A rapid detection method for Sudan red in chili products
The extraction, purification and separation steps of Sudan Red are optimized by thin-layer chromatography, and the time-consuming and labor-intensive detection of Sudan Red in chili products is solved, achieving rapid and accurate identification of Sudan Red dye.
Patent Information
- Application Number
- CN202411174169.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-26
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2044-08-26
AI Technical Summary
The detection method of Sudan Red in the prior art in chili products is time-consuming, expensive, poor reproducible and high detection limits, making it difficult to achieve fast and accurate detection.
The extraction, enrichment and separation of Sudan Red was performed by thin-layer chromatography by extraction of ethyl acetate-acetonitrile mixed solution, purification of silica gel and petroleum ether-ethyl acetate-acetonitrile-water-pyridine mixed solution developer, and each step was optimized to achieve rapid and effective detection.
It has achieved rapid and effective identification of Sudan Red dye in chili products, can effectively remove impurity interference, and achieve separation and accurate detection of four Sudan Red products.
Smart Images
Figure CN119043858B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food detection and relates to a rapid detection method for Sudan red in pepper products. Background Art
[0002] Sudan Red, also known as oil-soluble red and solvent red, is a type of artificial synthetic chemical dye with phenylazonaphthol as the main group. It is dark red in color, easily soluble in organic solvents, and difficult to dissolve in water. It is often used as a color enhancer for oil wax, motor oil, etc., and a brightener for leather, flooring, etc.
[0003] Animal experiments have shown that Sudan Red's primary metabolic pathway is oral ingestion, followed by metabolism by gastrointestinal microbial reductases, liver microsomes, and cytoplasmic reductases, producing the corresponding amines. Sudan Red's primary metabolites are aniline, naphthol, and their derivatives, all of which are carcinogenic and mutagenic. Aniline, primarily used as a raw material for dyes, pharmaceuticals, and resins, can affect the liver through skin contact or oral ingestion, causing toxic liver disease. It can also oxidize hemoglobin, leading to hypoxia and damage to the central nervous system and cardiovascular system, resulting in methemoglobinemia. Naphthol (carcinogenic, teratogenic, and mutagenic) is used as a chemical raw material for pharmaceuticals, dyes, and rubber. Contact with localized skin can cause peeling and even permanent pigmentation. It is also a strong irritant to the eyes, skin, and mucous membranes.
[0004] As early as 1995, the European Union explicitly classified it as a Class III carcinogen, prohibiting its addition to food, based on in vivo and in vitro animal studies. However, due to its vibrant coloring, excellent dyeing properties, stable chemical properties, and long-lasting color retention, some unscrupulous companies, seeking profit, have added it to foods to maintain a bright red color for extended periods. This is particularly prevalent in chili products. To ensure food safety, increased market oversight and related testing and research are crucial.
[0005] Conventional methods for determining Sudan Red in food primarily rely on liquid chromatography, gas chromatography, and their combination with mass spectrometry. Thin-layer chromatography, spectrophotometry, and polarography are also available. Traditional Sudan Red detection methods are time-consuming, labor-intensive, and expensive, with poor reproducibility and high detection limits. Therefore, developing a rapid, efficient, accurate, and simple-to-use method for detecting Sudan Red in food is highly desirable. Summary of the Invention
[0006] The main purpose of the present invention is to provide a rapid detection method for Sudan Red in chili products.
[0007] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:
[0008] A method for rapid detection of Sudan Red in chili products, wherein the Sudan Red is Sudan Red I, Sudan Red II, Sudan Red III, or Sudan Red IV; the detection method is thin-layer chromatography, specifically comprising the following steps:
[0009] Pretreatment of samples to be tested:
[0010] A. Extraction
[0011] Weigh the pepper product to be tested and place it in a conical flask. Add the extract at a solid-liquid ratio of 1:4.5-7.5 g / mL. Perform ultrasonic extraction, filter, and take the filtrate for later use. The extract is a mixed solution of ethyl acetate and acetonitrile.
[0012] B. Purification
[0013] The filtrate was concentrated to 2-3 mL, loaded onto a silica gel column, eluted with an elution solvent, and the eluate was collected and blown with nitrogen to 2 mL as the sample to be tested; wherein the eluate was a mixed solution of petroleum ether and n-hexane.
[0014] Thin layer chromatography
[0015] Spot the sample to be tested and the standard solution on the same silica gel G thin layer plate, use a mixed solution of petroleum ether-ethyl acetate-acetonitrile-water upper layer solution and pyridine as the developing agent, develop, remove, dry, and inspect under visible light.
[0016] Furthermore, the extract in the above-mentioned sample pretreatment extraction step is a mixed solution of ethyl acetate and acetonitrile (12:3, v / v).
[0017] Furthermore, the elution solvent in the above-mentioned pre-treatment and purification step of the sample to be tested is a mixed solution of petroleum ether and n-hexane (8:2, v / v).
[0018] Furthermore, in the above-mentioned thin layer chromatography detection step, the developing solvent is a mixed solution of the upper layer solution of petroleum ether-ethyl acetate-acetonitrile-water (95:5:15:85, / v / v / v) and pyridine.
[0019] Furthermore, the volume ratio of the upper layer solution to pyridine in the above-mentioned developing agent is 10:0.3.
[0020] In the above detection method, the preparation method of the standard solution is:
[0021] Weigh 100 mg each of Sudan Red I, Sudan Red II, Sudan Red III, and Sudan Red IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions; draw 1 mL of Sudan Red I, Sudan Red II, Sudan Red III, and Sudan Red IV standard stock solutions respectively, place them in a 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare 10 μg / mL standard solutions.
[0022] The present invention has the following beneficial effects:
[0023] Sudan Red is a non-ionic fat-soluble substance, which often causes a large amount of interfering components to be mixed into the extract during extraction. When Sudan Red is enriched in low-residue products, the endogenous substances in the sample are first concentrated, resulting in more serious interference.
[0024] The present invention optimizes Sudan red extraction, purification, and thin-layer chromatography detection. A mixed solution of ethyl acetate and acetonitrile is selected as an extraction solvent, thereby achieving effective extraction of Sudan red dye. The extract is enriched and purified by a silica gel column, and the components and usage ratio of the eluent are screened and optimized, thereby achieving effective enrichment and purification of Sudan red dye. In thin-layer chromatography detection, the developing agent is further screened, and a developing solution simply compounded between traditional organic solvents is abandoned. A polar system formed between an organic solvent with low polarity and water with high polarity is creatively used, and pyridine is added as a developing agent, thereby achieving effective separation between the four Sudan red dyes and identification of Sudan red dyes.
[0025] The method of the present invention can effectively identify Sudan red dyes in chili products. For commercially available dry chili products or chili sauces such as chili sauce, impurities can be effectively removed to avoid interference during detection. It can also effectively separate four Sudan red products, thereby achieving rapid and effective identification and detection of Sudan red dyes in chili products. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 : Thin layer chromatogram of chili powder, from left to right are respectively standard solution, sample to be tested in Example 1 containing standard, and sample to be tested in Example 1;
[0027] Figure 2 : Thin layer chromatogram of chili powder, from left to right are standard solution, comparative example 1 test sample containing standard, comparative example 1 test sample;
[0028] Figure 3 : Chili powder thin layer chromatogram, from left to right are standard solution, containing the standard sample to be tested in Comparative Example 2, Comparative Example 2 test sample. DETAILED DESCRIPTION
[0029] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope of protection of the claims of this application.
[0030] Example 1
[0031] 1. Pretreatment of the sample to be tested:
[0032] A. Extraction
[0033] Weigh 10 g of commercially available chili powder into a conical flask, add 45 mL of a mixed solution of ethyl acetate and acetonitrile (12:3, v / v), sonicate for 30 min, filter, and set aside the filtrate;
[0034] B. Purification
[0035] The filtrate was concentrated to 2 mL and the concentrate was set aside; 20 g of silica gel was weighed and wet-packed on a column; the concentrate was loaded and eluted with a mixture of petroleum ether and n-hexane (8:2, v / v) as the elution solution; the eluate was collected and purged with nitrogen to about 2 mL, which was used as the sample to be tested;
[0036] 2. Preparation of standard solution:
[0037] Weigh 100 mg each of Sudan I, Sudan II, Sudan III, and Sudan IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions. Pipette 1 mL of each standard stock solution of Sudan I, Sudan II, Sudan III, and Sudan IV, place them in the same 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 10 μg / mL mixed standard solution.
[0038] 3. Thin layer chromatography
[0039] Spot the sample to be tested and the mixed standard solution onto the same silica gel G thin layer plate. Use a mixed solution of 10 mL of the upper layer solution of petroleum ether-ethyl acetate-acetonitrile-water (95:5:15:85, / v / v / v) and 0.3 mL of pyridine as the developing solvent. Develop, remove, dry, and examine under visible light.
[0040] Example 2
[0041] 1. Pretreatment of the sample to be tested:
[0042] A. Extraction
[0043] Weigh 10 g of commercial chili sauce into a conical flask, add 75 mL of a mixed solution of ethyl acetate and acetonitrile (12:3, v / v), sonicate for 30 min, filter, and set aside the filtrate;
[0044] B. Purification
[0045] The filtrate was concentrated to 3 mL and the concentrate was set aside; 30 g of silica gel was weighed and wet-packed on a column; the concentrate was loaded and eluted with a mixed solution of petroleum ether and n-hexane (8:2, v / v) as the elution solution; the eluate was collected and purged with nitrogen to about 2 mL, which was used as the sample to be tested;
[0046] 2. Preparation of standard solution:
[0047] Weigh 100 mg each of Sudan I, Sudan II, Sudan III, and Sudan IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions. Pipette 1 mL of each standard stock solution of Sudan I, Sudan II, Sudan III, and Sudan IV, place them in the same 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 10 μg / mL mixed standard solution.
[0048] 3. Thin layer chromatography
[0049] Spot the sample to be tested and the mixed standard solution onto the same silica gel G thin layer plate. Use a mixed solution of 10 mL of the upper layer solution of petroleum ether-ethyl acetate-acetonitrile-water (95:5:15:85, / v / v / v) and 0.3 mL of pyridine as the developing solvent. Develop, remove, dry, and examine under visible light.
[0050] Example 3
[0051] 1. Pretreatment of the sample to be tested:
[0052] A. Extraction
[0053] Weigh 10 g of commercially available chili powder into a conical flask, add 60 mL of a mixture of ethyl acetate and acetonitrile (12:3, v / v), then add Sudan I, Sudan II, Sudan III, and Sudan IV standards at a concentration of 10 μg / mL. Ultrasonicate for 30 min, filter, and set aside the filtrate.
[0054] B. Purification
[0055] The filtrate was concentrated to 2 mL and the concentrate was set aside; 30 g of silica gel was weighed and wet-packed on a column; the concentrate was loaded and eluted with a mixture of petroleum ether and n-hexane (8:2, v / v) as the elution solution; the eluate was collected and purged with nitrogen to about 2 mL, which was used as the spiked sample;
[0056] 2. Preparation of standard solution:
[0057] Weigh 100 mg each of Sudan I, Sudan II, Sudan III, and Sudan IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions. Pipette 1 mL of each standard stock solution of Sudan I, Sudan II, Sudan III, and Sudan IV, place them in the same 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 10 μg / mL mixed standard solution.
[0058] 3. Thin layer chromatography
[0059] Spot the sample to be tested and the mixed standard solution onto the same silica gel G thin layer plate. Use a mixed solution of 10 mL of the upper layer solution of petroleum ether-ethyl acetate-acetonitrile-water (95:5:15:85, / v / v / v) and 0.3 mL of pyridine as the developing solvent. Develop, remove, dry, and examine under visible light.
[0060] Comparative Example 1
[0061] 1. Pretreatment of the sample to be tested:
[0062] Weigh 10 g of commercially available chili powder and place it in a conical flask. Add 45 mL of a mixed solution of ethyl acetate and acetonitrile (12:3, v / v). Ultrasonicate for 30 min, filter, and concentrate the filtrate to 2 mL to serve as the test sample.
[0063] 2. Preparation of standard solution:
[0064] Weigh 100 mg each of Sudan I, Sudan II, Sudan III, and Sudan IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions. Pipette 1 mL of each standard stock solution of Sudan I, Sudan II, Sudan III, and Sudan IV, place them in the same 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 10 μg / mL mixed standard solution.
[0065] 3. Thin layer chromatography
[0066] Spot the sample to be tested and the mixed standard solution on the same silica gel G thin layer plate, use a mixed solution of petroleum ether-ethyl acetate-acetonitrile-water (85:15:15:85, / v / v / v) as the developing solvent, develop, remove, dry, and inspect under visible light.
[0067] Comparative Example 2
[0068] Pretreatment of samples to be tested:
[0069] A. Extraction
[0070] Weigh 10 g of commercially available chili powder into a conical flask, add 45 mL of a mixed solution of ethyl acetate and acetonitrile (12:3, v / v), sonicate for 30 min, filter, and set aside the filtrate;
[0071] B. Purification
[0072] The filtrate was concentrated to 2 mL and the concentrate was set aside; 20 g of silica gel was weighed and wet-packed on a column; the concentrate was loaded and eluted with a mixture of petroleum ether and n-hexane (8:2, v / v) as the elution solution; the eluate was collected and purged with nitrogen to about 2 mL, which was used as the sample to be tested;
[0073] Preparation of standard solution:
[0074] Weigh 100 mg each of Sudan I, Sudan II, Sudan III, and Sudan IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions. Pipette 1 mL of each standard stock solution of Sudan I, Sudan II, Sudan III, and Sudan IV, place them in the same 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 10 μg / mL mixed standard solution.
[0075] Thin layer chromatography
[0076] Spot the sample to be tested and the mixed standard solution on the same silica gel G thin layer plate, use a mixed solution of petroleum ether-acetone-ethyl acetate (100:3:4, / v / v / v) as the developing solvent, develop, remove, dry, and examine under visible light.
[0077] Method validation
[0078] The samples to be tested and the standard solutions prepared in Example 1, Comparative Example 1 and Comparative Example 2 were subjected to thin layer chromatography. 0.5 mL of the standard solution was added to the sample to be tested and mixed thoroughly, and then used as the control solution.
[0079] since Figure 1 It can be seen that the test sample prepared by the method of the present invention contains fewer impurities and will not interfere with the detection of Sudan red. When the developing agent of the present invention is used for thin layer chromatography development, the four Sudan red dyes can be effectively separated, and the Sudan red dyes can be quickly and effectively identified.
[0080] since Figure 2 It can be seen that when the sample to be tested is prepared and the developing agent is used for thin layer chromatography detection in the manner described in Comparative Example 1, there are many impurities in the sample, and the four Sudan red dyes are not effectively separated, and effective identification cannot be achieved.
[0081] since Figure 3 It can be seen that when the developing agent in Comparative Example 2 is used for thin layer chromatography detection, the four Sudan red dyes cannot be effectively separated and effective identification cannot be achieved.
Claims
1. A rapid detection method for Sudan Red in chili products, characterized in that: The Sudan Red is Sudan Red I, Sudan Red II, Sudan Red III, and Sudan Red IV; the detection method is thin layer chromatography; the method comprises the following steps: Pretreatment of samples to be tested: A. Extraction Weigh the pepper product to be tested and place it in a conical flask. Add the extract at a solid-liquid ratio of 1:(4.5-7.5) g / mL, perform ultrasonic extraction, filter, and take the filtrate for later use. The extract is a mixed solution of ethyl acetate and acetonitrile in a volume ratio of 12:
3. B. Purification The filtrate was concentrated to 2-3 mL, loaded onto a silica gel column, eluted with an elution solvent, and the eluate was collected and purged with nitrogen to 2 mL as the sample to be tested; wherein the eluate was a mixed solution of petroleum ether and n-hexane in a volume ratio of 8:2; Thin layer chromatography detection: Spot the sample and standard solution onto the same silica gel G thin layer plate, use a mixed solution of petroleum ether-ethyl acetate-acetonitrile-water upper layer solution and pyridine as the developing solvent, develop, remove, dry, and inspect under visible light; the volume ratio of petroleum ether, ethyl acetate, acetonitrile, and water in the developing solvent is 95:5:15:85, and the volume ratio of the upper layer solution to pyridine is 10:0.
3.
2. The method according to claim 1, characterized in that The preparation method of the standard solution is: Weigh 100 mg each of Sudan Red I, Sudan Red II, Sudan Red III, and Sudan Red IV, dissolve them in acetonitrile, and prepare 1 mg / mL standard stock solutions; draw 1 mL of Sudan Red I, Sudan Red II, Sudan Red III, and Sudan Red IV standard stock solutions respectively, place them in a 100 mL volumetric flask, and dilute to volume with acetonitrile to prepare 10 μg / mL standard solutions.