300DA ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q pure water system (Millipore); Metler-Toledo electronic balance; Thermo Heraeus Multifuge X3 centrifuge (Thermo); Haier DW-25L262 medical cryogenic storage box (Qingdao Haier Co., Ltd.). Reagents: Ultrapure water; methanol, acetonitrile, and formic acid were all of mass spectrometry grade. Example 1 A rapid screening method for the illegal addition of antibiotics to cosmetics includes the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection data, and according to the logP values ​​of compounds in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or near-largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic column: Waters Cortecs UPLC T3 C18, 100 mm × 2.1 mm, 1.6 μm; mobile phase: A - 0.1% formic acid (methanol-acetonitrile), B - 0.1% formic acid solution; flow rate: 0.3 mL / min; column temperature: 35 ℃; injection volume: 1 μL; gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B. Mass spectrometry conditions: Electrospray ionization source mode: + / -; Capillary voltage: 2000V(+) / 3000V(-); Drying gas flow rate: 12.0L / min; Nebulizing gas pressure: 40psig; Drying gas temperature: 350℃; Fragmentation voltage: 125V; Scanning mode: Full scan primary and secondary mass spectrometry; Acquisition mode: MS(Auto); Mass spectrometry scan range: 50-1200. Result judgment: In the total ion chromatogram of the test sample, the [M+H] of all compounds in Table 2 were extracted. + [MH] - If no obvious chromatographic peaks are found in the EIC chromatogram, it can be determined that the sample does not contain any of the antibiotic components listed in Table 2. Inject the reference standard detection limit solution and calculate its detection concentration as a reference value for the sensitivity of this method to detect various antibiotics (see Table 14). It can be basically determined that at the 4 μg / g level, no antibiotics from the database in Table 2 were added to a certain batch of emulsion A. Example 2 A rapid screening method for the illegal addition of antibiotics to cosmetics includes the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection data, and according to the logP values ​​of compounds in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or near-largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic conditions: Chromatographic column: Waters Cortecs UPLC T3 C18, 100 mm × 2.1 mm, 1.6 μm; mobile phase: A - 0.1% formic acid (methanol-acetonitrile), B - 0.1% formic acid solution; flow rate: 0.3 mL / min; column temperature: 35 ℃; injection volume: 1 μL; gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B. Mass spectrometry conditions: Electrospray ionization source mode: + / -; Capillary voltage: 2000V(+) / 3000V(-); Drying gas flow rate: 12.0L / min; Nebulizing gas pressure: 40psig; Drying gas temperature: 350℃; Fragmentation voltage: 125V; Scanning mode: Full scan primary and secondary mass spectrometry; Acquisition mode: MS(Auto); Mass spectrometry scan range: 50-1200. Result judgment: In the total ion chromatogram of the test sample, [M+H] was extracted from Table 2. + [MH] - The EIC plot reveals [M+H]. + The chromatogram shows two peaks, with matching results for sulfamethoxazole ([M+H]+: 254.0599, molecular formula: C10H11N3O3S) and trimethoprim ([M+H]+: 291.1457, molecular formula: C14H18N4O3). Therefore, it is preliminarily determined that these two substances were added to the sample. (See attached image) Figure 11 , 13 ). Obtain sulfamethoxazole and trimethoprim reference standards. Prepare reference solutions with a concentration of 10 μg / mL according to the preparation of reference solutions in section S2. Inject the mixed reference solution, blank solution, and test solution sequentially. Collect chromatograms and mass spectra. Compare the chromatographic retention time, UV absorption spectrum, primary precursor ion, and secondary fragments of the compounds detected in the reference standards and samples. If all information is consistent with the sulfamethoxazole and trimethoprim reference standards, it confirms that sulfamethoxazole and trimethoprim have been added to the sample. Figure 12 , 14 ). Weigh 1 mg each of sulfamethoxazole and trimethoprim reference standards, process them according to step S2 above, dilute them to prepare a series of standard curve solutions (concentration of 0.5-10 μg / mL), inject them separately, and fit the equations of compound concentration and first-order mass spectrometry response. Weigh two portions of the test sample and process them according to step S3 above. Adjust the dilution factor of the test sample so that the concentrations of sulfamethoxazole and trimethoprim in the test solution are within the linear range. Inject the sample and calculate the content of the detected components in the sample according to the above fitting equation. The calculation shows that the content of sulfamethoxazole in a certain batch of face cream B is 59782 mg / kg, and the content of trimethoprim is 24077 mg / kg. Example 3 A rapid screening method for the illegal addition of antibiotics to cosmetics includes the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection data, and according to the logP values ​​of compounds in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or near-largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic column: Waters Cortecs UPLC T3 C18, 100 mm × 2.1 mm, 1.6 μm; mobile phase: A - 0.1% formic acid (methanol-acetonitrile), B - 0.1% formic acid solution; flow rate: 0.3 mL / min; column temperature: 35 ℃; injection volume: 1 μL; gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B. Mass spectrometry conditions: Electrospray ionization source mode: + / -; Capillary voltage: 2000V(+) / 3000V(-); Drying gas flow rate: 12.0L / min; Nebulizing gas pressure: 40psig; Drying gas temperature: 350℃; Fragmentation voltage: 125V; Scanning mode: Full scan primary and secondary mass spectrometry; Acquisition mode: MS(Auto); Mass spectrometry scan range: 50-1200. Result judgment: In the total ion chromatogram of the test sample, [M+H] was extracted from Table 2. + [MH] - The EIC chromatogram revealed peaks at [M+H]+: 292.2065 and 308.2014. Matching results showed terbinafine ([M+H]+: 292.2065, molecular formula: C21H25N), hydroxyterbinafine, and terbinafine N-oxide ([M+H]+: 308.2014, molecular formula: C21H25NO). Therefore, it was preliminarily determined that terbinafine, hydroxyterbinafine, or terbinafine N-oxide had been added to the sample. Terbinafine, hydroxyterbinafine, and terbinafine N-oxide reference standards were obtained. Following the preparation of the reference solution in section S2, single and mixed reference solutions with a concentration of 10 μg / mL were prepared. Under the conditions in section S4, the single reference solution, mixed reference solution, blank solution, and test solution were injected sequentially. Chromatograms and mass spectra were collected. The chromatographic retention times, primary precursor ions, and secondary fragments of the compounds detected in the reference standards and sample were compared. It was found that all characteristics were consistent with those of the terbinafine and terbinafine N-oxide reference standards, and the chromatographic peak resolution of hydroxyterbinafine and terbinafine N-oxide was good (>1.5). Therefore, it was confirmed that terbinafine and terbinafine N-oxide were added to the sample. (See attached...) Figure 15-19 ). Weigh 1 mg each of terbinafine and its N-oxide reference standard, process them according to step S2 above, dilute them to prepare a series of standard curve solutions (concentration of 0.5-10 μg / mL), inject them separately, and fit the equation between the compound concentration and the first-order mass spectrometry response. Weigh two portions of the test sample and process them according to step S3 above; adjust the dilution factor of the test sample so that the response of terbinafine and terbinafine N-oxide in the test solution is within the standard curve range, inject the sample, and calculate the content according to the above fitting equation. Calculations show that the terbinafine content in a certain batch of face cream C is 295 mg / kg, and the terbinafine N-oxide content is 10 mg / kg. The content of terbinafine in a certain batch of cream is 653 mg / kg. The content of terbinafine N-oxide is 3.8 mg / kg. Example 4 A rapid screening method for the illegal addition of antibiotics to cosmetics includes the following steps: S1. Method for constructing an antibiotic compound set: Based on the structural representativeness of the compounds and their previous detection status, and according to the logP values ​​of the components in the database of potentially added antibiotics in cosmetics (Table 2), 1-10 substances with the largest and smallest or close to the largest and smallest values ​​were selected according to their categories and combined. S2. Preparation of reference solution: Accurately weigh 1 mg of each component reference standard of the antibiotic compound set established by the method, add 1% formic acid methanol, sonicate to dissolve and make up to 100 mL to obtain a mixed reference solution with a concentration of 10 μg / mL. S3. Preparation of the test solution: Accurately weigh 1g of cosmetic product and place it in a 15mL graduated centrifuge tube. Add 7mL of methanol, vortex for 30s, and sonicate for 20min. Let stand, add methanol to 10mL, centrifuge at 3800r / min for 10min, transfer 4mL of the supernatant, add 1mL of cyclohexane, vortex for 1min, centrifuge at 3800r / min for 10min, and remove the lower methanol layer. Filter through a 0.22μm organic filter membrane to obtain the test solution. S4. Qualitative screening using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: Inject blank solution, test solution, and mixed reference solution into the liquid chromatography-mass spectrometry instrument, and perform detection according to the following chromatographic and mass spectrometric conditions: Chromatographic conditions: Chromatographic column: Waters Cortecs UPLC T3 C18, 100 mm × 2.1 mm, 1.6 μm; mobile phase: A - 0.1% formic acid (methanol-acetonitrile), B - 0.1% formic acid solution; flow rate: 0.3 mL / min; column temperature: 35 ℃; injection volume: 1 μL; gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B. Mass spectrometry conditions: Electrospray ionization source mode: + / -; Capillary voltage: 2000V(+) / 3000V(-); Drying gas flow rate: 12.0L / min; Nebulizing gas pressure: 40psig; Drying gas temperature: 350℃; Fragmentation voltage: 125V; Scanning mode: Full scan primary and secondary mass spectrometry; Acquisition mode: MS(Auto); Mass spectrometry scan range: 50-1200. Result judgment: In the total ion chromatogram of the test sample, [M+H] was extracted from Table 2. + [MH] - The EIC plot reveals [M+H]. + A peak was found in the chromatogram at 347.0718. The matching result showed miconazole impurity H ([M+H]+: 347.0702, molecular formula: C18H16Cl2N2O). Therefore, it was preliminarily determined that miconazole impurity H was added to the sample. Figure 20 ). To obtain miconazole impurity H reference standard, prepare reference solutions with a concentration of 10 μg / mL according to the preparation of reference solutions in section S2. Then, sequentially inject the reference solution, blank solution, and test solution, and collect chromatograms and mass spectra. Compare the chromatographic retention times, primary precursor ions, and secondary fragments of the compounds detected in the reference standard and the sample. If all information is consistent with the miconazole impurity H reference standard, then it is confirmed that miconazole impurity H has been added to the sample. (See attached...) Figure 21 ). Weigh 1 mg of miconazole impurity H reference standard, process it according to step S2 above, dilute it to prepare a series of standard curve solutions (concentration of 1-10 μg / mL), inject them separately, and fit the equation between the compound concentration and the first-order mass spectrometry response. Weigh two portions of the test sample, process them according to step S3 above, inject the samples, and calculate the content of the detected component in the samples based on the above fitting equation. The calculated content of miconazole impurity H in a certain batch of face cream D is 32 mg / kg. Although the present invention has been described in detail in the embodiments through general description, specific implementation and experiment, any modifications or improvements that can be made without departing from the core of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A rapid screening method for the addition of prohibited antibiotics in cosmetics, characterized by, Comprise the following steps: S1. Method of establishing the construction of the set of antibiotic compounds: according to the representative structure of the compounds and the previous detection, according to the logP value of the compounds in the database of possible antibiotic substances added in cosmetics in table 2, 1-10 kinds of maximum and minimum or close to maximum and minimum substances are selected according to the category, and combined; S2. Preparation of reference solution: accurately weigh 1 mg of each component of the set of antibiotic compounds in the method of establishing, add 1% formic acid methanol, ultrasonic dissolution and constant volume to 100 mL, and then the concentration of the mixed reference solution is 10 μg / mL; S3. Preparation of test solution: accurately weigh 1 g of cosmetic, add 7 mL of methanol in 15 mL graduated centrifuge tube, vortex for 30 s, ultrasonic extraction for 20 min, stand, add methanol to 10 mL, centrifuge at 3800 r / min for 10 min, remove 4 mL of supernatant, add 1 mL of cyclohexane, vortex for 1 min, centrifuge at 3800 r / min for 10 min, and then the lower layer of methanol liquid is absorbed, and then filtered through 0.22 μm organic filter membrane, and then the test solution is obtained; S4. Qualitative screening by using ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry: take the blank solution, test solution and mixed reference solution respectively, inject into the liquid chromatograph-mass spectrometer, and detect according to the following chromatographic and mass spectrometric conditions: Chromatographic conditions: Chromatographic column: Waters CORTECS UPLC HSS T3, 100 mm x 2.1 mm, 1.6 μm or C18 HPLC or UPLC chromatographic column with similar structure and capable of resisting high proportion of water phase; Mobile phase: A-0.1% formic acid methanol-acetonitrile solution, B-0.1% formic acid solution; Flow rate: 0.2-1.0 mL / min; Column temperature: 25-40℃; Injection volume: 0.5-50 μL; Gradient elution program: 0-2 min, 98% B; 10-13 min, 2% B; 13.2-15 min, 98% B; Spectral acquisition wavelength range: 210-400 nm; Mass spectrometric conditions: Electrospray positive and negative ion mode; Capillary voltage: 2000-3500 V (+) / 1500-3000 V (-); Dry gas flow rate: 12.0 L / min; Atomization gas pressure: 40 psig; Dry gas temperature: 350℃; Fragmentation voltage: 50-125 V; Scanning mode: full scan mass spectrometry; Acquisition mode: MS (Auto); Mass spectrometric scanning range: 50-1200.

2. The method of claim 1, wherein the method is characterized by: The specific construction method of the method of establishing the set of antibiotic compounds is: according to the category of the parent nucleus structure of each compound in the database of antibiotics to be screened, 1-10 kinds of maximum and minimum or close to maximum and minimum substances are selected, and combined.

3. The method of claim 1, wherein the method is characterized by: The test sample in step S3 includes ointment, cream, emulsion, film and water-based cosmetics.

4. The method of claim 1, wherein the method is a rapid screening method for detecting the presence of a prohibited antibiotic in a cosmetic product. In the ultra-high performance liquid chromatography-ultraviolet-time-of-flight mass spectrometry, the calibration of the time-of-flight mass spectrometer has a mass deviation of less than 2 ppm for positive and negative ions.

5. The method of claim 1, wherein the method is a rapid screening method for detecting the presence of a prohibited antibiotic in a cosmetic product. In S4, the ratio of methanol to acetonitrile in the 0.1% formic acid methanol-acetonitrile solution is 1:

1.

6. The method of claim 1, wherein the method is a rapid screening method for detecting the presence of a prohibited antibiotic in a cosmetic product. The column temperature in the mass spectrometry condition is 30-40 DEG C.

7. The method of claim 1, wherein the method is a rapid screening method for detecting the presence of a prohibited antibiotic in a cosmetic product. The chromatography condition is: using UPLC, the injection volume is 0.5-2 muL; using HPLC, the injection volume is 1-20 muL.

8. The method of claim 1, wherein the method is a rapid screening method for detecting the presence of a prohibited antibiotic in a cosmetic product. The capillary voltage in the mass spectrometry condition is 2000 V(+) / 3000 V(-).

9. The method of claim 1, wherein the method is a rapid screening method for the detection of illicit antibiotics in cosmetic products. The fragmentation voltage in the mass spectrometry condition is 100-125 V. The column temperature in the mass spectrometry condition is 30-40 DEG C. The chromatography condition is: using UPLC, the injection volume is 0.5-2 muL; using HPLC, the injection volume is 1-20 muL. The capillary voltage in the mass spectrometry condition is 2000 V(+) / 3000 V(-). The fragmentation voltage in the

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