Determination of six nitrobenzene compounds in cosmetics by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry

Abstract

The application discloses a method for determining six kinds of nitrobenzene compounds in cosmetics by using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, and belongs to the technical field of analysis and detection. The method comprises the following steps: S01, preparation of a standard solution; S02, sample treatment; S03, chromatographic conditions; S04, mass spectrometric conditions; and S05, establishment of a compound standard spectrum library. The six kinds of nitrobenzene compounds are 3,4-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,3-dinitrotoluene, 2,5-dinitrotoluene and 3,5-dinitrotoluene. The method fills the blank of the LC-MS method for determining nitrobenzene compounds, solves the pretreatment defects of the GC-MS method, improves the detection sensitivity, and provides a new technical selection for quality and safety monitoring of cosmetics.

Description

Technical Field

[0001] This invention relates to a method for determining six nitrobenzene compounds in cosmetics using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, belonging to the field of analytical detection technology. Background Technology

[0002] Nitrobenzene compounds are important raw materials or intermediates in the production of hair dyes (such as p-phenylenediamine, p-aminophenol, and toluene-2,5-diamine sulfate). If cosmetic manufacturers do not strictly review the quality of the raw materials they use, nitrobenzene substances can easily be introduced into hair dye cosmetics as residual impurities. During long-term and repeated use of hair dye products, nitrobenzene substances accumulate in the hair, scalp, and other tissues. Through transdermal absorption, they act on the blood, liver, and central nervous system, causing harm such as allergic dermatitis, teratogenicity, and carcinogenicity. Therefore, both EU and Chinese cosmetic regulations explicitly list six nitrobenzene compounds—3,4-dinitrotoluene, 2,3-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,5-dinitrotoluene, and 3,5-dinitrotoluene—as prohibited raw materials in cosmetics.

[0003] The main detection methods for nitrobenzene compounds include gas chromatography-flame ionization detector (GC-MS), gas chromatography-electron capture detector (GC-EM), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). Tested products or objects include water, air, soil, leather goods, textiles, and cosmetics. Given the easy vaporization of nitrobenzenes and the simple and rapid separation using capillary columns, GC-MS is the standard method for detecting nitrobenzene compounds in cosmetics. However, GC-MS has limitations such as simple sample preparation and relatively low detection sensitivity. For example, after extraction with a weakly polar solvent, the extract still contains a large amount of waxy components, which can easily contaminate the chromatographic column and mass spectrometer, reducing detection efficiency and shortening the lifespan of the column and instrument. Alternatively, excessive dilution of the extract can result in a low detection limit, leading to false positive results.

[0004] Therefore, there is an urgent need for an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method to determine six nitrobenzene compounds in cosmetics, which can overcome the pretreatment defects of GC-MS, improve detection sensitivity, and provide a new technical option for cosmetic quality and safety monitoring. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for determining six nitrobenzene compounds in cosmetics by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. This method is sensitive, has high recovery rate and good repeatability, and can be used for the detection of nitrobenzene compounds in cosmetics with various matrix types. It provides new ideas and technical support for ensuring the safety of cosmetic use and combating the illegal addition of prohibited raw materials in the cosmetics industry.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for determining six nitrobenzene compounds in cosmetics using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry includes the following steps:

[0008] S01, Preparation of standard solution;

[0009] S02, Sample Preparation:

[0010] Weigh 0.50 g of sample and place it in a 25 mL colorimetric tube. Add 10 mL of ethyl acetate, vortex to mix, and then extract ultrasonically for 15 min. Centrifuge at 5000 r / min for 5 min. Dehydrate the supernatant with anhydrous sodium sulfate and place it in a 50 mL round-bottom flask. Repeat the extraction once with 10 mL of ethyl acetate. Combine the extracts, add 5% palladium on carbon and 50 mg of Pd, and place the flask in a normal pressure hydrogenation apparatus. With the fume hood open, introduce hydrogen gas at a flow rate of 2 m / s and react at 40 °C for 2 h. Centrifuge the reaction solution at 5000 r / min for 5 min, collect the supernatant in a 25 mL nitrogen blow-off tube, and dry it with nitrogen gas in a 40 °C water bath. Add 1.0 mL of methanol and vortex to redissolve the solution. Filter the solution through a 0.22 μm microporous membrane. The filtrate is ready for analysis.

[0011] S03, chromatographic conditions:

[0012] Column: Agilent Poroshell 120EC-C 18 Column (100mm × 4.6mm, 2.7μm); Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile; Flow rate: 0.5mL / min; Injection volume: 5μL; Column temperature: 20℃; Gradient elution program: 0–10 min, 98% A, 2% B; 10–10.5 min, 98% A–5% A, 2% B–95% B; 10.2–14 min, 5% A, 95% B; 14–14.2 min, 5% A–98% A, 95% B–2% B; 14.2–17 min, 98% A, 2% B;

[0013] S04, Mass spectrometry conditions:

[0014] Ion source: Electrospray ionization source; positive ion mode; full scan range: m / z 50~1000; mass spectrometry data acquisition mode: information-dependent scanning source-in-source lysis Auto MS / MS CID; real-time calibration solution: 10 mmol / L sodium formate solution; drying gas temperature: 200℃; drying gas flow rate: 8.0 L / min; nebulizer gas pressure: 2.0 Bar; capillary voltage: 4500 V;

[0015] S05, Establishment of a standard spectral library of compounds;

[0016] The six nitrobenzene compounds are 3,4-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,3-dinitrotoluene, 2,5-dinitrotoluene, and 3,5-dinitrotoluene.

[0017] In S01, the standard solution is prepared as follows:

[0018] Standard stock solutions: Accurately weigh approximately 25.00 mg of 3,4-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,3-dinitrotoluene, 2,5-dinitrotoluene and 3,5-dinitrotoluene standards respectively, place them in a 10 mL volumetric flask, dissolve them in ethyl acetate and dilute to the mark, and store at 4°C for later use;

[0019] Mixed standard stock solution: Accurately transfer 0.10 mL of the standard stock solution into a 25 mL volumetric flask, add ethyl acetate to dissolve and dilute to the mark, and store at 4°C for later use;

[0020] Mixed standard reaction solution: Accurately transfer 0.50 mL of each standard stock solution into a 50 mL round-bottom flask, add 20 mL of ethyl acetate, sonicate to dissolve, add 5% palladium on carbon and 50 mg of Pd, place in a normal pressure hydrogenation apparatus, purge with hydrogen gas at a flow rate of 2 m / s, react at 40 °C for 2 h, centrifuge the reaction solution at 5000 r / min for 5 min, collect the supernatant into a 25 mL nitrogen blow-off tube, dry it with nitrogen gas in a 40 °C water bath, add 2 mL of methanol to vortex redissolve, transfer to a 10 mL volumetric flask, dilute to the mark with methanol, and store in a 4 °C refrigerator for later use;

[0021] Blank matrix mixed standard working solution: Accurately transfer an appropriate amount of the mixed standard reaction solution of the above 6 compounds, and prepare a series of mixed standard working solutions of different concentrations with blank matrix extract, ready for testing.

[0022] Accurately transfer 0.016 mL, 0.04 mL, 0.08 mL, 0.20 mL, 0.40 mL, 0.80 mL, 2.00 mL, and 4.00 mL of the mixed standard reaction solution into eight different 10 mL volumetric flasks, respectively. Dilute to the mark with blank matrix extract to obtain a series of mixed standard working solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL.

[0023] After the blank matrix is ​​treated with SO2, the extract is dried by nitrogen blowing and then dissolved in 10 mL of methanol to obtain the blank matrix extract.

[0024] In S02, if the sample is a cream, lipstick, or powder cosmetic, 2 mL of dichloromethane should be added first, and the sample should be vortexed to disperse it evenly before adding 10 mL of ethyl acetate and vortexing.

[0025] In S05, the method for establishing the compound standard spectral library is as follows: using the above chromatographic and mass spectrometric conditions, the mixed standard reaction solution of six nitrobenzene compounds with a mass concentration of 5 mg / L is injected and analyzed in information-dependent scanning mode to obtain the retention time, precise mass number of the primary parent ion, isotope peak shape and precise mass number of the secondary daughter ion of the compound, and to establish a compound standard spectral library containing the above information.

[0026] After processing, the sample is analyzed. The collected data is imported into Target Analysis software, and the sample mass spectrometry information is compared with the compound standard spectral library. Under the same experimental conditions, if the mass chromatogram of the sample containing the extracted molecular ion shows a chromatographic peak with the same retention time as the standard, the deviation of the accurate mass number of the primary and secondary ions is not higher than 5 mDa, the isotope peak shape matching degree is ≥85%, and the scoring standard is not lower than "++", then it is determined that the sample contains the corresponding nitrobenzene compound.

[0027] Cosmetics include creams, lotions, talcum powder, and lipsticks.

[0028] The detection concentrations of the six nitrobenzene compounds ranged from 0.03 mg / kg to 0.2 mg / kg, and the limits of quantification ranged from 0.1 mg / kg to 1.0 mg / kg.

[0029] The retention times for 2,6-dinitrotoluene were 2.2 min, 2,5-dinitrotoluene was 2.7 min, 3,5-dinitrotoluene was 3.6 min, 2,4-dinitrotoluene was 3.8 min, 3,4-dinitrotoluene was 8.0 min, and 2,3-dinitrotoluene was 8.3 min.

[0030] The method of this invention is applied to the qualitative and quantitative analysis and detection of nitrobenzene compounds in cosmetics.

[0031] The present invention has the following beneficial effects:

[0032] This invention selects ethyl acetate, a non-protonating solvent, to extract samples. The extract is then catalytically hydrogenated to convert it into easily ionizable aniline compounds. The reaction solution is concentrated by nitrogen blowing and reconstituted with methanol. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QFS-MS) is used for determination. A screening database is constructed based on retention time, precise mass number of the primary precursor ion, isotope abundance ratio, and precise mass number of the secondary daughter ion. The detection results are then retrieved to achieve screening and confirmation of six nitrobenzene compounds in cosmetics. Simultaneously, a matrix-matched standard curve external standard quantification method is established. The establishment of this method fills the gap in LC-MS determination of nitrobenzene substances, solves the pretreatment defects of GC-MS, and improves detection sensitivity, providing a new technical option for cosmetic quality and safety monitoring. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of an existing atmospheric pressure hydrogenation unit;

[0034] Figure 2 The base peak chromatograms of six nitrobenzene hydrogenation products were analyzed by UPLC-Qtof-MS.

[0035] Figure 3 To investigate the effect of extraction solvents on the hydrogenation reactions of six nitrobenzene compounds;

[0036] Figure 4 The chromatograms show the separation of six nitrobenzene compounds on a CAPCELL PAK MGII column. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0038] 1. Experimental Section

[0039] 1.1 Instruments, Reagents and Materials

[0040] UltiMate 3000 high-performance liquid chromatograph (HPLC), Thermo Fisher Scientific, USA; Impact HD quadrupole-time-of-flight mass spectrometer (QQ), Bruker GmbH, Germany; Milli-Q ultrapure water system, Millipore Scientific, USA; Talboys mixer, Henry Troemner Scientific, USA; 3-18K refrigerated centrifuge, Sigma-Aldrich, Germany; DF-101S magnetic stirrer, Yuhua Instruments Co., Ltd.; N-Evap 112 nitrogen evaporator, Organization Associates, USA; XS205DU electronic balance, Mettler AG, Switzerland.

[0041] Standards: 3,4-dinitrotoluene (3,4-DNT, 99.70%), 2,4-dinitrotoluene (2,4-DNT, 99.11%), 2,6-dinitrotoluene (2,6-DNT, 98.80%), Stanford Chemicals, USA; 2,3-dinitrotoluene (2,3-DNT, 98.1%), Dr. Ehrenstorfer, Germany; 2,5-dinitrotoluene (2,5-DNT, 95%), 3,5-dinitrotoluene (3,5-DNT, 95%), Jiangsu Aikon Biomedical R&D Co., Ltd.

[0042] Methanol, acetonitrile, isopropanol, n-butanol, toluene, ethyl acetate, dichloromethane, and formic acid were all chromatographically pure, from Fisher Scientific, USA; 5% palladium on carbon (Pd) was from Shanghai Maclean Biochemical Technology Co., Ltd. 67 batches of samples were purchased from local shopping malls and supermarkets. The blank matrix was Liangyan hair dye, approval number: G20150415; batch number: DC0601; product name: Liangyan hair dye (11); manufacturer: Wujiang Xingbolong Daily Chemical Products Co., Ltd.; according to the detection method of SN / T 4442-2016 for nitrobenzene, nitrobenzene, and dinitrotoluene in imported and exported cosmetics, Liangyan hair dye (11) did not contain nitrobenzene compounds.

[0043] 1.2 Preparation of Standard Solutions

[0044] Standard stock solution: Accurately weigh about 25.00 mg of the above standard, place it in a 10 mL volumetric flask, add ethyl acetate to dissolve it and dilute to the mark, and store it in a refrigerator at 4°C for later use.

[0045] Mixed standard stock solution: Accurately transfer 0.10 mL of the standard stock solution into a 25 mL volumetric flask, add ethyl acetate to dissolve and dilute to the mark, and store at 4°C for later use.

[0046] Mixed standard reaction solution: Accurately transfer 0.50 mL of each standard stock solution into a 50 mL round-bottom flask, add 20 mL of ethyl acetate, sonicate to dissolve, add 50 mg of 5% palladium on carbon (Pd), and place in an atmospheric pressure hydrogenation apparatus. [1] Hydrogen gas was introduced into the mixture (flow rate 2 m / s), and the reaction was carried out at 40 °C for 2 h. The reaction solution was centrifuged at 5000 r / min for 5 min, and the supernatant was collected into a 25 mL nitrogen blow-off tube. The supernatant was dried under nitrogen gas in a 40 °C water bath, and 2 mL of methanol was added for redissolution by vortexing. The solution was then transferred to a 10 mL volumetric flask, diluted to the mark with methanol, and stored in a 4 °C refrigerator for later use.

[0047] [1]Liu Xianxiang, YangYongjun, YinDulin, etal. Hydrogenation performance of o-chloronitrobenzene over Pd / C under atmospheric press[J]. Chemical Industry and Engineering Progress, 2016, 35(2):524-527.

[0048] Blank matrix mixed standard working solutions: Accurately transfer 0.016 mL, 0.04 mL, 0.08 mL, 0.20 mL, 0.40 mL, 0.80 mL, 2.00 mL, and 4.00 mL of the mixed standard reaction solution of the above 6 compounds into 8 different 10 mL volumetric flasks, respectively. Dilute to the mark with blank matrix extract to obtain a series of mixed standard working solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL, respectively, for testing.

[0049] After the blank matrix is ​​treated according to "1.3", the extract is dried by nitrogen blowing and then dissolved in 10 mL of methanol to obtain the blank matrix extract.

[0050] Specifically, weigh 0.50 g of blank matrix and place it in a 25 mL colorimetric tube. Add 10 mL of ethyl acetate (for creams, lipsticks, and powders, add 2 mL of dichloromethane first, then vortex to disperse the sample evenly). After vortexing and mixing, extract ultrasonically for 15 min, centrifuge at 5000 r / min for 5 min, dehydrate the supernatant with anhydrous sodium sulfate, and place it in a 50 mL round-bottom flask. Repeat the extraction once with 10 mL of ethyl acetate. Combine the extracts, add 50 mg of 5% palladium on carbon (Pd), and place in a normal pressure hydrogenation apparatus (e.g., ...). Figure 1As shown in the figure, with the fume hood open, hydrogen gas was introduced (flow rate of 2 m / s), and the reaction was carried out at 40°C for 2 h. The reaction solution was centrifuged at 5000 r / min for 5 min, and the supernatant was collected in a 25 mL nitrogen blow-off tube, dried under nitrogen gas in a 40°C water bath, and dissolved in 10 mL methanol to obtain the blank matrix extract.

[0051] 1.3 Sample Preparation

[0052] Weigh 0.50 g of the sample and place it in a 25 mL colorimetric tube. Add 10 mL of ethyl acetate (for creams, lipsticks, and powders, add 2 mL of dichloromethane first, then vortex to disperse the sample evenly). After vortexing and mixing, extract ultrasonically for 15 min, centrifuge at 5000 r / min for 5 min, dehydrate the supernatant with anhydrous sodium sulfate, and place it in a 50 mL round-bottom flask. Repeat the extraction once with 10 mL of ethyl acetate. Combine the extracts, add 50 mg of 5% palladium on carbon (Pd), and place in a normal pressure hydrogenation apparatus (e.g., Figure 1 As shown in the figure, with the fume hood open, hydrogen gas was introduced (flow rate range 2 m / s), and the reaction was carried out at 40°C for 2 h. The reaction solution was centrifuged at 5000 r / min for 5 min, and the supernatant was collected in a 25 mL nitrogen blow-off tube, dried under nitrogen gas in a 40°C water bath, and then redissolved by vortexing with 1.0 mL of methanol. The solution was filtered through a 0.22 μm microporous membrane, and the filtrate was prepared for analysis.

[0053] 1.4 Instrument Conditions

[0054] 1.4.1 Chromatographic conditions

[0055] Column: Agilent Poroshell 120EC-C 18 Column (100mm × 4.6mm, 2.7μm); Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile; Flow rate: 0.5mL / min; Injection volume: 5μL; Column temperature: 20℃. Gradient elution program: 0–10 min, 98% A, 2% B; 10–10.5 min, 98% A–5% A, 2% B–95% B; 10.2–14 min, 5% A, 95% B; 14–14.2 min, 5% A–98% A, 95% B–2% B; 14.2–17 min, 98% A, 2% B.

[0056] 1.4.2 Mass Spectrometry Conditions

[0057] Ion source: Electrospray ionization source; positive ion mode; full scan range: m / z 50~1000; mass spectrometry data acquisition mode: information-dependent scanning source fragmentation (Auto MS / MS CID); real-time calibration solution: 10 mmol / L sodium formate solution; drying gas temperature: 200℃; drying gas flow rate: 8.0 L / min; nebulizer gas pressure: 2.0 Bar; capillary voltage: 4500 V.

[0058] 1.4.3 Establishment of a standard spectral library of compounds

[0059] Using the above-mentioned instrument conditions, a mixed standard reaction solution of six analytes with a mass concentration of 5 mg / L was analyzed in information-dependent scanning mode to obtain the retention time, precise mass number of the primary precursor ion, isotope peak shape, and precise mass number of the secondary daughter ion of the compound. A standard spectral library of compounds containing the above information was established, and the specific information is shown in Table 1.

[0060] After processing, the experimental samples were analyzed using a mass spectrometer. The collected data were imported into Target Analysis software, and the sample mass spectrometry information was compared with the compound standard spectral library. Under the same experimental conditions, if the mass chromatogram of the sample containing the extracted molecular ion showed a peak with the same retention time as the standard, and the exact mass number deviation of the primary and secondary ions was no higher than 5 × 10⁻⁶, the sample was considered a good candidate. -6 If the peak shape matching degree (mSigma value) is ≥85% and the scoring standard is not lower than "++", then the corresponding compound can be determined to exist in the sample being tested.

[0061] Table 1. Mass spectrometric parameters of six nitrobenzene hydrogenation products

[0062]

[0063] 2 Results and Discussion

[0064] 2.1 Optimization of chromatographic conditions

[0065] After hydrogenation, six nitrobenzene compounds convert -NO2 into ionizable -NH2, which is then processed by ESI. + The mode yields [M+H]. +The molecular ion peak was detected, therefore a positive ion scanning mode was selected, and an attempt was made to add a certain amount of formic acid to the mobile phase to enhance the ionization efficiency. The separation and response intensity of the compounds were compared under 0.1% formic acid-water-methanol and 0.1% formic acid-water-acetonitrile mobile phase systems. The results showed that 0.1% formic acid-water-acetonitrile had better separation efficiency and stronger ion response for the six dinitrotoluene isomers. Therefore, 0.1% formic acid-water-acetonitrile was selected as the mobile phase in this experiment, and the elution program was optimized according to the elution order of the compounds, thus obtaining the best separation effect for the six dinitrotoluene compounds. The chromatograms are shown below. Figure 2 .

[0066] The experiment compared different types of chromatographic columns: Poroshell 120EC-C 18 (100mm×4.6mm, 2.7μm), Zorbax Eclipse plus RRHD C 18 (100mm×2.1mm, 1.8μm), xBridge phenyl (150mm×2.1mm, 3.5μm), Inertsil ODS 3 (100mm×2.1mm, 2μm), CAPCELL PAK MGII (5μm, 4.6mm×250mm), XBridge Shield RP18 (5μm, 4.6mm×250mm), Kinetex C8 (5μm, 4.6mm×250mm), ZORBAX SB-C 18 The separation effects of Purospher STAR RP-18 (5μm, 4.6mm × 250mm) on six dinitrotoluene compounds were evaluated. The results showed that the six dinitrotoluene isomers (2,6-dinitrotoluene, 2,5-dinitrotoluene, 3,5-dinitrotoluene, 2,4-dinitrotoluene, 3,4-dinitrotoluene, and 2,3-dinitrotoluene) were difficult to separate, except for those separated by Poroshell 120EC-C. 18 Except for the (100mm × 4.6mm, 2.7μm) column, all other columns exhibited varying degrees of peak overlap. Therefore, the Poroshell 120EC-C column was selected. 18 (100mm×4.6mm, 2.7μm) was used as the analytical column.

[0067] 2.2 Optimization of Sample Pretreatment Methods

[0068] Nitroaniline is generally prepared by reducing nitrobenzene in an acidic solution with metallic zinc. Nitrobenzene compounds are poorly soluble in water, and the extraction of these trace substances from cosmetics using hydrochloric acid / sulfuric acid solutions has the following limitations: poor sample dispersion, low extraction rate, and poor sensitivity. Therefore, this paper employs a green and environmentally friendly catalytic hydrogenation reduction method to reduce nitrobenzene to aniline for content determination.

[0069] In this experiment, 50 mg of palladium on carbon (5% Pd) was used as a catalyst (Pd to sample mass ratio was 1:10). The reaction was carried out at 40℃ for 120 min. Representative cream samples were selected, and the effects of methanol, isopropanol, n-butanol, toluene, and ethyl acetate as reaction solvents on the hydrogenation reaction of six nitrobenzene compounds were investigated, with conversion / yield as the index. The results are shown in […]. Figure 3 The results show that the solvent plays a crucial role in the reaction process, not only improving the mass transfer of the substrate but also suppressing the formation of byproducts. The conversion rate of the analyte increases accordingly with decreasing solvent polarity.

[0070] Nitrobenzene compounds are typically introduced into cosmetics in trace amounts as byproducts of raw materials. Using isopropanol and n-butanol as extraction solvents presents several challenges: firstly, the presence of co-extractants interferes with the determination; secondly, aqueous samples are difficult to concentrate; and thirdly, some creams, lotions, and lipsticks cannot be effectively dispersed, affecting extraction efficiency. Toluene is toxic to humans and also exhibits unsatisfactory dispersion effects on some creams, lotions, and lipsticks.

[0071] Therefore, in this experiment, a small amount of dichloromethane was first used to disperse creams, lipsticks, and powders in cosmetics. Then, ethyl acetate was used to extract or extract nitrobenzene compounds from the cosmetics. After hydrogenation, the extract was concentrated by nitrogen blowing and then re-dissolved in methanol. This process can remove most of the fat-soluble substances, reduce the matrix effect, and improve the detection sensitivity.

[0072] 2.3 Evaluation of matrix effect

[0073] Negative samples with four different matrices—creams, lotions, talcum powder, and lipsticks—were selected, and blank matrix solutions were prepared according to "1.3". Corresponding mixed standard series solutions and solvent standard curves of corresponding mass concentrations were prepared using the blank matrix solutions. The matrix effect was evaluated using the following formula: η = (slope of matrix matching curve - slope of solvent curve) / slope of solvent curve × 100%. The absolute value of η increases with the strengthening of the matrix effect; a positive η indicates matrix enhancement, while a negative value indicates matrix inhibition. When the absolute value of η is <20%, the matrix effect is weak; when the absolute value of η is in the range of 20% to 50%, it indicates a moderate matrix effect; and when the absolute value of the matrix effect is >50%, it indicates a strong matrix effect. The evaluation results are shown in Table 2. The results show that, except for powder matrices which exhibit a weak matrix effect, the other three matrices all show moderate to strong matrix effects, especially the matrix effect of cream samples, where the matrix enhancement effect of 3,4-dinitrotoluene and 2,3-dinitrotoluene is greater than 75%. Therefore, in order to obtain accurate quantitative results, this experiment used a blank matrix-matched standard curve to offset or compensate for matrix effects.

[0074] Table 2. Standard curves and matrix effects for quantification of six nitrobenzene hydrogenation products using primary precursor ions.

[0075]

[0076] 2.4 Linear range, detection concentration and limit of quantitation

[0077] Under optimized chromatographic conditions, a series of mixed standard working solutions with varying mass concentrations were determined. Linear regression analysis was performed with the mass concentration of each compound (X, mg / L) as the abscissa and the peak area of ​​the primary molecular ion (Y) as the ordinate. The results showed that the hydrogenation products of the six nitrobenzene compounds exhibited good linearity within their respective concentration ranges, with correlation coefficients (r) all greater than 0.999. Standard stock solutions were added to the blank matrix, with spiking concentrations decreasing incrementally. Sample processing was performed according to "1.3". The limit of detection (LOD) was calculated at a signal-to-noise ratio of 3, and the limit of quantitation (LOQ) was calculated at a signal-to-noise ratio of 10. The results are shown in Table 3. The results showed that due to matrix effects, the detection concentrations and LOQs varied for different sample types. The detection concentrations of each component ranged from 0.03 mg / kg to 0.2 mg / kg, and the LOQs ranged from 0.1 mg / kg to 1.0 mg / kg.

[0078] Table 3. Linear range, linear equation, correlation coefficient, detection concentration and limit of quantitation for six nitrobenzene compounds.

[0079]

[0080] 2.5 Recovery rate and precision

[0081] Standard stock solutions at low, medium, and high concentrations were added to blank matrices for creams, lotions, talcum powders, and lipsticks, respectively. Six parallel experiments were performed for each group. The quantitative results of spiked recoveries and relative standard deviations (RSDs) are shown in Table 4. The results showed that the spiked recoveries of each component in different matrices ranged from 85.2% to 105%, with RSDs ranging from 0.2% to 3.1%. The spiked recoveries at all three different levels met the needs of routine experimental detection.

[0082] Table 4. Spike recoveries and precision of six nitrobenzene compounds (n=6)

[0083]

[0084]

[0085] 2.6 This study investigated the separation of six dinitrotoluene compounds using high-performance liquid chromatography (HPLC). Chromatographic column: CAPCELL PAK MGII (5 μm, 4.6 mm × 250 mm); mobile phase: methanol-0.1% formic acid aqueous solution; flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 10 μL; detection wavelength: 254 nm. Gradient elution was used to separate the nitrobenzene compounds. Details of the gradient elution method are shown in Table 5. The separation chromatograms are shown in [Table 5]. Figure 4 Meanwhile, the XBridge Shield RP18 (5μm, 4.6mm×250mm), Kinetex C8 (5μm, 4.6mm×250mm), and ZORBAX SB-C were also examined. 18 The liquid chromatography separation of six dinitrotoluene compounds was performed using columns including 5μm (4.6mm×250mm), Purospher STAR RP-18 (5μm, 4.6mm×250mm), and XBridge Phenyl (3.5μm, 2.1mm×150mm). The results showed that under the above chromatographic conditions, at least 2-4 peaks of the four components (2,3-dinitrotoluene, 2,6-dinitrotoluene, 2,5-dinitrotoluene, and 2,4-dinitrotoluene) overlapped, failing to achieve baseline separation. This indicates that the use of liquid chromatography to determine these six compounds has certain limitations and low applicability.

[0086] Table 5 Gradient elution method

[0087]

[0088]

[0089] 2.7 Determination of actual samples

[0090] This method was used to test 67 commercially available cosmetic samples (including creams, lotions, lotions, gels, masks, and lipsticks, of which 45 were hair dye products). The results showed that no nitrobenzene compounds were detected in any of the samples, indicating that cosmetic manufacturers have relatively strict control over the quality of raw materials, avoiding the introduction of prohibited ingredients such as nitrobenzene and the resulting adverse reactions. Future research will further expand the number and scope of products tested to investigate potential risks.

[0091] 3. Conclusion

[0092] This study established a qualitative and quantitative analytical method for the simultaneous determination of six nitrobenzene compounds in cosmetics using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QT-MS). The chromatographic separation conditions, hydrogenation conditions, and extraction solvent were optimized, and a screening database was established. Experimental results show that this method is sensitive, has high recovery, and good reproducibility. It can be used for the detection of nitrobenzene compounds in cosmetics with various matrix types, providing new ideas and technical support for ensuring the safety of cosmetic use and combating the illegal addition of prohibited ingredients in the cosmetic industry.

[0093] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining six nitrobenzene compounds in cosmetics using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, characterized in that: Includes the following steps: S01, Preparation of standard solution; S02, Sample Preparation: Weigh 0.50 g of sample and place it in a 25 mL colorimetric tube. Add 10 mL of ethyl acetate, vortex to mix, and then extract ultrasonically for 15 min. Centrifuge at 5000 r / min for 5 min. Dehydrate the supernatant with anhydrous sodium sulfate and place it in a 50 mL round-bottom flask. Repeat the extraction once with 10 mL of ethyl acetate. Combine the extracts, add 50 mg of 5% palladium on carbon (Pd), and place the flask in a normal pressure hydrogenation apparatus. With the fume hood open, introduce hydrogen gas at a flow rate of 2 m / s and react at 40 °C for 2 h. Centrifuge the reaction solution at 5000 r / min for 5 min, collect the supernatant in a 25 mL nitrogen blow-off tube, and dry it with nitrogen gas in a 40 °C water bath. Add 1.0 mL of methanol and vortex to redissolve the solution. Filter the solution through a 0.22 μm microporous membrane. The filtrate is ready for analysis. S03, chromatographic conditions: Column: Agilent Poroshell 120EC-C 18 Column (100mm × 4.6mm, 2.7μm); Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile; Flow rate: 0.5mL / min; Injection volume: 5μL; Column temperature: 20℃; Gradient elution program: 0–10 min, 98% A, 2% B; 10–10.5 min, 98% A–5% A, 2% B–95% B; 10.2–14 min, 5% A, 95% B; 14–14.2 min, 5% A–98% A, 95% B–2% B; 14.2–17 min, 98% A, 2% B; S04, Mass spectrometry conditions: Ion source: Electrospray ionization source; positive ion mode; full scan range: m / z 50~1000; mass spectrometry data acquisition mode: information-dependent scanning, in-source fragmentation, Auto MS / MS CID; Real-time calibration solution: 10 mmol / L sodium formate solution; drying gas temperature: 200℃; drying gas flow rate: 8.0 L / min; nebulizing gas pressure: 2.0 Bar; capillary voltage: 4500 V; S05, Establishment of a standard spectral library of compounds; The six nitrobenzene compounds are 3,4-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,3-dinitrotoluene, 2,5-dinitrotoluene, and 3,5-dinitrotoluene.

2. The method according to claim 1, characterized in that: In S01, the standard solution is prepared as follows: Standard stock solutions: Accurately weigh approximately 25.00 mg of 3,4-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,3-dinitrotoluene, 2,5-dinitrotoluene and 3,5-dinitrotoluene standards respectively, place them in a 10 mL volumetric flask, dissolve them in ethyl acetate and dilute to the mark, and store at 4°C for later use; Mixed standard stock solution: Accurately transfer 0.10 mL of the standard stock solution into a 25 mL volumetric flask, add ethyl acetate to dissolve and dilute to the mark, and store at 4°C for later use; Mixed standard reaction solution: Accurately transfer 0.50 mL of each standard stock solution into a 50 mL round-bottom flask, add 20 mL of ethyl acetate, sonicate to dissolve, add 50 mg of 5% palladium on carbon (Pd), place in a normal pressure hydrogenation apparatus, purge with hydrogen gas at a flow rate of 2 m / s, react at 40 °C for 2 h, centrifuge the reaction solution at 5000 r / min for 5 min, collect the supernatant into a 25 mL nitrogen blow-off tube, dry it with nitrogen gas in a 40 °C water bath, add 2 mL of methanol to vortex redissolve, transfer to a 10 mL volumetric flask, dilute to the mark with methanol, and store in a 4 °C refrigerator for later use; Blank matrix mixed standard working solution: Accurately transfer an appropriate amount of the mixed standard reaction solution of the above 6 compounds, and prepare a series of mixed standard working solutions of different concentrations with blank matrix extract, ready for testing.

3. The method according to claim 2, characterized in that: Accurately transfer 0.016 mL, 0.04 mL, 0.08 mL, 0.20 mL, 0.40 mL, 0.80 mL, 2.00 mL, and 4.00 mL of the mixed standard reaction solution into eight different 10 mL volumetric flasks, respectively. Dilute to the mark with blank matrix extract to obtain a series of mixed standard working solutions with concentrations of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL. After the blank matrix is ​​treated with SO2, the extract is dried by nitrogen blowing and then dissolved in 10 mL of methanol to obtain the blank matrix extract.

4. The method according to claim 1, characterized in that: In S02, if the sample is a cream, lipstick, or powder cosmetic, 2 mL of dichloromethane should be added first, and the sample should be vortexed to disperse it evenly before adding 10 mL of ethyl acetate and vortexing.

5. The method according to claim 1, characterized in that: In S05, the method for establishing the compound standard spectral library is as follows: using the above chromatographic and mass spectrometric conditions, the mixed standard reaction solution of six nitrobenzene compounds with a mass concentration of 5 mg / L is injected and analyzed in information-dependent scanning mode to obtain the retention time, precise mass number of the primary parent ion, isotope peak shape and precise mass number of the secondary daughter ion of the compound, and to establish a compound standard spectral library containing the above information. After processing, the samples are analyzed on the instrument. The collected data are imported into Target Analysis software and compared with the sample mass spectrometry information and the compound standard spectral library. Under the same experimental conditions, if the sample tested shows a chromatographic peak with the same retention time as the standard in the mass chromatogram of the extracted molecular ion, the deviation of the accurate mass number of the primary and secondary ions is no higher than 5 mDa, the isotope peak shape matching degree is ≥85%, and the scoring standard is no lower than "++", then it is determined that the sample tested contains the corresponding nitrobenzene compound.

6. The method according to claim 1, characterized in that: Cosmetics include creams, lotions, talcum powder, and lipsticks.

7. The method according to claim 1, characterized in that: The detection concentrations of the six nitrobenzene compounds ranged from 0.03 mg / kg to 0.2 mg / kg, and the limits of quantification ranged from 0.1 mg / kg to 1.0 mg / kg.

8. The method according to claim 1, characterized in that: The retention times for 2,6-dinitrotoluene were 2.2 min, 2,5-dinitrotoluene was 2.7 min, 3,5-dinitrotoluene was 3.6 min, 2,4-dinitrotoluene was 3.8 min, 3,4-dinitrotoluene was 8.0 min, and 2,3-dinitrotoluene was 8.3 min.

9. The application of the method according to any one of claims 1 to 8 in the qualitative and quantitative analysis and detection of nitrobenzene compounds in cosmetics.

Images