Method for detecting aflatoxin B1 in vegetable oil based on concentrated sulfuric acid low-temperature sulfonation purification

Abstract

The invention relates to the technical field of food safety detection, and discloses a method for detecting aflatoxin B1 in vegetable oil based on concentrated sulfuric acid low-temperature sulfonation purification, which comprises the following steps: dissolving a vegetable oil sample by using n-hexane, slowly adding concentrated sulfuric acid under the strict control of ice-water bath (0-4 DEG C) to carry out sulfonation purification reaction, the aflatoxin B1 is protected from being degraded while the impurities are effectively removed; and after derivatization, detecting by using a liquid chromatography-fluorescence detector. The innovation of the invention lies in that a strict low-temperature reaction system is introduced, and the technical problem that a concentrated sulfuric acid sulfonation method may damage a target analyte is solved. According to the method, the detection limit can reach 0.05 mu g / kg, the quantification limit can reach 0.15 mu g / kg, the recovery rate is high (85-105%), the relative standard deviation is smaller than 5%, the cost is low, and the method is remarkably superior to an existing detection method.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and to a method for detecting harmful substances in vegetable oils, specifically a method for detecting aflatoxin B1 in vegetable oils based on low-temperature controlled hexane dissolution and concentrated sulfuric acid sulfonation purification. Background Technology

[0002] Aflatoxins are secondary metabolites produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. Among them, aflatoxin B1 (AFB1) is the most toxic and carcinogenic. AFB1 has been classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC), and long-term consumption of food containing AFB1 increases the risk of diseases such as liver cancer. Vegetable oil raw materials (such as peanuts, corn, and cottonseed) are easily contaminated with aflatoxins during planting, storage, and processing; therefore, the detection of AFB1 in vegetable oils is crucial.

[0003] Currently, common methods for detecting AFB1 in vegetable oils include enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS / MS). However, vegetable oils have complex matrices, containing various interfering substances such as triglycerides, fatty acids, pigments, and phospholipids. Direct detection is often severely affected by matrix interference, leading to poor accuracy. Existing technologies mainly employ sample pretreatment methods such as immunoaffinity column purification, solid-phase extraction purification, and liquid-liquid partitioning purification, as exemplified by the rapid quantitative detection method for aflatoxin B1 in vegetable oils disclosed in application number 202111482330.5. Immunoaffinity column purification offers good results but is costly; solid-phase extraction is cumbersome and has unstable recovery rates; while liquid-liquid partitioning is simple, its purification effect is unsatisfactory. Furthermore, the extraction solvents for AFB1 in vegetable oils often include acetonitrile, methanol-water solution, or chloroform, but these solvents have poor solubility in oils, requiring complex extraction steps.

[0004] Sulfonation is a purification method that uses concentrated sulfuric acid to react with impurities in a sample, thereby removing interferences. Concentrated sulfuric acid can carbonize organic matter, sulfonate unsaturated fatty acids, and remove pigments, but it is somewhat destructive to AFB1 at room temperature. Traditional sulfonation methods are mostly used for detoxification of edible oils, rather than for pre-detection purification, because the harsh reaction conditions can easily destroy the target analyte, leading to detection failure. Hexane is a commonly used organic solvent with good solubility in vegetable oils and exhibits good separation with concentrated sulfuric acid, facilitating subsequent separation. However, there are currently no reports on combining low-temperature control, hexane dissolution, and concentrated sulfuric acid sulfonation for pretreatment in the detection of AFB1 in vegetable oils.

[0005] Therefore, developing a highly efficient purification method that can suppress the destructive effects of concentrated sulfuric acid while retaining the target analyte is of great significance. This invention addresses the shortcomings of existing technologies by innovatively introducing a strict low-temperature reaction control system, organically combining hexane dissolution with concentrated sulfuric acid sulfonation, and establishing a novel, reliable, and low-cost method for detecting AFB1 in vegetable oils. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting AFB1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for detecting AFB1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid includes the following steps: S1: Sample pretreatment: Dissolve the vegetable oil sample in n-hexane to obtain a homogeneous sample solution; S2: Low-temperature sulfonation purification: The sample solution obtained in step S1 is placed in an ice-water bath to cool, and then concentrated sulfuric acid is slowly added dropwise to carry out the sulfonation reaction at a low temperature; S3: Extraction and separation: The reaction system after step S2 is vortexed at high speed to fully sulfonate it, and then placed in an ice-water bath for cooling treatment. S4: Derivatization treatment: Take the supernatant (n-hexane layer) obtained in step S3 into a derivatization bottle, blow it with nitrogen until it is almost dry, add the derivatization reagent, vortex to mix, and blow it with nitrogen until it is almost dry after the derivatization reaction; S5: Liquid Chromatography Analysis: The sample derivatized in step S4 was reconstituted with acetonitrile-methanol-water solution and detected by liquid chromatography-fluorescence detector; S6: Result Calculation: Calculate the AFB1 content in vegetable oil based on the standard curve.

[0008] Preferably, the volume ratio of n-hexane to vegetable oil sample in step S1 is 2~5:1.

[0009] Preferably, in step S2, the temperature of the ice-water bath is 0-4℃, and the cooling time is at least 5 minutes to ensure that the temperature of the sample solution drops to 0-4℃; the dropping rate of concentrated sulfuric acid is 2.0-4.0 mL / min, and 4 mL of concentrated sulfuric acid is added.

[0010] Preferably, the sulfonation reaction temperature in step S2 is maintained at 0-4°C.

[0011] Preferably, in step S3, after the concentrated sulfuric acid has been added, the mixture is immediately vortexed for 20 seconds. After vortexing, it is immediately placed back in an ice-water bath and left to stand for 5 minutes to allow the sulfonation reaction to be complete and the mixture to separate into layers.

[0012] Preferably, the derivatizing reagents in step S4 are n-hexane and trifluoroacetic acid, with a volume ratio of n-hexane to trifluoroacetic acid of 2:1, a derivatization temperature of 60~70℃, and a derivatization time of 10-20 minutes.

[0013] Preferably, in step S5, the volume ratio of acetonitrile-methanol-water solution is 2:2:6, and the amount added is 1 mL.

[0014] Preferably, the liquid chromatography conditions in step S5 are as follows: Chromatographic column: C18 reversed-phase column; Mobile phase: Acetonitrile-methanol-water; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 20 μL; Fluorescence detector parameters: excitation wavelength 360 nm, emission wavelength 440 nm.

[0015] Preferably, the volume ratio of acetonitrile-methanol-water in the mobile phase is 2:2:6.

[0016] This invention, by adopting the above technical solutions, has significant technical effects: (1) This invention innovatively introduces low temperature control. Through a strict 0-4℃ ice-water bath environment, the oxidation and destruction of AFB1 by concentrated sulfuric acid is greatly suppressed, which successfully solves the problem of applying the sulfonation method to the detection of trace toxins and achieves the dual purpose of efficient purification and target protection.

[0017] (2) The present invention has high sensitivity, with a method detection limit (LOD) of up to 0.05 μg / kg and a quantitation limit (LOQ) of up to 0.15 μg / kg, which are far below the limits specified in the national standard (10.0 μg / kg).

[0018] (3) The present invention has high accuracy, with an average recovery rate between 85% and 105% and a relative standard deviation (RSD) of less than 5%, which meets the requirements for trace analysis.

[0019] (4) The present invention has high-efficiency purification by using hexane dissolution combined with low-temperature concentrated sulfuric acid sulfonation, which effectively removes interfering substances such as fatty acids and pigments from vegetable oil and reduces matrix effect.

[0020] (5) The present invention is easy to operate and does not require expensive immunoaffinity columns or solid phase extraction columns in the pretreatment process, which greatly reduces the detection cost.

[0021] (6) The present invention is environmentally friendly. Compared with the traditional method which uses a large amount of organic solvents, the solvent usage of the present invention is reduced by more than 30%. Attached Figure Description

[0022] Figure 1 This is a flowchart of the detection method of the present invention.

[0023] Figure 2 This is the standard chromatogram of AFB1 in Embodiment 1 of the present invention.

[0024] Figure 3 This is a chromatogram of the peanut oil sample spiked in Example 1 of the present invention.

[0025] Figure 4 This describes the effect of different n-hexane dosages on the recovery rate in Example 2 of this invention.

[0026] Figure 5 This describes the effect of different sulfonation times on the recovery rate in Example 3 of the present invention.

[0027] Figure 6 This is a comparative data graph showing the effect of low temperature and room temperature operation on the recovery rate of AFB1 in Example 6 of the present invention. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0029] Example 1: Detection of AFB1 in peanut oil 1.1 Instruments and Reagents High-performance liquid chromatograph (equipped with a fluorescence detector); vortex mixer; nitrogen evaporator; analytical balance.

[0030] n-Hexane (chromatographic grade); concentrated sulfuric acid (analytical grade); acetonitrile (chromatographic grade); methanol (chromatographic grade); trifluoroacetic acid (derivative); AFB1 standard solution (100 μg / mL).

[0031] 1.2 Sample Pretreatment Accurately weigh 2.0 g of peanut oil sample into a 25 mL centrifuge tube, add 10 mL of n-hexane, and vortex for 5 min to completely dissolve the sample. Immerse the centrifuge tube in an ice-water bath (0-4℃) for at least 5 minutes. Using an adjustable pipette, slowly add 4.0 mL of concentrated sulfuric acid at a rate of 4.0 mL / min, keeping the centrifuge tube in the ice-water bath throughout the addition process. After the addition is complete, immediately vortex for 20 seconds, and then immediately place the tube back into the ice-water mixture for sulfonation (5 min). Allow it to stand and separate into layers, maintaining the temperature at 0-4℃ throughout the sulfonation process.

[0032] 1.3 Derivatization Process Take 5 mL of the supernatant (hexane layer) into a derivatization flask, blow it with nitrogen until nearly dry, add 0.4 mL of hexane and 0.2 mL of trifluoroacetic acid derivatization reagent, vortex to mix, react at 65 °C for 15 min, blow it with nitrogen until nearly dry, redissolve it with 1 mL of acetonitrile-methanol-water (20:20:60, v / v / v), filter it through a 0.22 μm organic filter membrane into a sample vial, and wait for detection.

[0033] 1.4 Liquid Chromatography Analysis Chromatographic conditions: C18 reversed-phase column (150 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-methanol-water (20:20:60, v / v / v); flow rate: 1.0 mL / min, isocratic elution; column temperature: 35℃; injection volume: 20 μL; fluorescence detector parameters: excitation wavelength 360 nm, emission wavelength 440 nm.

[0034] 1.5 Plotting Standard Curves AFB1 standard solutions of 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 μg / L were accurately prepared, and derivatization and determination were performed under the above conditions. Linear regression was performed on peak area (Y) against concentration (X, μg / L) to obtain the standard curve equation: Y = 4256.8X + 12.5, with a correlation coefficient R² = 0.9998. The chromatogram of the standard sample at a concentration of 5.0 μg / L is shown below. Figure 2 As shown.

[0035] 1.6 Spiked Recycling Spiking recovery tests were conducted on blank peanut oil samples at three concentration levels (0.5, 1.0, and 5.0 μg / kg). Each concentration level was measured in parallel six times. The results are shown in Table 1.

[0036] Table 1. Spiked recovery results of AFB1 in peanut oil (n=6) Spike level (μg / kg) Average measured value (μg / kg) Average recovery rate (%) RSD (%) 0.5 0.43 86 4.2 1 0.92 92 3.8 5 4.88 97.6 2.5 The results showed that the recovery rate of this method was between 86.0% and 97.6%, and the RSD was between 2.5% and 4.2%, meeting the requirements for trace analysis. The limit of detection (LOD, S / N=3) was 0.05 μg / kg, and the limit of quantitation (LOQ, S / N=10) was 0.15 μg / kg.

[0037] 1.7 Results of peanut oil sample analysis The results showed that the AFB1 content in peanut oil was 4.9 μg / kg.

[0038] Example 2: Optimization of n-hexane dosage To optimize the hexane dosage, while keeping other conditions constant, the volume ratio of hexane to vegetable oil samples was varied (1:1, 2:1, 3:1, 4:1, 5:1), and spiked recovery tests were conducted (spiking level 5.0 μg / kg). The results are shown in [Figure number missing]. Figure 4 .

[0039] The results showed that when the volume ratio of hexane to vegetable oil was less than 2:1, the amount of supernatant was small, making subsequent operations more difficult; when the ratio was greater than 3:1, the recovery rate tended to stabilize. Considering that excessive solvent volume would increase concentration time and solvent consumption, a volume ratio of 5:1 was chosen as the most suitable.

[0040] Example 3: Optimization of sulfonation time With other conditions kept constant, sulfonation time was varied (1, 3, 5, 7, 10 min), and spiked recovery tests were conducted (spiking level 1.0 μg / kg). The results are shown in [Figure number missing]. Figure 5 .

[0041] The results showed that when the sulfonation time was less than 3 min, the sulfonation reaction was incomplete and the impurities were not thoroughly removed; when the sulfonation time was greater than 5 min, the recovery rate tended to stabilize; however, excessively long sulfonation times (>10 min) may lead to the decomposition of AFB1 and a decrease in the recovery rate. Therefore, 5 min was selected as the optimal sulfonation time.

[0042] Example 4: Application of different vegetable oil matrices To verify the applicability of this method, five common vegetable oils—peanut oil, soybean oil, corn oil, sunflower oil, and olive oil—were selected for spiked recovery tests (spiking level 5.0 μg / kg). The results are shown in Table 2.

[0043] Table 2. Spiked recovery results of AFB1 in different vegetable oils (n=6) Types of vegetable oils Average recovery rate (%) RSD (%) Peanut oil 92 3.8 soybean oil 88.5 4.2 corn oil 90.2 3.5 Sunflower seed oil 93.1 4 olive oil 94.3 3.2 The results showed that the method is applicable to vegetable oils in different matrices, with a recovery rate between 88.5% and 94.3% and an RSD of less than 5%, indicating that the method has good applicability.

[0044] Example 5: Comparison with national standard method This method was compared with the second method of the national standard GB 5009.22-2016 "Determination of Aflatoxin B and G in Food". The AFB1 content in 10 vegetable oil samples was detected, and the results are shown in Table 3.

[0045] Table 3 Comparison of results obtained using this method and the national standard method (n=10) Sample number This method (μg / kg) National standard method (μg / kg) Relative deviation (%) 1 2.35 2.41 2.5 2 ND ND - 3 5.68 5.72 0.7 4 0.89 0.93 4.4 5 1.24 1.29 3.9 6 ND ND - 7 3.56 3.61 1.4 8 7.92 7.98 0.75 9 0.56 0.61 8.6 10 4.13 4.09 1.0 Note: ND indicates not detected.

[0046] The results showed that there was no significant difference in the determination results between the two methods, but the pretreatment of this method was simpler and the cost was lower.

[0047] Example 6: Comparative Experiment of Low Temperature Operation and Room Temperature Operation (Key Verification Example) 6.1 Experimental Methods Two spiked peanut oil samples from the same source (spiking level of 1.0 μg / kg) were processed in the following two ways: Experimental group: The sulfonation purification operation was carried out in an ice-water bath at 0-4℃, strictly following the requirements of this invention.

[0048] Control group: All other steps were exactly the same, except that concentrated sulfuric acid was added and sulfonation was performed at room temperature (25°C). Subsequent steps were identical, and the peak area of ​​AFB1 was determined by HPLC-FLD to calculate the recovery rate.

[0049] 6.2 Results and Discussion The results are shown in the table below: Table 6. Effect of low temperature and room temperature operation on AFB1 recovery (n=6) Group reaction temperature Average recovery rate (%) RSD (%) Phenomenon description experimental group 0-4℃ 94.5 3.1 The solution showed clear layers; the n-hexane phase was almost colorless, but slightly darker than the control group. control group 25℃ <15.0 >25 The solution is dark brown, and the n-hexane phase is almost colorless. The results show that sulfonation at room temperature almost completely destroys AFB1, resulting in extremely low recovery and unreliable results (extremely high RSD). However, at low temperatures of 0-4℃, AFB1 is effectively protected, with high and stable recovery. This fully demonstrates that low-temperature control is an absolute prerequisite for the success of this method and is the core innovation of this invention.

[0050] The AFB1 detection method in vegetable oil established in this invention, based on low-temperature hexane dissolution and concentrated sulfuric acid sulfonation purification, is not only suitable for supervision and spot checks by food safety regulatory agencies, but also for raw material and product quality control by vegetable oil production enterprises. Furthermore, it can be extended to the detection of mycotoxins in other oily foods. With the increasing demands for food safety and the growing need for rapid, accurate, and low-cost detection methods, this invention has broad industrial application prospects and market potential.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid, characterized in that, Includes the following steps: S1: Sample pretreatment: Dissolve the vegetable oil sample in n-hexane to obtain a homogeneous sample solution; S2: Low-temperature sulfonation purification: The sample solution obtained in step S1 is placed in an ice-water bath to cool, and then concentrated sulfuric acid is slowly added dropwise to carry out the sulfonation reaction at a low temperature; S3: Extraction and separation: The reaction system after step S2 is vortexed at high speed to fully sulfonate it, and then placed in an ice-water bath for cooling treatment. S4: Derivatization treatment: Take the supernatant obtained in step S3 into a derivatization bottle, blow it with nitrogen until it is almost dry, add the derivatization reagent, vortex to mix, and blow it with nitrogen until it is almost dry after the derivatization reaction. S5: Liquid Chromatography Analysis: The sample derivatized in step S4 was reconstituted with acetonitrile-methanol-water solution and detected by liquid chromatography-fluorescence detector; S6: Result Calculation: Calculate the content of aflatoxin B1 in vegetable oil based on the standard curve.

2. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S1, the volume ratio of n-hexane to vegetable oil sample is 2~5:

1.

3. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S2, the temperature of the ice-water bath is 0-4℃, and the sample solution temperature is cooled for at least 5 minutes to ensure that the temperature drops to 0-4℃; the dropping rate of concentrated sulfuric acid is 2.0-4.0 mL / min, and 4 mL of concentrated sulfuric acid is added.

4. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S2, the sulfonation reaction temperature is maintained at 0-4℃.

5. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S3, after the concentrated sulfuric acid has been added, immediately vortex for 20 seconds. After vortexing, immediately place it back in an ice-water bath and let it stand for 5 minutes to allow the sulfonation reaction to be complete and the layers to separate.

6. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S4, the derivatization reagents are n-hexane and trifluoroacetic acid, with a volume ratio of n-hexane to trifluoroacetic acid of 2:

1. The derivatization temperature is 60~70℃ and the derivatization time is 10-20 minutes.

7. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 1, characterized in that, In step S5, the volume ratio of acetonitrile-methanol-water solution is 2:2:6, and the amount added is 1 mL.

8. A method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to any one of claims 1-7, characterized in that, The liquid chromatography conditions in step S5 are as follows: Chromatographic column: C18 reversed-phase column; Mobile phase: Acetonitrile-methanol-water; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 20 μL; Fluorescence detector parameters: excitation wavelength 360 nm, emission wavelength 440 nm.

9. The method for detecting aflatoxin B1 in vegetable oil based on low-temperature sulfonation purification with concentrated sulfuric acid according to claim 8, characterized in that, The volume ratio of acetonitrile-methanol-water in the mobile phase is 2:2:6.

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