Thin layer chromatography method for high-throughput screening of glucocorticoid in cosmetics

Abstract

The invention discloses a thin layer chromatography method for high-throughput screening of glucocorticoid in cosmetics. The method comprises the following steps: establishing a database comprising a specific shift value Rf of known glucocorticoid of a characteristic control group, a tetrazole blue color development characteristic F1 and an anisaldehyde color development characteristic F2; carrying out thin layer chromatography on the characteristic control group glucocorticoid mixed standard working solution and a plurality of cosmetic to-be-detected solutions on the same thin layer plate, and carrying out tetrazole blue and anisaldehyde color development; qualitatively analyzing and judging whether the cosmetic solution to be detected is a negative sample or a positive sample; the method comprises the following steps: firstly, carrying out qualitative identification on a positive sample on the basis of an actual measurement ratio shift value, a tetrazole blue color development characteristic and an anisaldehyde color development characteristic of each glucocorticoid in a characteristic control group and the basis of Rf, F1 and F2 of a database. High-throughput screening of glucocorticoid in cosmetics is realized, expensive instruments and equipment are not needed, and the method has the advantages of simplicity in operation, low cost, high efficiency, easiness in popularization and the like.

Description

Technical Field

[0001] This invention relates to a method for screening glucocorticoids in cosmetics, and more particularly to a high-throughput thin-layer chromatography method for screening glucocorticoids in cosmetics. Background Technology

[0002] Glucocorticoids are widely used clinically and are commonly used drugs for treating skin diseases such as eczema and allergic dermatitis. They have anti-inflammatory, anti-allergic, immunosuppressive, and anti-proliferative effects. Short-term use of cosmetics containing glucocorticoids can create the illusion of smooth, fair skin. However, long-term use may lead to side effects such as dark spots, skin atrophy and thinning, and even steroid-dependent dermatitis, causing serious harm to the user's health. Therefore, the addition of glucocorticoids to cosmetics remains one of the most frequently discovered illegal practices in the current cosmetics regulatory field, requiring continuous strengthening of supervision and crackdown. However, glucocorticoids are a class of drugs with a basic structure containing a 21-carbon cyclopentane-polyhydrophenanthrene steroidal core, possessing multiple sites that can be derived and modified. Currently, more than 100 types of glucocorticoids have been reported on the market, and the continuous emergence of new hormones poses a significant challenge to regulation.

[0003] The "Cosmetic Safety Technical Specifications" (2015 edition) lists glucocorticoids as a prohibited ingredient in cosmetics, with Chapter 4, Method 2.34 including detection methods for 92 glucocorticoids. Two other national standards, GB / T 24800.2-2009 and GB / T 40145-2021, provide detection methods for 41 and 11 glucocorticoids, respectively. Currently, most glucocorticoid detection in cosmetics uses liquid chromatography-mass spectrometry (LC-MS / MS), with only GB / T 24800.2-2009 including a method for detecting glucocorticoids in cosmetics using thin-layer chromatography (TLC).

[0004] The advantages of liquid chromatography-mass spectrometry (LC-MS) are low detection limits, accurate qualitative and quantitative analysis, and the ability to achieve high-throughput screening through automated sample injection. The disadvantages are expensive equipment, the ability to analyze only one sample at a time (serial injection), and the difficulty in detecting hormones outside the screening range with targeted assays. Thin-layer chromatography (TLC) has the advantages of not requiring expensive equipment, being simple and easy to perform, and allowing for simultaneous analysis of multiple samples, often used as a primary screening method. The disadvantages are lower resolution and the limited R-value used for determination. fThe values ​​and colorimetric characteristics are easily affected by various factors, thus only preliminary qualitative analysis is possible. Furthermore, thin-layer chromatography (TLC) requires comparing the control and test samples on the same TLC plate. In addition to the test sample, all control substances need to be spotted, but each TLC plate has a limited capacity, making it generally unsuitable for high-throughput screening. For example, the detection method specified in GB / T 24800.2-2009 divides 41 glucocorticoids into 5 groups, each group forming a control. Each identification requires spotting the sample and 5 control substances together on the plate. Each 10 cm × 10 cm TLC plate can only simultaneously detect 3 samples, in addition to the 5 spots for the control group. Developing high-throughput, rapid screening technologies for glucocorticoids that do not rely on expensive equipment has become an urgent need for cosmetic industry regulation. Summary of the Invention

[0005] The present invention aims to address the problem that existing thin-layer chromatography techniques are difficult to achieve high-throughput screening, and provides a thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics.

[0006] The technical solution of this invention is a thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics, which is carried out according to the following steps: Step 1. Determine the initial translocation value, tetrazolium blue, and anisaldehyde colorimetric characteristics of known glucocorticoids under the same thin-layer chromatography conditions. Group the known glucocorticoids based on complete separation within the same developing distance. One group serves as a characteristic control group, which includes glucocorticoids with high colorimetric characteristic recognition and includes the two glucocorticoids with the lowest and highest initial translocation values. Establish the translocation value R for known glucocorticoids. f Databases of tetrazolium blue colorimetric characteristics F1 and anisaldehyde colorimetric characteristics F2; Step 2. Perform thin-layer chromatography on the same thin-layer chromatography plate with the mixed standard working solution of glucocorticoids in the characteristic control group and multiple cosmetic test solutions, and perform color development with tetrazolium blue and anisaldehyde to obtain the measured ratio shift value, tetrazolium blue color development characteristics and anisaldehyde color development characteristics of each glucocorticoid in the characteristic control group and each cosmetic test solution. Step 3. Qualitative analysis to determine whether the cosmetic sample solution is a negative or positive sample; Step 4. First, based on the measured ratio shift values, tetrazolium blue colorimetric characteristics, and anisaldehyde colorimetric characteristics of each glucocorticoid in the characteristic control group, then using the R data from the database... f Based on F1 and F2, positive samples are qualitatively identified.

[0007] Step 4 is described in detail below: First, the measured ratio shift R of the positive sample was... fy实测 Tetrazolium blue colorimetric characteristics F 1y实测and the colorimetric characteristics of anisaldehyde F 2y实测 Measured shift value R compared to the characteristic control group fb实测 Tetrazolium blue colorimetric characteristics F 1b实测 and the colorimetric characteristics of anisaldehyde F 2b实测 Perform a corresponding comparison; If the characteristic control group has R fb实测 With R fy实测 F 1b实测 With F 1y实测、 F 2b实测 With F 2y实测 If the glucocorticoids are all consistent, then it is determined to be the glucocorticoid present in the positive sample; If the characteristic control group has R fb实测 With R fy实测 Consistent but F 1b实测 With F 1y实测 or / and F 2b实测 With F 2y实测 Inconsistent glucocorticoids will lead to R fy实测 F 1y实测 and F 2y实测 With R database f Compare F1 and F2, and if R is found in the database... f With R fy实测 F1 and F 1y实测 and F2 and F 2y实测 If all glucocorticoids are consistent, then it is determined that it is a glucocorticoid present in the positive sample; otherwise, the glucocorticoid present in the positive sample is determined to be a glucocorticoid not contained in the database. If the characteristic control group does not have R fy实测 Consistent glucocorticoids, firstly, determine the characteristic control group with R fy实测 R of two adjacent points fb实测 With R in the database f If they match, then directly assign R. fy实测 F 1y实测 and F 2y实测 With R database f Compare F1 and F2, and if R is found in the database... f With R fy实测 F1 and F 1y实测 and F2 and F 2y实测 If the glucocorticoids present in the control group are consistent with those in the database, then the cosmetic is identified as a glucocorticoid present in the cosmetic; otherwise, the glucocorticoid present in the cosmetic is identified as a glucocorticoid not found in the database. fy实测 R of two adjacent points fb实测 With R in the database f If they are inconsistent, calculate the values ​​of R in the characteristic control group respectively. fy实测 R of two adjacent pointsfb实测 The corresponding R in the database f The ratio of the two ratios is then used as the calibration coefficient K for R. fy实测 Perform calibration to obtain R fy校准 =R fy实测 / K, will R fy校准 F 1y实测 and F 2y实测 Compare with the database, for example, if R is in the database. f With R fy校准 F1 and F 1y实测 and F2 and F 2y实测 If all glucocorticoids are consistent, then the sample is considered to contain glucocorticoids present in the positive sample; otherwise, the glucocorticoids present in the positive sample are considered to be glucocorticoids not found in the database.

[0008] Step 1 is described in detail as follows: First, several known glucocorticoids were subjected to single-point thin-layer chromatography, tetrazolium blue staining, and anisaldehyde staining to preliminarily determine the initial ratio shift value (R) of the known glucocorticoids. f初测 Tetrazolium blue colorimetric characteristics F 1初测 and the colorimetric characteristics of anisaldehyde F 2初测 Known glucocorticoids were grouped to obtain N groups of glucocorticoids, including a characteristic control group; it was determined whether N was greater than the maximum number of spots on a single thin-layer chromatography plate. If not, R was used as the maximum number of spots. f初测 F 1初测 and F 2初测 As R respectively f Databases were established for F1 and F2. Each group of glucocorticoids was prepared into a mixed standard working solution. The mixed standard working solution of the characteristic control group glucocorticoids was then subjected to thin-layer chromatography (TLC) on the same TLC plate, with tetrazolium blue and anisaldehyde staining. The measured ratio shift values ​​(Rf) of all glucocorticoids provided by multiple TLC plates were obtained. f实测 Tetrazolium blue colorimetric characteristics F 1实测 and the colorimetric characteristics of anisaldehyde F 2实测 If different thin-layer chromatography plates show characteristic control group glucocorticoid R f实测 All were consistent, based on the measured ratio shift value R of all glucocorticoids. f实测 F 1实测 and F 2实测 As R respectively f Establish databases for F1 and F2; otherwise, use one of the thin-layer chromatography plates as a standard plate and use the characteristic control group glucocorticoid R obtained from the standard plate. f实测 R was obtained by calibrating the ratio shift values ​​of glucocorticoids on other thin-layer chromatography plates. f校准 R of glucocorticoids on standard plates f实测R obtained from other thin-layer chromatography plates f校准 As R f F 1实测 and F 2实测 Create databases as F1 and F2 respectively.

[0009] Step 3 is as follows: Observe the thin-layer chromatography spectrum under ultraviolet light. If there are no dark spots, it can be determined that no glucocorticoids are detected in the cosmetic, i.e., it is a negative sample. If there are obvious dark spots, and no characteristic spots are found after color development with tetrazolium blue and anisaldehyde, it can be determined that the cosmetic is a negative sample. If characteristic spots appear after color development with tetrazolium blue or anisaldehyde, it can be determined that glucocorticoids are present in the cosmetic, i.e., it is a positive sample.

[0010] The thin-layer chromatography conditions are as follows: 1) Thin-layer chromatography plate: High-efficiency silica gel F254, activated in an oven at 110℃ for 1 hour before use, then placed in a desiccator to room temperature for later use; 2) Developing solvent: ethyl acetate + n-hexane, with a volume ratio of ethyl acetate to n-hexane of 11:10; 3) Tetrazolium blue colorimetric reagent: Weigh 20 mg of tetrazolium blue and dissolve it in 10 mL of methanol, then add 10 mL of 12% NaOH methanol solution. Prepare fresh before use. 4) Anisaldehyde colorimetric reagent: Add 5 mL of concentrated sulfuric acid and 1 mL of anhydrous acetic acid to 90 mL of anhydrous ethanol in an ice-water bath. After mixing and cooling, add 5 mL of anisaldehyde and mix well again. Set aside for use.

[0011] This invention establishes a database of glucocorticoid ratio shift values, tetrazolium blue, and anisaldehyde colorimetric characteristics containing a characteristic control group, and uses a thin-layer chromatography method calibrated with a set of characteristic control groups. This method requires only one set of characteristic control groups and the test sample to be detected on the same thin-layer chromatography plate, significantly increasing the number of cosmetic samples that can be detected simultaneously in a single run. This achieves high-throughput screening of glucocorticoids in cosmetics and overcomes the technical shortcomings of existing thin-layer chromatography methods in high-throughput screening. It requires no expensive instruments and equipment and has advantages such as simple operation, low cost, high efficiency, and ease of promotion. Attached Figure Description

[0012] Figure 1 This is a colorimetric result image of 105 glucocorticoids obtained in Example 1 of the present invention at 254nm.

[0013] Figure 2 This is a colorimetric result diagram of 105 glucocorticoids with anisaldehyde obtained in Example 1 of the present invention.

[0014] Figure 3 This is a diagram showing the results of the blue staining of 105 glucocorticoids obtained in Example 1 of this invention.

[0015] Figure 4 This is a 254nm colorimetric result image of each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 1 of the present invention.

[0016] Figure 5 This is a tetrazolium blue colorimetric result diagram of each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 1 of the present invention.

[0017] Figure 6 This is a colorimetric result diagram of anisaldehyde for each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 1 of the present invention.

[0018] Figure 7 This is a 254nm colorimetric result image of each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 2 of the present invention.

[0019] Figure 8 This is a colorimetric result diagram of anisaldehyde for each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 2 of the present invention.

[0020] Figure 9 This is a tetrazolium blue colorimetric result diagram of each glucocorticoid and each cosmetic test solution in the characteristic control group of Example 2 of the present invention. Detailed Implementation Example 1

[0021] The present invention provides a high-throughput thin-layer chromatography method for screening glucocorticoids in cosmetics, comprising the following steps: Step 1. First, the 105 known glucocorticoids, whose compound names, English names, and CAS numbers are shown in Table 1, were subjected to single-point thin-layer chromatography, tetrazolium blue staining, and anisaldehyde staining to preliminarily determine the initial ratio shift value (Rf) of the known glucocorticoids. f初测 Tetrazolium blue colorimetric characteristics F 1初测 and the colorimetric characteristics of anisaldehyde F 2初测 ; Table 1-105 names and CAS numbers of glucocorticoids (Chinese and English). ; ; ; Known glucocorticoids were grouped based on the condition that they were completely separated within the same unfolding distance. One group served as a characteristic control group, whose glucocorticoids exhibited high colorimetric characteristic recognition and included the initial measured ratio shift value R. f初测 The two lowest and highest levels of glucocorticoids were identified. The 105 glucocorticoids in Table 1 were divided into the following 13 groups: Group 1: Serial numbers 1, 14, 26, 43, 51, 65, 78, 93; Group 2: Serial numbers 2, 15, 27, 52, 66, 76, 79, 87, 94; Group 3: Serial numbers 3, 16, 28, 40, 53, 67, 81, 84; Group 4: Serial numbers 4, 24, 29, 41, 54, 68, 82, 97; Group 5: Serial numbers 5, 17, 30, 42, 55, 69, 83, 98; Group 6: Serial numbers 6, 18, 31, 56, 70, 86, 99; Group 7: Serial numbers 7, 19, 33, 44, 57, 71, 87, 100; Group 8: Serial numbers 8, 20, 34, 45, 58, 72, 88, 101; Group 9: Serial numbers 9, 21, 35, 46, 60, 73, 89, 102; Group 10: Serial numbers 10, 22, 36, 47, 61, 74, 90, 96; Group 11: Serial numbers 12, 23, 37, 49, 62, 75, 91, 103; Group 12: Serial numbers 13, 25, 38, 50, 63, 77, 92, 104; Characteristic control group: serial numbers 11, 32, 48, 59, 64, 79, 85, 95, 105.

[0022] The thin-layer chromatography plate of this invention has a size of 100 mm × 200 mm, with 100 mm as the spotting edge, and a maximum of 8 spotting samples. Since the number of groups (13) is greater than 8, two thin-layer chromatography plates are required to complete the chromatographic analysis of all known glucocorticoids. Each group of glucocorticoids is prepared into a mixed standard working solution. The mixed standard working solution of the characteristic control group glucocorticoids is chromatographically analyzed with the mixed standard working solutions of groups 1-6 on the same thin-layer chromatography plate, and then subjected to tetrazolium blue and anisaldehyde staining. The mixed standard working solution of the characteristic control group glucocorticoids is chromatographically analyzed with the mixed standard working solutions of groups 7-12 on another thin-layer chromatography plate, and then subjected to tetrazolium blue and anisaldehyde staining respectively.

[0023] The mixed standard working solution was prepared according to the following steps: ① Preparation of standard stock solution: (1) Accurately weigh 5 mg of each of the fluorometholone, flunisolone and triamcinolone epoxy standard substances into a 5 mL brown volumetric flask, and dilute to the mark with 50% methanol water to prepare a stock solution with a concentration of 1.0 mg / mL; (2) The remaining 102 glucocorticoid standard substances were diluted to the mark with methanol to prepare stock solutions with a concentration of 1.0 mg / mL.

[0024] All stock solutions should be stored frozen at -18°C in a sealed container for 12 months. Before use, they must be brought to room temperature. If crystallization occurs, shaking or sonication should be used to aid dissolution before use.

[0025] ② Preparation of mixed standard working solutions: Group 1: Take 100 μL of the standard stock solutions numbered 1, 14, 26, 43, 51, 65, 78, and 93 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 2: Take 100 μL of the standard stock solutions numbered 2, 15, 27, 52, 66, 76, 79, 87, and 94 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 3: Take 100 μL of the standard stock solutions numbered 3, 16, 28, 40, 53, 67, 81, and 84 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 4: Take 100 μL of the standard stock solutions numbered 4, 24, 29, 41, 54, 68, 82, and 97 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 5: Take 100 μL of the standard stock solutions numbered 5, 17, 30, 42, 55, 69, 83, and 98 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 6: Take 100 μL of the standard stock solutions numbered 6, 18, 31, 56, 70, 86, and 99 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 7: Take 100 μL of the standard stock solutions numbered 7, 19, 33, 44, 57, 71, 87, and 100 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 8: Take 100 μL of the standard stock solutions numbered 8, 20, 34, 45, 58, 72, 88, and 101 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 9: Take 100 μL of the standard stock solutions numbered 9, 21, 35, 46, 60, 73, 89, and 102 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 10: Take 100 μL of the standard stock solutions numbered 10, 22, 36, 47, 61, 74, 90, and 96 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 11: Take 100 μL of the standard stock solutions numbered 12, 23, 37, 49, 62, 75, 91, and 103 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Group 12: Take 100 μL of the standard stock solutions numbered 13, 25, 38, 50, 63, 77, 92, and 104 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. Characteristic control group: Take 100 μL of the standard stock solution with serial numbers 11, 32, 48, 59, 64, 79, 85, 95 and 105 in Table 1 respectively, and dilute to 1 mL with methanol to prepare a 100 μg / mL mixed standard working solution. The thin-layer chromatography conditions are as follows: 1) Thin-layer chromatography plate: high-efficiency silica gel F254, 100 mm × 200 mm, coating thickness 0.20 mm. Before use, activate in an oven at 110℃ for 1 hour, then cool to room temperature in a desiccator for later use. 2) Developing solvent: ethyl acetate + n-hexane (11 + 10, volume ratio); 3) Tetrazolium blue colorimetric reagent: Weigh 20 mg of tetrazolium blue and dissolve it in 10 mL of methanol, then add 10 mL of 12% NaOH methanol solution. Prepare fresh before use.

[0026] 4) Anisaldehyde colorimetric reagent: Add 5 mL of concentrated sulfuric acid and 1 mL of anhydrous acetic acid to 90 mL of anhydrous ethanol in an ice-water bath. After mixing and cooling, add 5 mL of anisaldehyde, mix well, and set aside for use. Thin-layer chromatography is performed as follows: Spot 20 μL of sample solution 1.0 cm from the bottom of the thin-layer chromatography plate. For each 100 mm × 200 mm thin-layer chromatography plate, leave a 1 cm margin on both sides, and spot one spot approximately every 1 cm. Each plate can simultaneously analyze 7 groups of samples. The 12 known glucocorticoid groups were sampled on two thin-layer chromatography plates (four plates in total, as subsequent anisaldehyde and tetrazolium blue staining are required). 20 μL of the characteristic control group glucocorticoid standard working solution was simultaneously spotted on each plate for calibration. In a dual-chamber chromatography tank, one chamber was filled with an appropriate amount of developing solvent, while the other chamber was left empty. The thin-layer chromatography plate was placed in the empty chamber for equilibration for 30 min, then placed in the chamber containing the developing solvent. Developed using the inclined upward method for approximately 18.0 cm. The thin-layer chromatography plate was removed from the developing chamber, allowed to air dry, and then photographed under a thin-layer photography system (or UV lamp) at 254 nm. The shift values ​​of all spots were recorded. Then, the anisaldehyde colorimetric reagent was evenly sprayed onto one of the thin-layer chromatography plates, removed and dried, and placed on a constant-temperature heating plate (105℃) until the spots were clearly visible. Immediately, photographs were taken to observe and record the anisaldehyde colorimetric characteristics of each standard substance. Next, the tetrazolium blue colorimetric reagent was evenly sprayed onto another thin-layer chromatography plate, and the tetrazolium blue colorimetric characteristics of each standard substance were immediately observed by direct photograph.

[0027] The results are as follows Figure 1 , Figure 2 , Figure 3 The two thin-layer chromatography plates shown provide the measured ratio shift values ​​(R0) of 105 glucocorticoids. f实测 Tetrazolium blue colorimetric characteristics F 1实测 and the colorimetric characteristics of anisaldehyde F 2实测 Of these, 13 (control) were the characteristic control group. Because the characteristic control group on the two thin-layer chromatography plates contained glucocorticoid R... f实测 They are all consistent, therefore R is used. f实测 F 1实测 and F 2实测 As R respectively f Databases were established for F1 and F2 as shown in Table 2. However, if the characteristic control group of glucocorticoid R on two thin-layer chromatography plates was used... f实测 Since they are not all identical, one of the thin-layer chromatography plates is used as the standard plate, and the characteristic control group of glucocorticoid R obtained from the standard plate is used. f实测 The ratio shift of glucocorticoids on another thin-layer chromatography plate was calibrated to obtain R. f校准 R of glucocorticoids on standard plates f实测 R obtained from another thin-layer chromatography plate f校准 As R f F 1实测 and F 2实测 Create databases as F1 and F2 respectively.

[0028] Table 2 Reference R for 105 glucocorticoids f Reference colorimetric characteristics of α-value, tetrazolium blue, and anisaldehyde. ; ; ; ;

[0029] Note: with * The compounds are used to prepare mixed standard solutions of characteristic glucocorticoids.

[0030] Step 2. Perform thin-layer chromatography on the same thin-layer chromatography plate with the mixed standard working solution of glucocorticoids in the characteristic control group and multiple cosmetic test solutions, and perform color development with tetrazolium blue and anisaldehyde to obtain the measured ratio shift value, tetrazolium blue color development characteristics and anisaldehyde color development characteristics of each glucocorticoid in the characteristic control group and each cosmetic test solution. The cosmetic test solution was prepared according to the following steps: ① Accurately weigh 0.5 g of the cosmetic sample into a 10 mL stoppered graduated centrifuge tube; ② For non-oil-based cosmetics: first add 4 mL of saturated sodium chloride solution containing 0.2% acetic acid and vortex mix well, then add 5 mL of acetonitrile containing 0.2% acetic acid. For oil-based cosmetics: first add 5 mL of acetonitrile containing 0.2% acetic acid, vortex mix well, then add 4 mL of saturated sodium chloride solution containing 0.2% acetic acid. ③ Vortex or shake thoroughly to mix well, then centrifuge at 5000 r / min for 10 min; ④ Transfer 4 mL of the supernatant to a 50 mL centrifuge tube, accurately add 40 mL of water to the extract, and mix well; ⑤ Add 0.25 mL of 10% potassium ferrocyanide solution and mix well. Add 0.25 mL of 20% zinc acetate solution containing 0.4% acetic acid, mix well, and centrifuge at 5000 r / min for 10 min. ⑥ Take the supernatant and pass it through an Oasis HLB solid-phase extraction column (60 mg / 3 mL specification, pre-activated with 5 mL methanol and 10 mL water). First, wash the column with 10 mL of 10% acetonitrile aqueous solution, then rinse the column with 8 mL of methanol and collect the eluent. ⑦ Blow the sample to near dryness with nitrogen in a 60℃ water bath, accurately add 0.1 mL of methanol, mix well, and use as the test solution for the cosmetic sample.

[0031] The thin-layer chromatography and tetrazolium blue and anisaldehyde color development were performed on the same thin-layer chromatography plate, and the specific operation is as follows: ① Thin-layer chromatography conditions 1) Thin-layer chromatography plate: high-efficiency silica gel F254, 100 mm × 200 mm, coating thickness 0.20 mm. Before use, activate in an oven at 110℃ for 1 hour, then cool to room temperature in a desiccator for later use. 2) Developing solvent: ethyl acetate + n-hexane (11 + 10, volume ratio); 3) Anisaldehyde colorimetric reagent: Add 5 mL of concentrated sulfuric acid and 1 mL of anhydrous acetic acid to 90 mL of anhydrous ethanol in an ice-water bath. After mixing and cooling, add 5 mL of anisaldehyde, mix well, and set aside for use. 4) Tetrazolium blue colorimetric reagent: Weigh 20 mg of tetrazolium blue and dissolve it in 10 mL of methanol, then add 10 mL of 12% NaOH methanol solution. Prepare fresh before use.

[0032] ② Thin-layer chromatography and colorimetric test For each 100 mm × 200 mm thin-layer chromatography plate, leave a 1 cm margin on both sides. Draw a horizontal line 1.0 cm from the bottom of the plate, and mark a spot approximately every 1.5 cm, for a total of 6-7 spots per plate. First, apply 15-20 μL of the characteristic control group glucocorticoid standard working solution to each plate for calibration. Then, apply 15-20 μL of the test sample to the other 6 spots, simultaneously on two plates. Add an appropriate amount of developing solvent to one tank of the dual-chamber chromatography tank, leaving the other tank empty. Place the thin-layer chromatography plate in the empty tank for equilibration for 30 min, then place it in the tank containing the developing solvent. Develop using the inclined upward method for approximately 18.0 cm. Remove the thin-layer chromatography plate from the developing tank, allow it to air dry, and observe it under a 254 nm UV lamp. Record any suspicious spots (dark spots) with a pencil. The results are as follows: Figure 4 As shown. Then, the anisaldehyde colorimetric reagent is evenly sprayed onto one of the thin-layer chromatography plates, removed and dried, and placed on a constant-temperature heating plate (105℃) until the spots are clearly visible. The colorimetric results are immediately observed visually. Figure 5 As shown. Next, evenly spray the tetrazolium blue reagent onto another thin-layer chromatography plate, and immediately observe the colorimetric results visually. Figure 6 As shown.

[0033] ③ Qualitative judgment Under ultraviolet light, if no dark spots are observed, the cosmetic is considered negative if no glucocorticoids are detected. Alternatively, if obvious dark spots are present, but no characteristic spots appear after development with tetrazolium blue and anisaldehyde, the cosmetic is considered negative. If characteristic spots appear after development with tetrazolium blue or anisaldehyde, the cosmetic is considered positive if glucocorticoids are present. Figure 4-6As shown, the qualitative results indicate that channel 1 is the characteristic control group, channels 2 and 3 are positive samples, and channel 4 is a negative sample.

[0034] ④ Qualitative identification First, the measured ratio shift R of the positive sample was... fy实测 Tetrazolium blue colorimetric characteristics F 1y实测 and the colorimetric characteristics of anisaldehyde F 2y实测 Measured shift value R compared to the characteristic control group fb实测 Tetrazolium blue colorimetric characteristics F 1b实测 and the colorimetric characteristics of anisaldehyde F 2b实测 Perform a corresponding comparison. For example... Figure 4-6 As shown, the qualitative identification result is that R exists in the characteristic control group. fb实测 With R fy实测 F 1b实测 With F 1y实测、 F 2b实测 With F 2y实测 Consistent glucocorticoid levels were observed in positive samples from channel 2, with levels R detected at points 1 and 7 in the characteristic control group. f The positive sample from channel 2 contained glucocorticoids with consistent colorimetric characteristics, namely glucocorticoids number 11 and 85 from the characteristic control group. These could be directly identified as prednisolone and hydrocortisone acetate propyl ester, and this result was confirmed by mass spectrometry.

[0035] Example 2: Example 2 is basically the same as Example 1, except that the cosmetics to be tested are different. Therefore, thin-layer chromatography was performed on the same thin-layer chromatography plate, and after color development with tetrazolium blue and anisaldehyde, the 254nm color development results of each glucocorticoid in the characteristic control group and each cosmetic test solution are as follows: Figure 7 As shown, the colorimetric results of anisaldehyde are as follows: Figure 8 As shown, the results of the tetrazolium blue staining are as follows: Figure 9 As shown.

[0036] like Figure 7-9 As shown, the qualitative judgment results are: channel 1 is the characteristic control group, channel 2 is the positive sample, and channel 3 is the negative sample.

[0037] From such Figure 7-9 It can be seen that the substance detected in the positive sample appears between point 5 and point 6 of the characteristic control group, R fy实测 =0.45, meaning there was no R in the characteristic control group. fy实测 Consistent glucocorticoids. Therefore, qualitative identification first determines the characteristic control group that is consistent with R. fy实测 R of two adjacent points fb实测 With R in the database f Whether they are consistent, i.e., R at points 5 and 6 of the characteristic control group.fb实测 With R in the database f Whether they are consistent, point 5 R fb实测 =0.40, point 6 R fb实测 =0.55. According to the database shown in Table 2, the glucocorticoid in point 5 is serial number 64, hydrocortisone acetate, R. f The value is 0.43; the glucocorticoid in point 6 is ancinonide, serial number 79 in the database, R. f It is 0.58, which is the 5th point R. fb实测 And point 6 R fb实测 Each corresponds to point R in the database. f And point 6 R f Inconsistent. Therefore, the values ​​of R in the characteristic control group were calculated separately. fy实测 R of two adjacent points fb实测 The corresponding R in the database f The ratios are 0.93 and 0.95, respectively. The average of the two values, 0.94, is taken as the calibration coefficient K for R. fy实测 Perform calibration to obtain R fy校准 =R fy实测 / K=0.45 / 0.94=0.48, so R fy校准 F 1y实测 and F 2y实测 Comparison with the database revealed that dexamethasone acetate, serial number 68, had the following R... f With R fy校准 F1 and F 1y实测 and F2 and F 2y实测 If all results are consistent, then the sample is determined to be a positive sample containing dexamethasone acetate, a result confirmed by mass spectrometry.

[0038] If, during testing, the glucocorticoid present in a positive sample is determined to be a glucocorticoid not found in the database, it can be qualitatively identified by mass spectrometry.

Claims

1. A thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics, characterized in that... Follow these steps: Step 1. Determine the initial translocation value, tetrazolium blue, and anisaldehyde colorimetric characteristics of known glucocorticoids under the same thin-layer chromatography conditions. Group the known glucocorticoids based on complete separation within the same developing distance. One group serves as a characteristic control group, which includes glucocorticoids with high colorimetric characteristic recognition and includes the two glucocorticoids with the lowest and highest initial translocation values. Establish the translocation value R for known glucocorticoids. f Databases of tetrazolium blue colorimetric characteristics F1 and anisaldehyde colorimetric characteristics F2; Step 2. Perform thin-layer chromatography on the same thin-layer chromatography plate with the mixed standard working solution of glucocorticoids in the characteristic control group and multiple cosmetic test solutions, and perform color development with tetrazolium blue and anisaldehyde to obtain the measured ratio shift value, tetrazolium blue color development characteristics and anisaldehyde color development characteristics of each glucocorticoid in the characteristic control group and each cosmetic test solution. Step 3. Qualitative analysis to determine whether the cosmetic sample solution is a negative or positive sample; Step 4. First, based on the measured ratio shift values, tetrazolium blue colorimetric characteristics, and anisaldehyde colorimetric characteristics of each glucocorticoid in the characteristic control group, then using the R data from the database... f Based on F1 and F2, positive samples are qualitatively identified.

2. The thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics according to claim 1, characterized in that... Step 4 is as follows: First, the measured ratio shift R of the positive sample was... fy实测 Tetrazolium blue colorimetric characteristics F 1y实测 and the colorimetric characteristics of anisaldehyde F 2y实测 Measured shift value R compared to the characteristic control group fb实测 Tetrazolium blue colorimetric characteristics F 1b实测 and the colorimetric characteristics of anisaldehyde F 2b实测 Perform a corresponding comparison; If the characteristic control group has R fb实测 With R fy实测 F 1b实测 With F 1y实测、 F 2b实测 With F 2y实测 If the glucocorticoids are all consistent, then it is determined to be the glucocorticoid present in the positive sample; If the characteristic control group has R fb实测 With R fy实测 Consistent but F 1b实测 With F 1y实测 or / and F 2b实测 With F 2y实测 Inconsistent glucocorticoids will lead to R fy实测 F 1y实测 and F 2y实测 With R database f Compare F1 and F2, and if R is found in the database... f With R fy实测 F1 and F 1y实测 and F2 and F 2y实测 If all glucocorticoids are consistent, then it is determined that it is a glucocorticoid present in the positive sample; otherwise, the glucocorticoid present in the positive sample is determined to be a glucocorticoid not contained in the database. If the characteristic control group does not contain R fy实测 Consistent glucocorticoids, firstly, determine the characteristic control group with R fy实测 R of two adjacent points fb实测 With R in the database f If they match, then directly assign R. fy实测 F 1y实测 and F 2y实测 With R database f Compare F1 and F2, and if R is found in the database... f With R fy实测 F1 and F 1y实测 and F2 and F 2y实测 If the glucocorticoids present in the control group are consistent with those in the database, then the cosmetic is identified as a glucocorticoid present in the cosmetic; otherwise, the glucocorticoid present in the cosmetic is identified as a glucocorticoid not found in the database. fy实测 R of two adjacent points fb实测 With R in the database f If they are inconsistent, calculate the values ​​of R in the characteristic control group respectively. fy实测 R of two adjacent points fb实测 The corresponding R in the database f The ratio of the two ratios is then used as the calibration coefficient K for R. fy实测 Calibration was performed to obtain R. fy校准 =R fy实测 / K, will R fy校准 F 1y实测 and F 2y实测 Compare with the database, for example, if R is in the database. f With R fy校准 F1 and F 1y实测 and F2 and F 2y实测 If all glucocorticoids are consistent, then the sample is considered to contain glucocorticoids present in the positive sample; otherwise, the glucocorticoids present in the positive sample are considered to be glucocorticoids not found in the database.

3. The thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics according to claim 1 or 2, characterized in that... Step 1 is described in detail as follows: First, several known glucocorticoids were subjected to single-point thin-layer chromatography, tetrazolium blue staining, and anisaldehyde staining to preliminarily determine the initial ratio shift value (R) of the known glucocorticoids. f初测 Tetrazolium blue colorimetric characteristics F 1初测 and the colorimetric characteristics of anisaldehyde F 2初测 Known glucocorticoids were grouped to obtain N groups of glucocorticoids, including a characteristic control group; it was determined whether N was greater than the maximum number of spots on a single thin-layer chromatography plate. If not, R was used as the maximum number of spots. f初测 F 1初测 and F 2初测 As R respectively f Databases were established for F1 and F2. Each group of glucocorticoids was prepared into a mixed standard working solution. The mixed standard working solution of the characteristic control group glucocorticoids was then subjected to thin-layer chromatography (TLC) on the same TLC plate, with tetrazolium blue and anisaldehyde staining. The measured ratio shift values ​​(Rf) of all glucocorticoids provided by multiple TLC plates were obtained. f实测 Tetrazolium blue colorimetric characteristics F 1实测 and the colorimetric characteristics of anisaldehyde F 2实测 If different thin-layer chromatography plates show characteristic control group glucocorticoid R f实测 All were consistent, based on the measured ratio shift value R of all glucocorticoids. f实测 F 1实测 and F 2实测 As R respectively f Establish databases for F1 and F2; otherwise, use one of the thin-layer chromatography plates as a standard plate and use the characteristic control group glucocorticoid R obtained from the standard plate. f实测 R was obtained by calibrating the ratio shift values ​​of glucocorticoids on other thin-layer chromatography plates. f校准 R of glucocorticoids on standard plates f实测 R obtained from other thin-layer chromatography plates f校准 As R f F 1实测 and F 2实测 Create databases as F1 and F2 respectively.

4. The thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics according to claim 3, characterized in that... Step 3 is as follows: Observe the thin-layer chromatography spectrum under ultraviolet light. If there are no dark spots, it can be determined that no glucocorticoids are detected in the cosmetic, i.e., it is a negative sample. If there are obvious dark spots, and no characteristic spots are found after color development with tetrazolium blue and anisaldehyde, it can be determined that the cosmetic is a negative sample. If characteristic spots appear after color development with tetrazolium blue or anisaldehyde, it can be determined that glucocorticoids are present in the cosmetic, i.e., it is a positive sample.

5. The thin-layer chromatography method for high-throughput screening of glucocorticoids in cosmetics according to claim 4, characterized in that... The thin-layer chromatography conditions are as follows: 1) Thin-layer chromatography plate: High-efficiency silica gel F254, activated in an oven at 110℃ for 1 hour before use, then placed in a desiccator to room temperature for later use; 2) Developing solvent: ethyl acetate + n-hexane, with a volume ratio of ethyl acetate to n-hexane of 11:10; 3) Tetrazolium blue colorimetric reagent: Weigh 20 mg of tetrazolium blue and dissolve it in 10 mL of methanol, then add 10 mL of 12% NaOH methanol solution. Prepare fresh before use. 4) Anisaldehyde colorimetric reagent: Add 5 mL of concentrated sulfuric acid and 1 mL of anhydrous acetic acid to 90 mL of anhydrous ethanol in an ice-water bath. After mixing and cooling, add 5 mL of anisaldehyde and mix well again. Set aside for use.

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