Method for detecting content of catechol and pyrogallol in cosmetics

Abstract

The application provides a method for detecting the content of catechol and pyrogallol in cosmetics, and belongs to the technical field of analytical chemistry.The method for detecting the content of catechol and pyrogallol in cosmetics provided by the application promotes the separation of catechol and pyrogallol in cosmetics by adding sodium chloride in the extraction solvent to adjust the osmotic pressure and ionic strength of the extraction solution, has high extraction efficiency, and is especially suitable for extracting low-concentration catechol and pyrogallol; then, solid-phase extraction is performed on an extraction column filled with polystyrene-divinylbenzene, methanol is used for elution, catechol and pyrogallol are enriched and separated from various organic matters, the accuracy of detection is improved, and low-concentration detection is realized.The detection limit of catechol and pyrogallol is 0.01 mg / kg, and the quantitative limit is 0.03 mg / kg.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for detecting the content of catechol and pyrogallol in cosmetics. Background Technology

[0002] Catechol, also known as catechol, is a strong reducing agent and an important chemical intermediate used as a rubber hardener and skin preservative. On October 27, 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization published a preliminary list of carcinogens, classifying catechol as a Group 2B carcinogen with an acute toxicity LD50 of 260 mg / kg (oral, rat). Pyrogallol, also known as pyrogallol, is a strong reducing agent used for leather coloring, dyeing fur and hair, and as an intermediate in pharmaceuticals and dyes. It is extremely irritating to the skin and mucous membranes; there have been cases of death from skin absorption poisoning. Ingestion can damage the digestive organs, liver, and kidneys, causing hemolysis, drowsiness, collapse, and even death. Its acute toxicity LD50 is 789 mg / kg (oral, rat). my country's "Cosmetic Safety Technical Specifications" (2015 edition) and the EU Cosmetic Regulation CE1223 / 2009 explicitly prohibit the use of catechol and pyrogallol in cosmetics.

[0003] With the development of online shopping and purchasing agents, a large number of foreign cosmetics have entered the domestic market. To ensure the quality and safety of cosmetics, it is necessary to monitor the content of catechol and pyrogallol in cosmetics. Currently, the only method for detecting the content of catechol and pyrogallol in cosmetics is liquid chromatography, which uses a mixture of sodium bisulfite aqueous solution and methanol and formic acid to pretreat the sample. However, this method has poor extraction efficiency, resulting in low detection results and low accuracy. In particular, it is no longer sufficient to meet the detection requirements for low concentrations of catechol and pyrogallol. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting the content of catechol and pyrogallol in cosmetics. The detection method provided by this invention has high extraction rate and high accuracy, meeting the needs for trace detection of catechol and pyrogallol in cosmetics.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting the content of catechol and pyrogallol in cosmetics, comprising the following steps:

[0007] 1) The sample to be tested is mixed with the extraction solvent and extracted, and then solid-liquid separation is performed to obtain a clear liquid; the extraction solvent is a mixture of organic solvent, sodium chloride and water; the organic solvent includes methanol, ethanol, formic acid or acetic acid;

[0008] 2) The clarified liquid obtained in step 1) is subjected to solid-phase extraction and then eluted with methanol to obtain the eluent as the test solution; the packing material for the solid-phase extraction is polystyrene-divinylbenzene;

[0009] 3) The test solution obtained in step 2) is subjected to high performance liquid chromatography-tandem mass spectrometry to detect the content of catechol and pyrogallol in the cosmetic.

[0010] Preferably, the mass concentration of sodium chloride in the extraction solvent in step 1) is 5-10%.

[0011] Preferably, the concentration of the organic solvent in the extraction solvent in step 1) is 0.05–0.1 mol / L.

[0012] Preferably, in step 1), the mass ratio of the sample to the extraction solvent is 1:(5-20).

[0013] Preferably, the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions for detection in step 3) include: a C18 column, a mobile phase of formic acid aqueous solution-methanol, gradient elution, a flow rate of 0.2–0.5 mL / min, a column temperature of 20–40 °C, and an injection volume of 5–20 μL.

[0014] Preferably, the formic acid in the formic acid aqueous solution has a mass percentage of 0.05% to 0.2%.

[0015] Preferably, the tandem mass spectrometry detection conditions for high performance liquid chromatography-tandem mass spectrometry detection in step 3) include: an electrospray ion source, a negative ion scanning mode, a cone voltage of 15–25 V, an ion source temperature of 120–220 °C, a desolvation temperature of 500–650 °C, a capillary voltage of 1.5–3.0 V, and a multiple reaction monitoring (MRM) mode.

[0016] Preferably, the standard solution used in step 3) for high performance liquid chromatography-tandem mass spectrometry detection is a mixed solution of catechol standard and pyrogallol standard.

[0017] Preferably, the solvent of the standard solution includes methanol, ethanol, formic acid, or acetic acid.

[0018] Preferably, the solid-liquid separation method in step 1) is centrifugal separation, and the centrifugal separation speed is 8000-12000 r / min.

[0019] This invention provides a method for detecting the content of catechol and pyrogallol in cosmetics, comprising the following steps: 1) mixing the sample to be tested with an extraction solvent and then separating the solid and liquid phases to obtain a clear liquid; the extraction solvent is a mixture of an organic solvent and an aqueous solution of sodium chloride; the organic solvent includes methanol, ethanol, formic acid, or acetic acid; 2) performing solid-phase extraction on the clear liquid obtained in step 1) and then eluting with methanol to obtain an eluent as the test solution; the packing material for the solid-phase extraction is polystyrene-divinylbenzene; 3) performing high-performance liquid chromatography-tandem mass spectrometry on the test solution obtained in step 2) to detect the content of catechol and pyrogallol in the cosmetics. This invention promotes the separation of catechol and pyrogallol in cosmetics by adding sodium chloride to the extraction solvent, adjusting the osmotic pressure and ionic strength of the extraction solution, resulting in high extraction efficiency, especially for low concentrations of catechol and pyrogallol. Solid-phase extraction is then performed using an extraction column packed with polystyrene-divinylbenzene, followed by methanol elution, which enriches and separates catechol and pyrogallol from various organic substances, improving detection accuracy and enabling the detection of low concentrations of catechol and pyrogallol. The results of the examples show that the detection method provided by this invention has a detection limit of 0.01 mg / kg and a quantitation limit of 0.03 mg / kg for catechol and pyrogallol. Attached Figure Description

[0020] Figure 1 The total ion chromatogram of catechol and pyrogallol standard substances in Example 1 of the present invention is shown, where 1 is catechol and 2 is pyrogallol.

[0021] Figure 2 The standard curve of catechol plotted in Example 1 of this invention;

[0022] Figure 3 The standard curve of pyrogallol was plotted in Example 1 of this invention;

[0023] Figure 4 The image shows the total ion chromatogram of catechol and pyrogallol in the sample to be tested, where 1 represents pyrogallol and 2 represents catechol. Detailed Implementation

[0024] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0025] This invention provides a method for detecting the content of catechol and pyrogallol in cosmetics, comprising the following steps:

[0026] 1) The sample to be tested is mixed with the extraction solvent and extracted, and then solid-liquid separation is performed to obtain a clear liquid; the extraction solvent is a mixture of organic solvent, sodium chloride and water; the organic solvent includes methanol, ethanol, formic acid or acetic acid;

[0027] 2) The clarified liquid obtained in step 1) is subjected to solid-phase extraction and then eluted with methanol to obtain the eluent as the test solution; the packing material for the solid-phase extraction is polystyrene-divinylbenzene;

[0028] 3) The test solution obtained in step 2) is subjected to high performance liquid chromatography-tandem mass spectrometry to detect the content of catechol and pyrogallol in the cosmetic.

[0029] This invention involves mixing the sample to be tested with an extraction solvent for extraction, followed by solid-liquid separation to obtain a clear liquid.

[0030] This invention does not have any particular requirements regarding the type of sample to be tested; any type of cosmetic can be tested using the detection method of this invention. As one embodiment of this invention, the sample to be tested can be a powder, hair care product, or hair dye.

[0031] In this invention, the extraction solvent is a mixture of an organic solvent, sodium chloride, and water; the organic solvent includes methanol, ethanol, formic acid, or acetic acid. The extraction solvent extracts and separates catechol and pyrogallol from the sample to be tested. By selecting and preparing the extraction solvent, this invention can effectively extract catechol and pyrogallol from the sample to be tested, improving the accuracy of detection.

[0032] In this invention, the mass concentration of sodium chloride in the extraction solvent is preferably 5-10%, more preferably 6-8%. As one embodiment of this invention, the mass concentration of sodium chloride in the extraction solvent can be 5%, 6%, 7%, 8%, 9%, or 10%. By adding sodium chloride to the extraction solvent, this invention adjusts the osmotic pressure and ionic strength of the solution. Maintaining a sodium chloride concentration within the above-mentioned range is beneficial for further improving the extraction efficiency of catechol and pyrogallol.

[0033] In this invention, the concentration of the organic solvent in the extraction solvent is preferably 0.05–0.1 mol / L, more preferably 0.06–0.08 mol / L. As one embodiment of this invention, the mass concentration of the organic solvent in the extraction solvent can be 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or 0.1 mol / L. Concentrations of the organic solvent within the above ranges are beneficial for further improving the extraction efficiency of catechol and pyrogallol.

[0034] In this invention, the mass ratio of the sample to the extraction solvent is preferably 1:(5-20), more preferably 1:(7-18), and even more preferably 1:(10-15). A mass ratio of the sample to the extraction solvent within the above range is beneficial for further improving the extraction efficiency of catechol and pyrogallol.

[0035] The present invention does not impose any particular requirements on the specific mixing method; any mixing method well known to those skilled in the art can be used. In an embodiment of the present invention, vortex mixing is used, and the mixing time is 1 minute.

[0036] In this invention, the extraction is preferably ultrasonic extraction; the ultrasonic extraction time is preferably 15-25 minutes. This invention does not have special requirements for other parameters of the ultrasonic extraction; conventional ultrasonic parameters in the art can be used. This invention, by using ultrasonic extraction on the mixed system, facilitates the separation of catechol and pyrogallol from the sample to be tested.

[0037] In this invention, the solid-liquid separation method is preferably centrifugation, and the centrifugation speed is preferably 8000-12000 r / min, more preferably 9000-11000 r / min. The centrifugation time is preferably 8-15 min, more preferably 10-12 min. Centrifugation in this invention is beneficial for further improving the extraction efficiency of catechol and pyrogallol.

[0038] After obtaining the clarified liquid, the present invention performs solid-phase extraction and elution on the clarified liquid to obtain the eluent as the test solution.

[0039] In this invention, the packing material for solid-phase extraction is polystyrene-divinylbenzene. Using polystyrene-divinylbenzene as the packing material for solid-phase extraction effectively adsorbs catechol and pyrogallol from the solution, thus improving the accuracy of detection.

[0040] The present invention does not have special requirements for the specific operation of the solid-phase extraction; any operation method known to those skilled in the art can be used. As one embodiment of the present invention, the solid-phase extraction operation can be as follows: transfer 5 mL of the clarified liquid to a solid-phase extraction column, rinse with 5 mL of deionized water, and dry under reduced pressure.

[0041] In this invention, the eluent used for elution is methanol. The specific elution operation is not particularly demanding; conventional procedures in the art can be employed. In one embodiment of this invention, the amount of methanol used is 50% of the clarified liquid, and the elution flow rate is 1 mL / min.

[0042] After elution, the eluted solution is preferably filtered through a microporous membrane to obtain the eluent as the test solution. In one embodiment of the invention, a 0.2 μm microporous membrane can be used. This invention removes suspended particles and microorganisms from the test solution using a microporous membrane, which helps to further improve the accuracy of the detection.

[0043] After obtaining the test solution, the present invention performs high performance liquid chromatography-tandem mass spectrometry on the test solution to obtain the content of catechol and pyrogallol in the cosmetic.

[0044] In this invention, the standard solution used in the high performance liquid chromatography-tandem mass spectrometry detection is preferably a mixed solution of catechol standard and pyrogallol standard.

[0045] The present invention does not have special requirements for the specific preparation process of the standard solution; any preparation method commonly used by those skilled in the art can be adopted. As one embodiment of the present invention, the preparation process of the standard solution can be as follows: accurately weigh 10 mg each of catechol and pyrogallol standard substances, place them in 100 mL brown volumetric flasks, dissolve and dilute with methanol to prepare a standard stock solution with a mass concentration of 100 μg / mL; accurately transfer 1 mL each of the catechol and pyrogallol standard stock solutions to 100 mL brown volumetric flasks, dilute and dilute with methanol to prepare a mixed standard intermediate solution with a mass concentration of 1 μg / mL; accurately transfer 1 mL of the mixed standard intermediate solution to volumetric flasks of different sizes, dilute and dilute with methanol to prepare a series of mixed standard working solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0046] In this invention, the preferred high-performance liquid chromatography (HPLC) conditions for HPLC-tandem mass spectrometry detection include: a C18 column, a mobile phase of formic acid aqueous solution-methanol, gradient elution, a flow rate of 0.2–0.5 mL / min, a column temperature of 20–40 °C, and an injection volume of 5–20 μL.

[0047] In this invention, the mass percentage of formic acid in the formic acid aqueous solution is preferably 0.05-0.2%, more preferably 0.1-0.15%.

[0048] In this invention, the gradient elution method is preferably performed according to Table 1.

[0049] Table 1 Gradient Elution of Mobile Phase

[0050] 0～6 0.2～0.5 90 5％～60％ 6～6.01 0.2～0.5 70 60％～5％ 6.01～10 0.2～0.5 70 5％

[0051] This invention limits the conditions of high performance liquid chromatography to the above-mentioned range, which is beneficial to further improve the accuracy of detection.

[0052] In this invention, the preferred tandem mass spectrometry detection conditions for high performance liquid chromatography-tandem mass spectrometry detection include: an electrospray ion source, a negative ion scanning mode, a cone voltage of 15–25 V, an ion source temperature of 120–220 °C, a desolvation temperature of 500–650 °C, a capillary voltage of 1.5–3.0 V, and a multiple reaction monitoring (MRM) mode.

[0053] In this invention, the specific conditions for the multiple reaction monitoring are preferably as shown in Table 2.

[0054] Table 2 Mass spectrometry parameters of catechol and pyrogallol

[0055]

[0056] This invention limits the conditions for tandem mass spectrometry detection to the above-mentioned range, which is beneficial to further improve the accuracy of detection.

[0057] This invention promotes the separation of catechol and pyrogallol from cosmetics by adding sodium chloride to the extraction solvent, thereby adjusting the osmotic pressure and ionic strength of the extraction solution and achieving high extraction efficiency, especially for low concentrations. Solid-phase extraction is then performed using an extraction column packed with polystyrene-divinylbenzene, followed by methanol elution. This enriches catechol and pyrogallol and separates them from various organic substances, improving the accuracy of detection and enabling the detection of low concentrations.

[0058] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0059] Example 1

[0060] A method for detecting catechol and pyrogallol in cosmetics, comprising the following steps:

[0061] 1) Instruments and reagents

[0062] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS): charged spray ionization source; ultrasonic cleaner; high-speed centrifuge: maximum speed 15000 r / min; electronic analytical balance: sensitivity of 0.1 mg and 0.001 g; solid-phase extraction instrument (with solid-phase extraction column: packing material is polystyrene-divinylbenzene, 500 mg, 6 mL); catechol and pyrogallol standards: purity greater than 99.5%; methanol (chromatographic grade); formic acid (chromatographic grade); glacial acetic acid (analytical grade); sodium chloride (analytical grade); ultrapure water (18.2 MΩ·cm).

[0063] 2) Preparation of standard solutions

[0064] Standard stock solutions (100 μg / mL): Accurately weigh 10 mg each of catechol and pyrogallol standards (accurate to 0.1 mg), place them in 100 mL brown volumetric flasks, dissolve them in methanol and dilute to the mark to prepare standard stock solutions with a mass concentration of 100 μg / mL; store at -18℃ protected from light.

[0065] Mixed standard intermediate solution (1.0 μg / mL): Accurately transfer 1 mL each of catechol and pyrogallol standard stock solutions into 100 mL brown volumetric flasks, dilute to the mark with methanol, and prepare a mixed standard intermediate solution with a mass concentration of 1 μg / mL; store at -18℃ protected from light.

[0066] Mixed standard series working solutions: Accurately transfer an appropriate amount of the mixed standard intermediate solution, dilute with methanol and shake well to prepare the mixed standard series working solutions. The mass concentrations of each analyte are 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively. Prepare fresh before use.

[0067] 3) Preparation of the test solution

[0068] Accurately weigh 1.0 g (accurate to 0.1 mg) of an imported hair dye cosmetic sample into a 50 mL polypropylene centrifuge tube, accurately add 10 mL of 0.05 mol / L acetic acid solution and 0.6 g of sodium chloride, vortex mix for 1 min, ultrasonically extract for 20 min, centrifuge at 10000 r / min for 10 min, take 5.0 mL of the clear liquid and transfer it to a solid phase extraction column, rinse with 5 mL of deionized water, dry under reduced pressure, and elute with 2.5 mL of methanol. The flow rate of the entire elution process is controlled at 1 mL / min. The collected eluent is filtered through a 0.2 μm microporous membrane to obtain the test solution.

[0069] 4) High-performance liquid chromatography-tandem mass spectrometry detection

[0070] Gradient elution was performed using 0.1% formic acid solution-methanol as the mobile phase, and the determination was carried out using multiple reaction monitoring (MRM) mode of high performance liquid chromatography-tandem mass spectrometry with an electrospray ionization source.

[0071] The chromatographic conditions for high performance liquid chromatography were as follows: column: C18 column, 100 mm × 2.1 mm (particle size 1.7 μm); mobile phase: 0.1% formic acid solution-methanol (elution according to the gradient in Table 1); flow rate: 0.3 mL / min; column temperature: 30 ℃; injection volume: 10 μL.

[0072] Tandem mass spectrometry measurement conditions: ion source was electrospray ion source; scanning mode: negative ion scanning; cone voltage: 20V; ion source temperature: 150℃; desolvation temperature: 600℃; capillary voltage: 2.0V; scanning mode: multiple reaction monitoring (MRM). Specific conditions are shown in Table 2.

[0073] 5) Qualitative and quantitative determination

[0074] Qualitative determination

[0075] If the retention time of the chromatographic peak detected in the test solution is consistent with the retention time of a certain component peak in the mixed standard series working solution, and the mass-to-charge ratio of the two selected pairs of ions (Table 2) is consistent in the mass spectrum of the sample after background subtraction, and the relative deviation between the relative abundance of the qualitative ions in the sample and the relative abundance of the qualitative ions in the standard working solution with equivalent concentration does not exceed the range specified in Table 3, then it can be determined that the component exists in the sample.

[0076] Table 3. Maximum permissible deviation of relative ion abundance during qualitative confirmation.

[0077] Permissible relative deviation / % ±20 ±25 ±30 ±50

[0078] Quantitative determination

[0079] Preparation of standard curves: The mixed standard series working solutions were injected separately into a liquid chromatography-tandem mass spectrometer (LC-MS / MS) to determine the corresponding analytes, obtaining the total ion chromatograms of the standard substances catechol and pyrogallol, as shown below. Figure 1 As shown.

[0080] A standard curve is plotted with the concentration of the analyte on the x-axis and the peak area of ​​the analyte on the y-axis, as shown below. Figure 2 , Figure 3 As shown, the linear regression equation for catechol is:

[0081] y1=8.94068x1+20.5876 formula (1);

[0082] The linear regression equation for pyrogallol is as follows:

[0083] y2=22.5973x2-37.147 formula (2);

[0084] The method detection limits for catechol and pyrogallol were 0.01 mg / kg, and the method quantification limits were 0.03 mg / kg.

[0085] Determination of the test solution: Inject the above-mentioned sample test solution for determination, and obtain the total ion chromatogram of catechol and pyrogallol in the sample, as shown below. Figure 4As shown. Based on the peak area measured by the software, and according to the linear regression equation obtained above, the instrument automatically calculates the concentrations of catechol and pyrogallol in the test solution.

[0086] The contents of catechol and pyrogallol in the sample are calculated according to formula (3).

[0087]

[0088] In the formula:

[0089] Xi: Content of catechol and pyrogallol in the sample, in micrograms per kilogram (μg / kg);

[0090] Ci: The concentrations of catechol and pyrogallol in the test sample solution are obtained from the standard curve, in micrograms per milliliter (μg / mL);

[0091] V: Volume of the sample solution, in milliliters (mL);

[0092] m: Sample mass, in grams (g);

[0093] b: Dilution factor.

[0094] The sample contained 100 μg / kg of catechol and 550 μg / kg of pyrogallol.

[0095] As can be seen from the above embodiments, the detection method provided by the present invention can detect microgram levels of catechol and pyrogallol in cosmetics, thus meeting the detection requirements.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the content of catechol and pyrogallol in cosmetics, comprising the following steps: 1) The sample to be tested is mixed with the extraction solvent and extracted, and then solid-liquid separation is performed to obtain a clear liquid; the extraction solvent is a mixture of organic solvent, sodium chloride and water; the organic solvent is acetic acid; 2) The clarified liquid obtained in step 1) is subjected to solid-phase extraction and then eluted with methanol to obtain the eluent as the test solution; the packing material for the solid-phase extraction is polystyrene-divinylbenzene; 3) The test solution obtained in step 2) is subjected to high performance liquid chromatography-tandem mass spectrometry to detect the content of catechol and pyrogallol in the cosmetics; The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions for step 3) include: a C18 column, a formic acid aqueous solution-methanol mobile phase, gradient elution, a flow rate of 0.2–0.5 mL / min, a column temperature of 20–40 °C, and an injection volume of 5–20 μL; the gradient elution method is as follows: The formic acid aqueous solution contains formic acid at a mass percentage of 0.05-0.2%. The tandem mass spectrometry detection conditions for high performance liquid chromatography-tandem mass spectrometry in step 3) include: the ion source is an electrospray ion source, the scanning mode is negative ion scanning, the cone voltage is 15~25V, the ion source temperature is 120~220℃, the desolventizing temperature is 500~650℃, the capillary voltage is 1.5~3.0V, and the scanning mode is multiple reaction monitoring.

2. The detection method according to claim 1, characterized in that, In step 1), the mass concentration of sodium chloride in the extraction solvent is 5-10%.

3. The detection method according to claim 1, characterized in that, In step 1), the concentration of the organic solvent in the extraction solvent is 0.05~0.1mol / L.

4. The detection method according to claim 1, 2 or 3, characterized in that, In step 1), the mass ratio of the sample to the extraction solvent is 1:(5~20).

5. The detection method according to claim 1, characterized in that, In step 3), the standard solution used for high performance liquid chromatography-tandem mass spectrometry detection is a mixed solution of catechol standard and pyrogallol standard.

6. The detection method according to claim 5, characterized in that, The solvent for the standard solution includes methanol, ethanol, formic acid, or acetic acid.

7. The detection method according to claim 1, characterized in that, In step 1), the solid-liquid separation method is centrifugal separation, and the centrifugal separation speed is 8000~12000 r / min.

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