A method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry

Abstract

The invention discloses a method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics by ultrahigh pressure liquid chromatography-triple quadrupole tandem mass spectrometry. The method is characterized in that the method comprises the following steps: S1, extraction: mixing a sample with an extraction solvent and performing ultrasonic extraction, centrifuging, taking the supernatant and blowing nitrogen to obtain a liquid to be purified containing the extract; S2, purification: taking the liquid to be purified in step S1, passing it through an activated solid phase extraction column, then eluting with water and methanol in sequence, and then eluting with an ammonia-methanol solution, collecting the eluate, blowing the eluate to dryness with nitrogen in a 45°C water bath, and finally dissolving the eluate with an organic solvent, fixing the volume, mixing uniformly, and filtering the eluate through a membrane to obtain a purified liquid; S3, detection: using ultrahigh pressure liquid chromatography-triple quadrupole tandem mass spectrometry to detect and analyze the purified liquid obtained in step S2. The method realizes the detection of three methylimidazole compounds in cosmetics with different matrices for the first time, and has the advantages of good separation, high sensitivity, accuracy and precision.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics detection, and in particular relates to a method for determining 1-methylimidazole, 2-methylimidazole and 4-methylimidazole in cosmetics by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry. Background Art

[0002] Methylimidazoles are byproducts of caramel color production, which is permitted for use in various cosmetics. The byproduct 4-methylimidazole is produced during the production of caramel color. 4-Methylimidazole has toxic properties similar to those of pyridine and pyrrole, inhibiting cytochrome P450 isoenzymes. It catalyzes reactions in the human liver and produces numerous low-molecular-weight carcinogens. These compounds can induce seizures in animals and cause thyroid tumors, posing a risk of cancer and leukemia.

[0003] 2-Methylimidazole is an intermediate in the synthetic drug metronidazole, a nitroimidazole derivative. As an antiprotozoal and antibacterial drug, it is clinically used to treat skin diseases such as scabies, tinea pedis, acne vulgaris, and bedsores. It is a commonly used antibacterial and anti-inflammatory drug in modern medicine. These drugs are inexpensive and have significant short-term effects. Unscrupulous vendors illegally add these ingredients to certain cosmetics (such as acne-removing cosmetics) to claim immediate acne-removing effects, engage in false advertising, and profiteering. my country's "Technical Specifications for Safety of Cosmetics" (2015 edition) explicitly stipulates that metronidazole is a banned substance in cosmetics and cannot be added as a raw material or component in cosmetics. The illegal addition of metronidazole during the cosmetics production process can result in residues of its synthetic intermediate, 2-methylimidazole. Repeated use of cosmetics containing these banned substances can induce skin flora imbalance and cause allergic reactions such as contact dermatitis and antibiotic allergies. Long-term use can also lead to serious internal organ damage. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer (IARC) published a list of carcinogens, defining both 2-methylimidazole and 4-methylimidazole as Class 2B carcinogens. 1-Methylimidazole, 2-methylimidazole, and 4-methylimidazole are isomers with similar physical properties. Currently, testing for methylimidazoles is primarily conducted in food, with no reports on testing methods for cosmetics.

[0004] Therefore, obtaining a method that can quickly detect 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics is one of the research directions in the field of cosmetics testing. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry. This method realizes the detection of three methylimidazole compounds in cosmetics with different matrices, and has the advantages of good separation, high sensitivity, accuracy, and precision.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for determining 1-methylimidazole (1-MEI), 2-methylimidazole (2-MEI), and 4-methylimidazole (4-MEI) in cosmetics by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, the method comprising the following steps:

[0008] S1. Extraction: Mix the sample with the extraction solvent and perform ultrasonic extraction, centrifuge, and take the supernatant and blow nitrogen to obtain a solution to be purified containing the extract;

[0009] S2, purification: the liquid to be purified in step S1 is passed through an activated solid phase extraction column, and then sequentially rinsed with water and methanol, and then eluted with an ammonia-methanol solution, the eluate is collected, and the eluate is blown dry with nitrogen in a 45° water bath, and finally dissolved with an organic solvent and fixed to volume, mixed evenly, and filtered through a membrane to obtain a purified liquid;

[0010] S3. Detection: The purified liquid obtained in step S2 is detected and analyzed using ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry.

[0011] The present invention establishes for the first time a method for determining three methylimidazole compounds in cosmetics in different matrices using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry. The samples are extracted with an extraction solvent, purified using a solid-phase extraction column, and then tested on the machine. The statistical results of the present invention show that the average recovery rate of this method ranges from 80.9% to 107.9%, and the relative standard deviation (RSD) is from 1.16 to 12.75%, both of which meet the requirements of SN / T 0001-2016. The limits of detection (LOD) are 10 to 30 μg / kg, and the limits of quantification (LOQ) are 25 to 100 μg / kg, respectively.

[0012] The method provided by the present invention has good separation of three methylimidazole compounds, high sensitivity, accuracy and precision, and a simple and rapid pretreatment method. The method can be used for risk assessment and screening monitoring of 1-MEI, 2-MEI and 4-MEI in cosmetics, and has broad application prospects.

[0013] Furthermore, the solid phase extraction column is any one of a mixed cationic solid phase extraction column (MCX, 3mL / 60mg), a mixed weak cationic solid phase extraction column (WCX, 3mL / 60mg), an aminopropyl extraction column (NH2 column, 6mL / 500mg), or an enhanced lipid removal solid phase extraction column (EMR-Lipid, 3mL / 300mg).

[0014] Preferably, the solid phase extraction column is a mixed cationic solid phase extraction column (MCX, 3 mL / 60 mg).

[0015] In the present invention, when a mixed cationic solid phase extraction column (MCX, 3 mL / 60 mg) is preferably used for purification, the recovery rates of 1-MEI, 2-MEI, and 4-MEI in cosmetics with different matrices are between 80.0% and 95.3%, and the recovery effect is good.

[0016] Furthermore, the specific extraction steps of step S1 are: adding an extraction solvent to the sample, shaking for 2-2.5 minutes to fully mix the sample and the extraction solvent, extracting under ultrasound for 10-20 minutes, centrifuging at 9000-11000 r / min for 5-10 minutes, taking all the supernatant into a 15 mL test tube, blowing nitrogen to 2 mL in a 45°C water bath, and obtaining a liquid to be purified.

[0017] Furthermore, the sample in step S1 is specifically one of a water-based emulsion, essential oil or cream cosmetic, and the specific extraction steps of step S1 are: weigh 0.20g (accurate to 0.001g) of sample into a 50mL centrifuge tube, add 10mL of acetonitrile, shake for 2min to fully mix the sample and the extraction solvent, ultrasonically extract for 15min, centrifuge at 10000r / min for 5min, take all the supernatant into a 15mL test tube, blow nitrogen to about 2mL in a 45°C water bath to obtain the liquid to be purified.

[0018] Furthermore, the sample in step S1 is specifically a powder-based cosmetic, and the specific extraction steps of step S1 are: adding a saturated sodium chloride solution to the sample to uniformly disperse the sample, then adding an extraction solvent, oscillating for 2-2.5 minutes to fully mix the sample and the extraction solvent, extracting under ultrasound for 10-20 minutes, centrifuging at 9000-11000 r / min for 5-10 minutes, taking the supernatant into a 15 mL test tube, blowing nitrogen to 2 mL in a 45°C water bath, and obtaining a liquid to be purified.

[0019] Furthermore, the sample in step S1 is specifically a powder-based cosmetic, and the specific extraction steps of step S1 are: weigh 0.20 g (accurate to 0.001 g) of the sample into a 50 mL centrifuge tube, first add 1 mL of saturated sodium chloride solution to evenly disperse the sample, then add 10 mL of acetonitrile, shake for 2 minutes to fully mix the sample and the extraction solvent, ultrasonically extract for 15 minutes, centrifuge at 10,000 r / min for 5 minutes, take the supernatant into a 15 mL test tube, blow nitrogen to 2 mL in a 45°C water bath to obtain a liquid to be purified.

[0020] When the sample is a powder-based cosmetic, simply using an extraction solvent may not be able to fully disperse the sample. The dispersion solvent has stronger penetration ability and will make the sample more uniform. Choosing to first disperse it with a saturated sodium chloride solution and then extract it with an extraction solvent can optimize the recovery rate of the three compounds in powder-based cosmetics.

[0021] Furthermore, the specific purification step of step S2 is: activating the solid phase extraction column with 1 mL of methanol and 1 mL of water in sequence, taking the liquid to be purified and passing it through the column, controlling the flow rate to 1 drop / 3 to 4 s, eluting with 1 mL of water and 1 mL of methanol in sequence to remove impurities, and then eluting with 3 mL of ammonia-methanol solution (5:95, V / V), collecting the eluate, and drying the eluate with nitrogen in a 45°C water bath, dissolving the dried substance with acetonitrile, and fixing the volume to 1 mL. After mixing evenly, passing through a 0.22 μm organic filter membrane to obtain a purified liquid.

[0022] Furthermore, in order to meet the extraction requirements of the three methylimidazole compounds in cosmetics with different bases, the extraction solvent in step S1 is acetonitrile.

[0023] Furthermore, the detection conditions of the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry are as follows: The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: chromatographic column: Shied RP18 chromatographic column (3.5 μm, 150 mm × 4.6 mm); mobile phase A: 20 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid), mobile phase B: acetonitrile; elution program: 0-5 min, 98% A, flow rate 0.6 mL / min; injection volume: 20.0 μL; column temperature: 45°C.

[0024] The mass spectrometry conditions are as follows: electrospray ionization source (ESI); positive ion scanning; multiple reaction monitoring (MRM) mode; curtain gas pressure: 15 psi; nebulizing gas pressure: 50 psi; heating auxiliary gas pressure: 50 psi; collision gas pressure; electrospray voltage: 5500 V; ion source temperature: 550°C.

[0025] Compared with the prior art, the present invention has achieved beneficial technical effects:

[0026] The present invention establishes for the first time a method for detecting 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics. The method provides good separation of 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole, with high sensitivity, accuracy, and precision. The method also features a simple and rapid pretreatment method. The method can be used for risk assessment and screening monitoring of 1-MEI, 2-MEI, and 4-MEI in cosmetics, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1-4 The effect of the chromatographic column on the separation and response of the three compounds ( Figure 1 and Figure 2 For Atlantis HILICSilica; Figure 3 It is ZORBAX SB-Aq; Figure 4 for shied RP18);

[0028] Figure 5-Figure 9 The effects of different mobile phases on the separation and response of three compounds (mobile phase: Figure 5 0.1% formic acid-acetonitrile; Figure 6 5mmol / L ammonium formate solution-acetonitrile containing 0.1% formic acid; Figure 7 20mmol / L ammonium formate solution-acetonitrile containing 0.1% formic acid; Figure 8 5mmol / L ammonium formate solution-acetonitrile; Figure 9 is 20mmol / L ammonium formate solution-acetonitrile);

[0029] Figure 10-13 Effects of extraction solvents on the recovery of three compounds in different matrices ( Figure 10 It is a water-based cosmetic; Figure 11 Essential oil cosmetics; Figure 12 It is a cream cosmetic; Figure 13 for loose powder cosmetics);

[0030] Figure 14-16 The effect of solid phase extraction column on three compounds in different matrices ( Figure 14 It is a water-based cosmetic; Figure 15 Essential oil cosmetics; Figure 16 Cream cosmetics);

[0031] Figure 17 is the elution curve;

[0032] Figures 18-20 The matrix effect on the three compounds in different matrices ( Figure 18 It is a water-based cosmetic; Figure 19 Essential oil cosmetics; Figure 20For cream cosmetics). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. However, the scope of protection claimed in the present invention is not limited to the following specific examples, and the reagents or instruments used in the detection method of the present invention can be purchased from the market.

[0034] Example 1

[0035] This embodiment provides a method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics (the cosmetics in this embodiment are toners) by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, the method comprising the following steps:

[0036] S1. Extraction: Weigh 0.20 g (accurate to 0.001 g) of sample into a 50 mL centrifuge tube, add 10 mL of acetonitrile, and oscillate for 2 min to fully mix the sample and the extraction solvent. Ultrasonic extraction was performed for 15 min. Centrifuge at 10,000 rpm for 5 min. All the supernatant was placed in a 15 mL glass test tube and blown with nitrogen to 2 mL to obtain the solution to be purified.

[0037] S2. Purification: The MCX solid phase extraction column was activated with 1 mL of methanol and 1 mL of water, respectively. The solution to be purified obtained in step S1 was passed through the column at a flow rate of 1 drop / 3-4 s. The column was then rinsed with 1 mL of water and 1 mL of methanol, respectively. The column was then eluted with 3 mL of ammonia-methanol solution (5:95, V / V). The eluate was collected and dried with nitrogen in a 45°C water bath. Finally, the dried substance was dissolved in acetonitrile and the volume was adjusted to 1 mL. After uniform mixing, the solution was passed through a 0.22 μm microporous organic filter membrane to obtain a purified solution.

[0038] S3. Detection: The purified liquid obtained in step S2 is detected and analyzed using ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry.

[0039] The chromatographic conditions of the ultra-high performance liquid chromatography of this embodiment are as follows: chromatographic column: Shied RP18 chromatographic column (3.5 μm, 150 mm × 4.6 mm); mobile phase A: 20 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid), mobile phase B: acetonitrile; elution program: 0-5 min, 98% A, flow rate 0.6 mL / min; injection volume: 20.0 μL; column temperature: 45°C.

[0040] The mass spectrometry conditions were as follows: API 4000Q Trap UPLC-MS / MS (AB Company, USA), equipped with an ESI ion source; positive ion scanning; multiple reaction monitoring (MRM) mode; curtain gas pressure: 15 psi; nebulizer gas pressure: 50 psi; heating auxiliary gas pressure: 50 psi; collision gas pressure; electrospray voltage: 5500 V; and ion source temperature: 550°C.

[0041] Examples 2-5

[0042] With reference to Example 1, the difference from Example 1 is that the cosmetics in Examples 2-5 are essence lotion, essential oil, facial cream, and lipstick in order.

[0043] Example 6

[0044] This embodiment provides a method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics (the cosmetics in this embodiment are loose powder) by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, the method comprising the following steps:

[0045] S1. Extraction: Weigh 0.20 g (accurate to 0.001 g) of sample into a 50 mL centrifuge tube, add 1 mL of saturated sodium chloride solution to evenly disperse the sample, then add 10 mL of acetonitrile and shake for 2 min to fully mix the sample and extraction solvent. Ultrasonic extraction for 15 min, centrifuge at 10,000 rpm for 5 min, take all the supernatant into a 15 mL glass test tube, and blow nitrogen to 2 mL to obtain the solution to be purified.

[0046] S2. Purification: An MCX solid-phase extraction column was activated with 1 mL of methanol and 1 mL of water, respectively. The solution to be purified obtained in step S1 was passed through the column at a flow rate of 1 drop / 3-4 s. The column was then eluted with 1 mL of water and 1 mL of methanol, respectively. The column was then eluted with 3 mL of an aqueous ammonia-methanol solution (5:95, V / V). The eluate was collected and dried with nitrogen in a 45°C water bath. The volume was then adjusted to 1 mL with acetonitrile. The dried substance was dissolved in acetonitrile and the volume was adjusted to 1 mL. After uniform mixing, the column was passed through a 0.22 μm microporous organic filter membrane to obtain a purified solution.

[0047] S3. Detection: The purified liquid obtained in step S2 is detected and analyzed using ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry.

[0048] The conditions for ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection in this example are the same as those in Example 1.

[0049] The following examples investigate the effects of different factors on the detection of 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics with different bases.

[0050] Among them, the preparation of 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole standard working solutions is as follows:

[0051] (1) Preparation of standard stock solutions: Accurately weigh 10.0 mg (accurate to 0.00001 g) of 1-MEI, 2-MEI, and 4-MEI standards, place them in 10 mL volumetric flasks, dissolve them in acetonitrile and dilute to the mark, shake well, and prepare 1000 mg / L standard stock solutions. Store in a refrigerator at 0-4°C away from light.

[0052] (2) Preparation of mixed standard intermediate solution: 0.10 mL of each standard stock solution of 1-MEI, 2-MEI and 4-MEI was measured and placed in a 10 mL volumetric flask. The solution was diluted with acetonitrile and made up to the mark. The solution was shaken to prepare a 10 mg / L mixed standard intermediate solution. The solution was stored in a refrigerator at 0-4 °C away from light.

[0053] (3) Preparation of matrix standard solutions: Before use, dilute the mixed standard intermediate solution with blank matrix solution (the liquid obtained by treating the cosmetic sample without 1-MEI, 2-MEI and 4-MEI as described in S1 and S2 in the example) to a working solution of appropriate concentration, and prepare it for use immediately.

[0054] Example 7 Optimization of liquid chromatography and mass spectrometry conditions

[0055] (1) Optimization of mass spectrometry conditions

[0056] The molecular weights of the three methylimidazoles were queried using their CAS numbers. Full scan results of the parent ions revealed that the highest response was achieved in positive ion mode. Product ion scans were used to obtain the optimal mass spectrometric parameters for the target compounds. Quantitative and qualifier ions were determined based on the response intensities of the methylimidazole compounds. The mass spectrometric parameters for the three methylimidazole compounds are shown in Table 1.

[0057] Table 1 Mass spectrometry parameters of methylimidazole compounds

[0058]

[0059] * indicates quantification ion

[0060] (2) Optimization of liquid phase conditions

[0061] 2.1 Column selection

[0062] This example compares The separation of 1-MEI, 2-MEI, and 4-MEI was performed using Shied RP18 (3.5 μm, 150 mm × 4.6 mm), ZORBAX SB-Aq (3.5 μm, 100 mm × 4.6 mm), and Atlantis HILIC Silica (3.0 μm, 100 mm × 3.0 mm) columns. The injection volume was 20.0 μL, the column temperature was 45°C, the mobile phase A was 20 mmol / L aqueous ammonium formate (containing 0.1% formic acid), and the mobile phase B was acetonitrile. The results are shown in the figure. Figures 1-4 .

[0063] Depend on Figure 1 It can be seen that the Atlantis HILIC Silica column has good retention for highly polar compounds under hydrophilic interaction. In the literature reports on the detection of methylimidazole compounds, HILIC columns are mostly used. However, the inventors found in experimental research that Figure 2 As shown in the figure, although the HILIC column can separate the three compounds well, the chromatographic peak stability and reproducibility are poor, and the chromatographic peak bifurcation phenomenon occurs later. Figure 3 As shown in the figure, the ZORBAX SB-Aq column has a hydrophilic surface and is an HPLC alkyl reversed-phase bonded phase that has good retention for hydrophilic compounds. However, the chromatographic peaks are found to be broadened and tailing, and the peak shape is poor. Figure 4 As shown, shied RP18 contains polar embedded C 18 Chain can effectively improve the peak shape of chromatographic peaks. Experimental verification shows that the Shied RP18 column can achieve good baseline separation for the three compounds, and the chromatographic peak shape is good and the stability is good. Therefore, the choice Shied RP18 (3.5 μm, 150 mm×4.6 mm) was used as the chromatographic column of the present invention.

[0064] 2.2 Selection of mobile phase

[0065] The chromatographic column is Shied RP18 (3.5μm, 150mm×4.6mm), injection volume: 20.0μL, column temperature 45℃, using five different aqueous phases (0.1% formic acid solution, 5mmol / L ammonium formate solution containing 0.1% formic acid, 20mmol / L ammonium formate solution containing 0.1% formic acid, 5mmol / L ammonium formate solution, 20mmol / L ammonium formate solution) on the separation and response of 1-methylimidazole, 2-methylimidazole and 4-methylimidazole. The organic phase was acetonitrile. Chromatographic analysis was performed. The results are shown in the figure. Figure 5-9 .

[0066] Depend on Figure 5-9It can be seen that the peak shape and separation of the analyte in the aqueous phase containing 0.1% formic acid are better than those without 0.1% formic acid. Moreover, as the concentration of ammonium formate increases, the peak shape and separation of the chromatographic peaks become better. This is because the addition of ammonium formate makes the ionization degree of the mass spectrometry process higher and the mass spectrometry response stronger. Therefore, a 20mmol / L ammonium formate solution containing 0.1% formic acid was selected.

[0067] Acetonitrile was used as the mobile phase.

[0068] Example 8 Effect of Different Extraction Solvents on the Recovery of 1-MEI, 2-MEI and 4-MEI in Cosmetics

[0069] (1) When the cosmetic sample is a lotion, cream, or essential oil

[0070] 0.05 mL of a 1000 ng / mL mixed standard solution of 1-MEI, 2-MEI, and 4-MEI was added to a blank cosmetic sample (emulsion, cream, and essential oil cosmetics), and four extraction solvents, water, methanol, acetonitrile, and anhydrous ethanol, were selected, respectively. After treatment in steps S1 and S2 of Reference Example 1, a purified solution was obtained, and then detection and analysis were performed using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, and the recoveries of 1-MEI, 2-MEI, and 4-MEI were calculated, respectively. The results are shown in FIG. Figure 10-13 shown.

[0071] from Figure 10 The results showed that except for the low recovery rates of 1-MEI (74.0% and 70.2%) when methanol and anhydrous ethanol were used to extract water-based cosmetics, the other recovery rates were between 82.7% and 99.6%. Therefore, for water-based cosmetics, the extraction effects of methanol and anhydrous ethanol were not as good as those of acetonitrile and water. Figure 11 The results showed that when essential oil cosmetics were extracted with anhydrous ethanol, the recovery rate of 4-MEI was only 79.5%, and the rest were between 80.0% and 97.1%. The extraction effect of anhydrous ethanol was slightly worse. Figure 12 The results showed that the recovery rate of 1-MEI in creams and ointments was only 76.3% when extracted with water, with the remaining recovery rates ranging from 80.1% to 95.3%. Water extraction was inferior to acetonitrile, methanol, and anhydrous ethanol. Furthermore, considering that water extraction solvents are difficult to dry, and methanol and anhydrous ethanol extraction solvents cause flocculent material in creams and ointments, making it difficult to pass through the column naturally and requiring prolonged column run times, acetonitrile was selected as the extraction solvent for these three types of cosmetics.

[0072] (2) When the cosmetic sample is a powdered matrix

[0073] 0.05 mL of a 1000 ng / mL mixed standard solution of 1-MEI, 2-MEI and 4-MEI was added to a blank cosmetic sample (powdered matrix cosmetics), and four extraction solvents, saturated sodium chloride solution-acetonitrile, acetonitrile, methanol and anhydrous ethanol, were selected respectively. After treatment in steps S1 and S2 of Reference Example 1, a purified solution was obtained. After treatment in steps S1 and S2 of Reference Example 6, a purified solution was obtained. Ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry was used for detection and analysis, and the recoveries of 1-MEI, 2-MEI and 4-MEI were calculated, as shown in FIG. Figure 13 As shown in the figure, when using acetonitrile, methanol and anhydrous ethanol as the three extraction solvents, the recovery rates of the three compounds ranged from 11.5% to 45.5%, 55.1% to 102.4%, and 40.1% to 87.4%, respectively, which all failed to achieve good extraction results. Considering that powdered cosmetics may not be fully dispersed by simply using organic solvents for extraction, and that dispersing solvents have strong penetrating power and will make the sample more uniform, a common saturated sodium chloride solution was used as the dispersing solvent, followed by acetonitrile extraction to improve its recovery rate. The results are shown in the figure. Figure 13 The results show that the recovery rate of this method is between 81.2% and 86.5%, which meets the requirements, and the extraction is simple and fast. Therefore, the method of first dispersing with saturated sodium chloride solution and then extracting with extraction solvent was selected.

[0074] Example 9 Investigating the Effect of Different SPE Columns on the Recovery of 1-MEI, 2-MEI, and 4-MEI in Cosmetics

[0075] 0.05 mL of a 1000 ng / mL mixed standard solution of 1-MEI, 2-MEI, and 4-MEI was added to a blank cosmetic sample (emulsion, cream, essential oil), and after extraction in step S1 of Reference Example 1, four solid phase extraction columns, MCX, WCX, NH2, and EMR, were used (due to the different performances of the four solid phase extraction columns, different purification methods were required. The MCX, WCX, and NH2 solid phase extraction columns were pre-activated with methanol and water and then loaded with the sample, followed by elution with water and methanol in sequence. Finally, MCX was washed with 5 mL of ammonia methanol (5:95, V / V) ) was eluted, WCX and NH2 were eluted with 5mL acetonitrile-water-formic acid (88:10:2, V / V / V), and the eluate was collected. The EMR solid phase extraction column has a strong selective adsorption for lipids. The column does not need to be activated. After extraction, water is added to make the water content in the liquid to be purified reach 20%, and then the column can be passed and the eluate is collected. The eluate is dried with nitrogen in a 45°C water bath), purified with reference to the step S2 in the embodiment, and then detected and analyzed by ultra-high liquid chromatography-triple quadrupole tandem mass spectrometry, and the recoveries of 1-MEI, 2-MEI and 4-MEI are calculated respectively, as shown in FIG. Figure 14-16The results show that when MCX was used for cleanup, the recoveries of 1-MEI, 2-MEI, and 4-MEI for the three cosmetic categories ranged from 80.0% to 95.3%, demonstrating excellent recovery. However, when WCX was used for cleanup, the matrix of essential oils and creams significantly affected the recovery of the three compounds, resulting in recoveries of less than 10%. The recovery of 1-MEI for emulsions was also low, at 71.4%. When NH₂ was used for cleanup, the matrix of essential oils and creams similarly significantly affected the recovery, ranging from 21.1% to 326.0%. The matrix of emulsions showed no effect, with recoveries ranging from 84.8% to 98.0%. Although the EMR cleanup method is simple, it strongly adsorbs the three compounds, resulting in recoveries of 0 to 58.2% for the three cosmetic categories, which did not meet the requirements. Therefore, MCX was ultimately selected as the cleanup column for this method.

[0076] Example 10 Effect of eluent volume on the recovery of 1-MEI, 2-MEI and 4-MEI

[0077] 0.10 mL of a 1000 ng / mL mixed standard solution of 1-MEI, 2-MEI, and 4-MEI was added to a blank cosmetic sample (water emulsion), and the purified solution was obtained by treatment with reference to step S2 in Example 1. When collecting the eluate, one tube was collected for every 1 mL of elution, and a total of 5 mL was collected. The eluate was detected and analyzed by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, and the recoveries of 1-MEI, 2-MEI, and 4-MEI were calculated respectively. The results are shown in FIG. Figure 17 The results showed that 4-MEI was almost completely eluted in the 1st mL, 2-MEI was almost completely eluted in the 2nd mL, and 1-MEI was still partially eluted in the 3rd mL. Therefore, the elution volume was determined to be 3 mL.

[0078] Example 11 Influence of Matrix Effect on the Recovery of 1-MEI, 2-MEI and 4-MEI in Cosmetics

[0079] In this example, acetonitrile was used to prepare a standard curve for quantitative analysis of the extraction solvent of the cosmetic sample with a spike concentration of 50 ng / mL to obtain the quantitative concentration of a group of compounds; then a standard curve was prepared using the matrix solution extracted from the blank cosmetic sample, and the recovery rate was calculated for the spiked sample extraction solvent. The results are shown in FIG. Figure 18-20 The results showed that when the standard curve was prepared using a blank matrix extraction solvent, the recoveries of 1-MEI, 2-MEI, and 4-MEI ranged from 80.0% to 95.3%. However, when the standard curve was prepared using acetonitrile, the recoveries of the three compounds were significantly lower, ranging from 33.3% to 88.1%. Therefore, this method adopted the use of a blank matrix extraction solvent for the standard curve to eliminate matrix effects.

[0080] Examples 12 and 13 are used to verify the accuracy, sensitivity and repeatability of the detection method of the present invention.

[0081] Example 12

[0082] The mixed standard solution was diluted into different gradient concentrations using blank matrix extraction solvent, and the sample was injected and analyzed according to the optimized instrument method. The standard curve was drawn with the quantitative ion peak area as the ordinate (y) and the corresponding concentration as the abscissa (x). See Table 2 for details.

[0083] Table 2 Detection limit, quantification limit, linear range, linear equation and correlation coefficient of 1-MEI, 2-MEI and 4-MEI

[0084]

[0085]

[0086] As shown in Table 2, the three compounds 1-MEI, 2-MEI and 4-MEI have a good linear relationship within their corresponding mass concentration ranges, with a correlation coefficient r greater than 0.9971, a detection limit (LOD) of 10-30 μg / kg, and a quantification limit (LOQ) of 25-100 μg / kg.

[0087] Example 13

[0088] Blank matrices of four cosmetics were spiked with 1-MEI, 2-MEI, and 4-MEI at three concentration levels, and six parallel tests were performed. The recoveries (%) and relative standard deviations (RSDs) of the three spiked concentration levels and the test results are shown in Table 3.

[0089] Table 3 Average recoveries and relative standard deviations of 1-MEI, 2-MEI and 4-MEI in cosmetics with different matrices (n=6)

[0090]

[0091]

[0092] As shown in Table 33, the average recoveries of 1-MEI, 2-MEI, and 4-MEI ranged from 80.9% to 107.9%, and the relative standard deviations (RSDs) were from 1.16% to 12.75%, all meeting the requirements of SN / T 0001-2016.

[0093] Application Examples

[0094] The detection methods of Examples 1-6 were used to test 48 cosmetic samples on the market, including 15 emulsions, 8 essential oils, 19 creams, and 6 loose powders. The emulsions, essential oils, and creams were tested using the detection methods of Examples 1-5, respectively, and the loose powders were tested using the detection method of Example 6. The test results showed that 4-MEI was detected in 9 samples, including 2 emulsions with detection levels of 69.5 and 361.0 μg / kg, respectively, 2 hair dyes with detection levels of 89.0 and 434.0 μg / kg, and 5 creams with detection levels of 1000.0, 26.6, 26.3, 26.4, and 25.7 μg / kg, respectively. 2-MEI was detected in 2 hair dye samples with detection levels of 239.5 and 267.0 μg / kg, respectively.

[0095] Based on the disclosure and teachings of the above description, those skilled in the art may also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and any modifications and variations of the invention should also fall within the scope of protection of the claims of the present invention. In addition, although certain specific terms are used in this description, these terms are for convenience of description only and do not constitute any limitation to the invention.

Claims

1. A method for determining 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole in cosmetics by ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, characterized in that: The method comprises the following steps: S1, extraction; S2, purification; S3, detection; When the sample in step S1 is specifically a cosmetic of the emulsion type, essential oil type, or cream type, the specific extraction steps of step S1 are: adding acetonitrile as an extraction solvent to the sample, shaking to fully mix the sample and the extraction solvent acetonitrile, extracting under ultrasound, centrifuging, and nitrogen blowing the entire supernatant to obtain a liquid to be purified; When the sample in step S1 is a powder-based cosmetic, the specific extraction steps of step S1 are: adding a saturated sodium chloride solution to the sample to uniformly disperse the sample, then adding the extraction solvent acetonitrile, shaking to fully mix the sample and the extraction solvent acetonitrile, extracting under ultrasound, centrifuging, and taking the supernatant and nitrogen blowing to obtain a liquid to be purified; The specific purification steps of step S2 are as follows: activating a mixed cationic solid phase extraction column with 1 mL of methanol and 1 mL of water in sequence, passing the liquid to be purified through the column, eluting with 1 mL of water and 1 mL of methanol in sequence to remove impurities, and then eluting with 3 mL of ammonia-methanol solution with a volume ratio (V / V) of 5:95, collecting the eluate, blowing the eluate dry with nitrogen in a 45°C water bath, dissolving the dried substance with acetonitrile, and diluting the volume to 1 mL, mixing evenly, and passing through an organic filter membrane to obtain a purified liquid; The detection conditions of ultra-high pressure liquid chromatography-triple quadrupole tandem mass spectrometry are: The chromatographic conditions of ultra-high pressure liquid chromatography are: chromatographic column: XBridge ® Shied RP18 column, 3.5 μm, 150 mm × 4.6 mm; mobile phase A: 20 mmol / L aqueous ammonium formate containing 0.1% formic acid; mobile phase B: acetonitrile; elution program: 0–5 min, 98% A, flow rate 0.6 mL / min; injection volume: 20.0 μL; column temperature: 45°C; The mass spectrometry conditions were as follows: electrospray ionization source (ESI) positive ion scanning; multiple reaction monitoring (MRM) mode; curtain gas pressure: 15 psi; nebulizer gas pressure: 50 psi; heating auxiliary gas pressure: 50 psi; collision gas pressure; electrospray voltage: 5500 V; and ion source temperature: 550°C.

Images