Method for detecting aflatoxin in silkworm chrysalis meal

Abstract

The invention discloses a method for detecting aflatoxin in silkworm chrysalis meal. According to the invention, a salting-out auxiliary liquid-liquid extraction technology and a supramolecular solvent extraction technology are integrated and are executed by an automatic salting-out extraction device specially designed for a pretreatment process, so that a system and a scheme for detecting aflatoxin in silkworm chrysalis meal, which integrate a special device, a core reagent and a detection method, are provided; a detection method which is green, environment-friendly, rapid, efficient, low in cost, high in sensitivity and good in accuracy is established. The preparation method comprises the following specific steps: preparing a supermolecular solvent consisting of n-heptanol, tetrahydrofuran and water and a saturated aqueous solution; the method comprises the following steps: mixing a silkworm chrysalis meal sample with a supramolecular solvent and ammonium sulfate, and carrying out vortex, centrifugation, constant volume and filtration to obtain a test solution; and carrying out quantitative analysis by adopting a matrix matching standard curve. The method is simple and convenient to operate, low in organic solvent consumption and low in cost, and is suitable for high-throughput rapid screening and quantitative detection of trace aflatoxin in silkworm chrysalis meal.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for detecting aflatoxin in silkworm pupa powder. Background Technology

[0002] Silkworm pupa powder is a powdered product made from dried and pulverized silkworm pupae. It is rich in protein and fat, making it a highly nutritious animal protein resource. However, precisely because of its high protein and fat content, it is highly susceptible to contamination by toxin-producing molds such as Aspergillus flavus during processing and storage, especially in high-temperature and high-humidity environments, which can lead to the production of aflatoxins.

[0003] Aflatoxins (AFTs) are a class of fungal toxins with strong carcinogenic, teratogenic, and mutagenic properties. AFTs B1, B2, G1, and G2 are the most common and have the highest toxicity, posing a serious threat to human and animal health. The International Agency for Research on Cancer (IARC) has classified AFT B1 as a Group 1 carcinogen. Therefore, establishing a sensitive and accurate detection method for aflatoxins in silkworm pupa powder is crucial to ensuring its safety for consumption.

[0004] Currently, the main pretreatment methods for detecting aflatoxin in food include immunoaffinity chromatography, solid-phase extraction, and QuEChERS. While immunoaffinity column methods offer good selectivity, the antibodies used are expensive, the procedures are cumbersome and time-consuming, and the column capacity is limited, making it difficult to meet the high-throughput, rapid detection needs of large batches of samples. Solid-phase extraction also suffers from cumbersome procedures and high consumption of organic solvents. Traditional liquid-liquid extraction methods often use large amounts of toxic and harmful organic solvents, which are environmentally unfriendly, and have limited extraction efficiency and selectivity for trace targets in complex matrices.

[0005] Supramolecular solvents are a novel, green extraction medium developed in recent years. They are formed by the self-assembly of amphiphilic molecules through non-covalent intermolecular forces, offering advantages such as simple preparation, low cost, and high extraction efficiency. Salting-out assisted liquid-liquid extraction (SLIE) can effectively improve the extraction recovery rate of target analytes through the salting-out effect. However, a complete method for the detection of multiple aflatoxins in the complex matrix of silkworm pupa powder, including the optimal composition of the supramolecular solvent, the selection of the salting-out agent, and the optimal ratio of various materials, has not yet been reported in existing technologies. Therefore, the development of a rapid, green, accurate, and efficient method specifically for the detection of aflatoxins in silkworm pupa powder is particularly urgent. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting aflatoxin in silkworm pupa powder, addressing the problems of existing technologies such as immunoaffinity chromatography, including cumbersome operation, high cost, high toxicity of organic solvents, and difficulty in meeting high-throughput detection requirements. This invention integrates salting-out assisted liquid-liquid extraction technology with supramolecular solvent extraction technology, and executes the process using an automated salting-out extraction device specifically designed for this pretreatment process. This provides a system and solution for detecting aflatoxin in silkworm pupa powder that integrates a dedicated device, core reagents, and a detection method, establishing a detection method that is environmentally friendly, rapid, efficient, low-cost, highly sensitive, and accurate. This method not only optimizes the composition, ratio, and extraction process of the supramolecular solvent, significantly improving pretreatment efficiency and reducing environmental burden, but also, by combining it with high-sensitivity mass spectrometry, provides a reliable technical solution for the rapid and accurate screening and quantitative analysis of trace aflatoxins in silkworm pupa powder. It rapidly and accurately detects aflatoxins B1, B2, G1, and G2 in silkworm pupa powder, and is particularly suitable for the monitoring and quality control of large-scale samples.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for detecting aflatoxin in silkworm pupa powder, the core of which lies in integrating salting-out assisted liquid-liquid extraction with supramolecular solvent extraction technology to construct an efficient and green pretreatment system, and combining it with modern chromatography-mass spectrometry technology for precise quantification. The method specifically includes the following steps: S1. Preparation of supramolecular solvent: Long-chain alkyl alcohol, tetrahydrofuran (THF), and ultrapure water are mixed in a specific volume ratio. After vortexing and centrifugation, the system separates into two phases. The upper clear organic phase is used as the supramolecular solvent for extraction and is sealed for later use. At the same time, the lower saturated aqueous phase solution is collected for subsequent sample wetting.

[0008] S2. Preparation of the test solution: Accurately weigh a certain mass of silkworm pupa powder sample, and add the saturated supramolecular solvent aqueous solution obtained in step S1, the supramolecular solvent, and the specific salting-out agent sequentially to a centrifuge tube. Vortex the solution to completely dissolve the salting-out agent and achieve thorough mixing, followed by high-speed centrifugation. After centrifugation, accurately transfer a certain volume of the supernatant, dilute to volume with a methanol-water mixture, and filter through a filter membrane to obtain the test solution.

[0009] S3. Plotting matrix matching standard curves: Using blank silkworm pupa powder matrix solution that has been confirmed to be free of the target aflatoxin, the aflatoxin mixed standard stock solution is gradually diluted to prepare a series of matrix matching standard working solutions of different concentrations, so as to effectively correct the interference caused by complex matrices.

[0010] S4. UPLC-MS / MS Detection: The test solution prepared in step S2 and the series of matrix-matched standard working solutions prepared in step S3 are sequentially injected into the ultra-high performance liquid chromatography-tandem mass spectrometry system. Separation and detection are performed under optimized chromatographic and mass spectrometric parameters.

[0011] S5. Quantitative Analysis: Plot a standard curve with the concentration of the matrix-matched standard working solution on the x-axis and the corresponding peak area of ​​the quantitative ion on the y-axis. Substitute the peak areas of each aflatoxin B1, B2, G1, and G2 in the test sample solution into the corresponding standard curve to calculate their specific content in the silkworm pupa powder.

[0012] Preferably, in step S1, the long-chain alkyl alcohol is a straight-chain alkyl alcohol with 6 to 9 carbon atoms, preferably n-heptanol; the volume ratio of the long-chain alkyl alcohol, tetrahydrofuran and water is 1:(4~6):(1~3), preferably 1:5:2.

[0013] Preferably, in step S2, the salting-out agent is ammonium sulfate.

[0014] Preferably, in step S2, the weight of the silkworm pupa powder sample is 0.5g, the amount of the saturated supramolecular solvent aqueous solution added is 0.8~1.5mL, preferably 1.0mL; the amount of the supramolecular solvent added is 0.8~1.5mL, preferably 1.2mL; and the amount of the salting-out agent added is 0.4~0.6g, preferably 0.5g.

[0015] Preferably, in step S2, the solvent used for volume adjustment is a mixture of methanol and water in a volume ratio of 1:1.

[0016] Preferably, in step S4, the chromatographic conditions of the UPLC-MS / MS include: a C18 reversed-phase column; mobile phase A being a mixture of methanol and acetonitrile, and mobile phase B being an aqueous solution of ammonium acetate; gradient elution is used.

[0017] Preferably, in step S4, the mass spectrometry conditions of the UPLC-MS / MS include: ionization mode of electrospray positive ion mode; and scanning mode of multiple reaction monitoring mode.

[0018] Preferably, the preparation of the test solution in step S2 is carried out in a salting-out extraction apparatus to achieve automated quantitative addition of liquid, salt, and homogenization stirring. The apparatus includes a worktable, support legs, an extraction mechanism, and a rotating assembly. The extraction mechanism includes a turntable rotatably connected to the worktable, with a sample-carrying cylinder mounted on the turntable. A stirring assembly for stirring the mixture in the sample cylinder is mounted on the worktable. Support frame one and support frame two are also fixed on the worktable. A first feeder is mounted on support frame one, and a second feeder is mounted on support frame two.

[0019] Furthermore, the feeder includes a first feeder for adding the supramolecular solvent and a second feeder for adding the salting-out agent, and the feeder is equipped with a solenoid valve for controlling the amount added.

[0020] In the sample solution preparation stage of the detection method, this invention employs a dedicated salting-out extraction device to automate and standardize the pretreatment process. Specifically, the weighed silkworm pupa powder sample is placed in the device's feed cylinder, and the rotating disc sequentially transfers it to various stations: first, a saturated supramolecular solvent aqueous solution is quantitatively added by the first feeder to wet the sample; then, the supramolecular solvent and ammonium sulfate salting-out agent are precisely added by the second feeder; finally, at the stirring station, the stirring assembly efficiently and uniformly extracts the mixture in the feed cylinder. This integrated application of the device achieves precise control of the entire process from adding liquid and salt to mixing, greatly improving the reproducibility, efficiency, and high-throughput detection capability of the method.

[0021] As a preferred embodiment, the salting-out assisted supramolecular solvent extraction kit for implementing the method for detecting aflatoxin in silkworm pupa powder comprises: a supramolecular solvent prepared by n-heptanol, tetrahydrofuran and water in a volume ratio of 1:(4~6):(1~3), a saturated supramolecular solvent aqueous solution, and ammonium sulfate salting-out agent, preferably in a ratio of 1:5:2.

[0022] In summary, the present invention has the following beneficial effects: 1. Unlike traditional liquid-liquid extraction or expensive immunoaffinity columns that rely on large amounts of toxic organic solvents (such as chloroform and n-hexane), this invention utilizes a supramolecular solvent system composed of n-heptanol, tetrahydrofuran, and water. This solvent is inexpensive and readily available, and its toxicity and environmental impact are far lower than those of traditional solvents. It also avoids the high cost of hundreds of yuan per immunoaffinity column, significantly reducing the material cost per detection and aligning with the development concept of green chemistry, providing an environmentally friendly new option. 2. Compared to the cumbersome pretreatment process of existing immunoaffinity chromatography or solid phase extraction, which requires more than 60 minutes, this invention organically combines salting-out-assisted extraction with supramolecular solvent extraction technology to form a one-step synergistic extraction process. The entire pretreatment process can be completed within 10 minutes, greatly simplifying the operation steps and reducing the total amount of organic solvent used by more than 85%. This makes the method particularly suitable for high-throughput rapid screening of large batches of samples in scenarios such as market supervision and enterprise quality control. 3. Compared to traditional methods, which are susceptible to interference and have large fluctuations in recovery rates in complex matrices, this invention, through systematic optimization research, has determined the optimal parameter range for the entire set of parameters, including alkyl alcohol type, solvent ratio, and salting-out agent dosage. Quantification using a matrix-matched standard curve effectively corrects for the ion inhibition effect caused by the high-protein, high-fat matrix of silkworm pupa powder. This results in a method with low detection limits (0.03–0.12 μg / kg) and high sensitivity (quantitation limit 0.1–0.4 μg / kg), while achieving high accuracy (spiking recovery rate 77.9%–99.0%) and excellent reproducibility (RSD 0.6%–5.2%). 4. To address the shortcomings of existing technologies, which heavily rely on manual operation and have poor reproducibility, this invention matches the optimized method with a dedicated salting-out extraction device. This device automates and quantifies the addition of liquid, salt, and stirring, completely changing the uncontrollability of traditional manual operation and ensuring high reproducibility of experimental data. Furthermore, this method can be further developed into a standardized reagent kit, greatly reducing the operational threshold and facilitating the standardized promotion and application of this technology in testing institutions at all levels. 5. Unlike traditional high testing costs, this invention reduces the pretreatment cost of a single test to an extremely low level by using inexpensive alternatives and reducing solvent usage. This "low cost and high performance" characteristic provides an economic foundation for large-scale and routine application in the daily safety monitoring of silkworm pupa powder and similar high-value agricultural products. It provides strong technical support for ensuring food safety and promoting the healthy development of the industry, demonstrating huge potential for industrial application and market economic value. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the alkyl alcohol / acid type screening method of the present invention; Figure 2 This is the phase diagram of the ternary supramolecular solvent system of this invention; Figure 3 This is a schematic diagram illustrating the screening of supramolecular solvent volume ratios in this invention. Figure 4 This is a schematic diagram illustrating the screening of salting-out agents according to the present invention; Figure 5 This is a schematic diagram illustrating the screening of supramolecular solvent dosage in this invention; Figure 6 This is a schematic diagram illustrating the screening of the salting-out agent dosage in this invention; Figure 7 This is a chromatogram of the aflatoxin mixed standard solution of the present invention; Figure 8 This is a schematic diagram of the salting-out extraction apparatus specifically designed for this invention. The following are labels in the attached diagram: 1. Workbench; 2. Support leg; 3. Extraction mechanism; 4. Turntable; 5. Material cylinder; 6. Stirring assembly; 7. Rotating assembly; 8. Support frame one; 9. First feeder; 10. Support frame two; 11. Second feeder; 12. Solenoid valve. Detailed Implementation

[0024] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0025] 1. Experimental Section 1.1 Instruments and Reagents 1260 high-performance liquid chromatograph, 6470 triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); XPR205DUE electronic analytical balance (accuracy 0.0001 g, Mettler Toledo Instruments GmbH, Switzerland); Genius 3 vortex mixer (IKA GmbH, Germany); Sorvall St16 high-speed centrifuge (Thermo Fisher Scientific, USA); Simplicity ultrapure water system (Millipore, USA).

[0026] All silkworm pupa powder samples used in the experiment were commercially available. AFT B1, B2, G1, and G2 mixed solution standard substances (100 μg / mL, Qingdao Purybang Biotechnology Co., Ltd.); the experimental water was ultrapure water; 0.22 μm Nylon syringe filter (Tianjin Jinteng Experimental Equipment Co., Ltd.). Ammonium acetate (≥97%), ammonium chloride (≥99.5%), ammonium sulfate (≥99%), ammonium sulfite monohydrate (92%), acetonitrile (chromatographic grade), methanol (chromatographic grade), tetrahydrofuran (chromatographic grade), n-pentanol (≥99%), n-hexanol (≥99%), n-heptanol (≥99.5%), n-octanol (≥99%), n-nonanol (≥98%), n-decanol (≥98%), n-undecyl alcohol (97%), n-dodecanol (≥98%), n-valeric acid (≥99%), n-hexanoic acid (≥99%), n-heptanoic acid (≥99%), n-octanoic acid (≥99%) (Merck, Germany).

[0027] 1.2 Solution Preparation 1.2.1 Preparation of mixed reference solution Accurately pipette an appropriate amount of the aflatoxin mixed solution standard, dilute with acetonitrile and bring to volume to prepare a mixed standard stock solution with a mass concentration of 1 μg / mL, and store at -20℃ protected from light. The stock solution was serially diluted with blank matrix solution to obtain a series of mixed standard working solutions with concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 ng / mL. All standard solutions were filtered through a 0.22 μm nylon filter before instrumental analysis.

[0028] 1.2.2 Preparation of supramolecular solvents Accurately measure 5.0 mL of n-heptanol and 25.0 mL of tetrahydrofuran, quickly transfer them to a 50 mL centrifuge tube, add 10.0 mL of ultrapure water, vortex for 5 min, centrifuge at 10000 r / min for 10 min, transfer the upper organic phase and seal it at 4 ℃ for later use, and collect the lower water layer saturated with supramolecular solvent for the extraction of aflatoxin from silkworm pupa powder.

[0029] 1.2.3 Preparation of the test solution Weigh 0.50 g of silkworm pupa powder (accurate to 0.01 g) into a 15 mL centrifuge tube, add 1.0 mL of saturated supramolecular solvent aqueous solution, 1.2 mL of supramolecular solvent and 0.5 g of ammonium sulfate in sequence, vortex until the ammonium sulfate solid is completely dissolved (about 4 min), then centrifuge at 10000 r / min for 5 min, collect 0.6 mL of supernatant and make up to 1 mL with methanol-water (v / v: 1 / 1) solution, filter through a 0.22 μm nylon membrane and analyze with the instrument.

[0030] 1.3 Plotting the Standard Curve A standard curve was plotted using the external standard method: 5 μL of a mixed standard working solution of four aflatoxins at five concentration levels was sequentially injected into the UPLC-MS / MS system for analysis. The concentration of the standard working solution was plotted on the x-axis (X), and the peak area of ​​the corresponding quantitative ion was plotted on the y-axis (Y). Linear regression was performed by the instrument software to automatically calculate the linear equation and correlation coefficient.

[0031] 1.4 Chromatographic and Mass Spectrometric Conditions Chromatographic conditions: ZORBAX SB-C18 column (100 mm × 2.1 mm, 1.8 μm); column temperature 40℃; flow rate 0.3 mL / min; mobile phase A was a methanol / acetonitrile mixture (50:50, V:V), mobile phase B was 5 mmol / L ammonium acetate solution; gradient elution program: 0.0–0.5 min, 40% A; 0.5–3.0 min, 40%–60% A; 3.0–4.0 min, 60% A; 4.0–4.2 min, 60%–95% A; 4.2–5.0 min, 95% A; 5.0–5.5 min, 95%–40% A; 5.5–8.0 min, 40% A; injection volume 5 μL.

[0032] Mass spectrometry conditions: Electrospray ionization (ESI+) with multiple reaction monitoring (MRM) mode; capillary voltage 3.5 kV; drying gas temperature 300℃; drying gas flow rate 7 L / min; nebulizer pressure 45 psi; desolventizing temperature 70℃; desolventizing gas flow rate 11 L / min; relevant mass spectrometry parameters for AFT B1, B2, G1, and G2 are detailed in Table 1.

[0033] Table 1. Monitoring ion pairs and optimal mass spectrometry parameters for the analytes

[0034] 1.5 Data Processing The contents of the four aflatoxins in the sample were calculated according to formula (1):

[0035] In formula (1): Xi—The content of aflatoxin in the test sample, μg / kg; Ci—aflatoxin content in the sample solution, ng / mL; V—Volume of the sample solution after final volume adjustment, mL; m—mass of the test sample, in grams.

[0036] f—Dilution factor.

[0037] 1.6 Data Statistics and Analysis Data processing and graph creation were performed using Origin 9.0 software. Statistical analysis was conducted using SPSS 26.0 software. One-way ANOVA was used to compare differences between groups, with a significance level of α=0.05. A p-value less than 0.05 was considered statistically significant.

[0038] 1.7 Matrix effect This study assessed the matrix effect using the relative response value method, which is calculated based on the ratio of the slope of the blank solution standard curve to the slope of the matrix standard curve. The formula is: ME = k2 / k1 × 100%, where k1 and k2 represent the slopes of the blank solution and matrix-matched standard curves, respectively.

[0039] 1.8 Optimization of Salting-out Supramolecular Solvent Extraction Method 1.8.1 Screening of Alkyl Alcohols / Acids Supramolecular solvents (SUPRAS) possess structural tunability, and their properties vary with the type and content of amphiphilic compounds within them, thus affecting the extraction rate of the target analyte. Therefore, this study investigated the extraction capabilities of supramolecular solvents formed from different long-chain alcohols / acids for aflatoxin. Twelve common long-chain alcohols / acids were selected for comparative experiments: n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol, n-undecylol, n-valeric acid, n-hexanoic acid, n-heptanoic acid, and n-octanoic acid. The results are shown in [Figure 1]. Figure 1 The results showed that the average recovery rate of alkyl alcohols was higher than that of alkyl acids. This is because the low pH environment provided by long-chain acids affected the extraction efficiency of supramolecular solvents. In addition, the extraction efficiency of supramolecular solvents was the highest when the number of carbon atoms in the alkyl chain was 7 (heptanol), and there were significant differences among the four aflatoxins (P < 0.05). This is because the supramolecular composition of heptanol reached a balance in viscosity and hydrophobic interaction with the target analyte. Therefore, heptanol was selected to prepare supramolecular solvents for subsequent experimental studies.

[0040] 1.8.2 Screening of supramolecular solvent proportions Supramolecular solvents generally consist of three solvents that form supramolecular solvents within a specific range. Therefore, this study plotted a phase diagram for preparing supramolecular solvents using heptanol, THF, and water. For example... Figure 2 As shown, a supramolecular solvent can be formed when the volume percentage of THF is below 63.52%. Based on the phase diagram, this study selected eight solvent systems with different composition ratios within the supramolecular solvent formation region to explore the effect of varying the composition ratios of heptanol, THF, and water on the extraction rate of the target analyte. Results are shown below. Figure 3 The experimental results showed that the higher the THF content, the higher the recovery rate of the four aflatoxins, with a significant difference for AFT B1 (P < 0.05). This is because the ratio of THF to water affects the three-dimensional structure of the supramolecular solvent. Higher THF content results in larger supramolecular solvent vesicles, thus enhancing the extraction capacity of the supramolecular solvent. Simultaneously, higher THF content leads to lower viscosity of the supramolecular solvent, which is more conducive to increasing mass transfer efficiency. Therefore, a heptanol:THF:water volume ratio of 1:5:2 was selected to prepare the supramolecular solvent for subsequent experiments.

[0041] 1.8.3 Screening of Salting-out Agents In liquid-liquid extraction with salting-out, the introduction of a salting-out agent can improve the extraction efficiency of the target analyte. However, the use of a large amount of salting-out agent can lead to residues in the extraction solvent, reducing the ion abundance of the target analyte in mass spectrometry, and also corroding and contaminating the mass spectrometry hardware. To avoid this drawback, this study selected volatile ammonium salts as salting-out agents to improve the extraction efficiency of supramolecular solvents. The extraction efficiencies of four ammonium salts—ammonium acetate, ammonium chloride, ammonium sulfate, and ammonium bicarbonate—for aflatoxin were investigated. The results are shown in [Figure number missing]. Figure 4 The results showed that ammonium sulfate had a higher extraction recovery rate for all four aflatoxins than other salts, with significant differences compared to other salts (P < 0.05). It also showed significant differences in extraction efficiency for AFT B2, G1, and G2 compared to no salt added (P < 0.05). Due to its better water solubility and stronger salting-out ability, ammonium sulfate was selected as a salting-out aid for subsequent studies.

[0042] 1.8.4 Screening of supramolecular solvent dosage In liquid-liquid extraction (Li-Liquid Extraction) of salting-out analytes, the volume of the extraction solvent has a significant impact on the extraction efficiency and enrichment factor. Too low a volume of extraction solvent reduces the extraction efficiency, while too high a volume affects the enrichment factor. Therefore, this study investigated the effect of the amount of supramolecular solvent (0.4–1.6 mL) on the extraction efficiency of the target analyte. The results are shown in [Figure 1]. Figure 5 When the supramolecular solvent volume was 0.4 mL, the silkworm pupa powder concentrated at the interface between the two phases, easily interfering with the absorption of the supramolecular solvent above. This resulted in less supramolecular solvent being available for absorption after salting out, leading to a large error. Therefore, this volume data was discarded. Increasing the supramolecular solvent volume from 0.8 mL to 1.2 mL significantly improved the recovery rates of all four aflatoxins (P < 0.05). Further increasing the supramolecular solvent volume showed no significant difference in the recovery rates of the four aflatoxins (P > 0.05). Therefore, to save organic solvent, 1.2 mL was determined to be the optimal extraction volume.

[0043] 1.8.5 Screening of Salting-out Agent Dosage The dosage of salting-out agent needs to balance the salting-out effect with the degree of interference with the ionization of the target analyte. This study investigated the effect of ammonium sulfate dosage (0.3, 0.4, 0.5, 0.6, 0.7 g) on ​​the recovery rate of four aflatoxins. The results are shown in [Figure number missing]. Figure 6Studies showed that ammonium sulfate dissolved completely within the tested range, and the extraction solvent was clear and easy to absorb. The recovery rates of the four aflatoxins increased significantly when the ammonium sulfate dosage increased from 0.3 g to 0.5 g (P < 0.05), but the increase in recovery rate was not significant with further increases in ammonium sulfate dosage (P > 0.05). Therefore, 0.5 g was determined as the optimal salting-out mass based on the principle of reagent conservation.

[0044] 1.9 Standard Curve, Linear Range, and Limit of Detection Given the strong matrix effect exhibited by silkworm pupa powder, this study used matrix-matched standard curves for quantitative analysis. The matrix effect of the four aflatoxins ranged from 51.47% to 86.56% as assessed by the relative response value method. Among them, AFTB1, B2, and G1 showed significant matrix inhibition effects (P < 0.05). Subsequently, a series of matrix-matched standard working solutions at various concentrations were analyzed. Within the range of 0.1–10 ng / mL, all four AFTs showed good linearity, with correlation coefficients (r) greater than 0.999 (Table 2). Figure 7 The chromatograms of the mixed standard solution with a concentration of 5.0 ng / mL are shown. Based on signal-to-noise ratios S / N=3 and S / N=10, the limits of detection and quantitation of the method were calculated, with ranges of 0.03–0.12 μg / kg and 0.1–0.4 μg / kg, respectively. The specific results are summarized in Table 2.

[0045] Table 2. Regression equations, linear ranges, limits of detection, and limits of quantitation for four aflatoxins.

[0046] 1.10 Recovery and Precision Experiments A 0.5 g blank silkworm pupa powder was used for a spiked recovery experiment. The spiked concentrations of the four aflatoxins at three levels were 0.5 μg / kg, 1.0 μg / kg, and 5.0 μg / kg, respectively. Six parallel experiments were conducted to examine the recovery rate and precision at the three levels. The results are shown in Table 3. The average recovery rate of the four aflatoxins was in the range of 77.9% to 99.0%, and the RSD was in the range of 0.6% to 5.2%. The results meet the requirements for recovery rate in GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food".

[0047] Table 3. Spiking recoveries and precision of four aflatoxins in silkworm pupa powder (n=6)

[0048] 2. Implementation Part 2.1 Experimental Materials Sample: Uniform silkworm pupa powder sample from the same batch.

[0049] Standard: AFT B1, B2, G1, G2 mixed standard solution.

[0050] Main reagents: n-Heptanol, tetrahydrofuran, ammonium sulfate, methanol, acetonitrile, etc. (specifications as above).

[0051] 2.2 Example 1: The method of the present invention (salting-out assisted supramolecular solvent extraction-device method) Accurately weigh 0.50 g of silkworm pupa powder sample into a 15 mL centrifuge tube and place it in the feed cylinder of the salting-out extraction device described in this invention. The salting-out extraction device described in this invention is used to automate the preparation of the test solution, and its structure is as follows: Figure 8 As shown. The device includes a workbench 1 and support legs 2; an extraction mechanism 3 and a rotating assembly 7 are arranged on the workbench 1; the extraction mechanism 3 includes a turntable 4, which is connected to the output shaft of the rotating assembly 7 and can rotate on the workbench 1; multiple material cylinders 5 for carrying sample centrifuge tubes are arranged on the turntable 4; a first support frame 8 and a second support frame 10 are also fixedly arranged on the workbench 1; a first feeder 9 is installed on the first support frame 8 for quantitatively adding saturated supramolecular solvent aqueous solution to the sample in the material cylinder 5; a second feeder 11 is installed on the second support frame 10 for quantitatively adding supramolecular solvent and / or ammonium sulfate salting agent to the material cylinder 5; both the first feeder 9 and the second feeder 11 are equipped with solenoid valves 12 to control the amount added; in addition, a stirring assembly 6 is also arranged on the workbench 1, whose stirring shaft can be raised and lowered to correspond to a certain position of the turntable 4, for fully stirring the mixture in the material cylinder 5 at that position to complete the extraction. The device is started and automatically completes the following process: 1.0 mL of saturated supramolecular solvent aqueous solution is added through the first feeder; 1.2 mL of supramolecular solvent and 0.5 g of ammonium sulfate are added through the second feeder; the turntable is rotated to the stirring position, and the stirring component automatically stirs for 4 minutes.

[0052] Subsequent centrifugation, volume adjustment, filtration, and UPLC-MS / MS detection steps are the same as described above. Record the pretreatment time and solvent volume, and calculate the recovery rate and RSD.

[0053] 2.3 Comparative Example 1: Solid Phase Extraction-HPLC Method This comparative example uses solid-phase extraction based on an immunoaffinity column combined with high-performance liquid chromatography (HPLC) for detection. The specific steps are as follows: Accurately weigh 5.0 g of silkworm pupa powder sample into a 50 mL centrifuge tube, add 1.0 g of sodium chloride and 20.0 mL of acetonitrile-water solution (70:30, V / V), vortex for 3 minutes, and then centrifuge at 5000 r / min for 5 minutes. Accurately transfer 4.0 mL of the supernatant, dilute and mix with 46.0 mL of phosphate buffer solution, and filter through glass fiber filter paper. Pass all the filtrate through an aflatoxin immunoaffinity column at a flow rate of approximately 2–3 mL / min. After the surface of the liquid has dried, rinse twice with 10 mL of ultrapure water and discard the eluent. The solution was then eluted with 1.5 mL of chromatographically pure methanol at a flow rate of 1–2 mL / min, and the eluent was collected in a vial. After drying with nitrogen at 50 °C, the solution was reconstituted by vortexing with 1.0 mL of methanol-water solution (3:7, V / V) for 30 seconds. Finally, the solution was filtered through a 0.22 μm nylon membrane to obtain the test solution. HPLC analysis was performed under the following conditions: C18 column (150 mm × 4.6 mm, 5 μm), column temperature 30 °C, mobile phase methanol-acetonitrile-water (20:20:60, V / V / V), flow rate 1.0 mL / min, injection volume 50 μL, and fluorescence detector parameters: excitation wavelength 360 nm, emission wavelength 440 nm. The total pretreatment time and total organic solvent consumption were recorded, and the recovery rate was calculated.

[0054] 2.4 Comparative Example 2: Solid Phase Extraction-HPLC-MS / MS Method This comparative example uses solid-phase extraction based on a multifunctional purification column combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for detection. The specific steps are as follows: Accurately weigh 2.0 g of silkworm pupa powder sample into a 50 mL centrifuge tube, add 10.0 mL of acetonitrile-water-formic acid solution (80:19:1, V / V / V), vortex for 5 minutes, and then centrifuge at 8000 r / min for 5 minutes. Accurately transfer 5.0 mL of the supernatant to an activated multifunctional purification column, push the column stopcock to allow the extract to pass through the packing material at a flow rate of approximately 1–2 mL / min, and collect approximately 2.0 mL of purified eluent. Accurately transfer 1.0 mL of the eluent to a vial, add 1.0 mL of ultrapure water, vortex to mix, and filter through a 0.22 μm nylon syringe filter to obtain the test solution. HPLC-MS / MS was used for analysis. The chromatographic conditions were as follows: C18 column (100 mm × 2.1 mm, 1.7 μm), column temperature 40℃, mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid, with gradient elution, a flow rate of 0.3 mL / min, and an injection volume of 5 μL. The mass spectrometry conditions were electrospray ionization in positive ion mode and multiple reaction monitoring mode. The total pretreatment time and total organic solvent consumption were recorded, and the recovery rate was calculated.

[0055] 2.5 Comparative Example 3: Manual Extraction Method (without using the apparatus of this invention) Except for the preparation of the test solution, the remaining steps were exactly the same as in Example 1. Specific procedures: Accurately weigh 0.50 g of silkworm pupa powder sample into a 15 mL centrifuge tube. Using a graduated cylinder and balance, manually add 1.0 mL of saturated supramolecular solvent aqueous solution, 1.2 mL of supramolecular solvent, and 0.5 g of ammonium sulfate sequentially. Then, vortex the centrifuge tube on a vortex mixer until the salt is completely dissolved (approximately 4 minutes). Subsequent steps are the same as in Example 1. Record the pretreatment time and focus on the reproducibility (RSD).

[0056] 2.6 Comparison Results and Analysis The above examples were systematically compared with the comparative examples in terms of pretreatment time, organic solvent usage, operational reproducibility (RSD), and cost per test. The results are summarized in Table 4.

[0057] Table 4 Comparison of key performance indicators between the examples and comparative examples

[0058] Results analysis: In terms of pretreatment efficiency and environmental friendliness, the method of this invention (Example 1) demonstrates an absolute advantage. As shown in Table 4, using the salting-out extraction device specifically designed for this invention, the pretreatment time for a single sample is only about 10 minutes. In contrast, Comparative Example 1, based on immunoaffinity column purification, and Comparative Example 2, based on a multifunctional purification column, both involve complex pretreatment processes including extraction, dilution, column chromatography, rinsing, elution, and concentration, with a total time exceeding 60 minutes. This indicates that this invention improves pretreatment efficiency by more than 6 times. Furthermore, regarding organic solvent consumption, the total solvent consumption of this invention is only about 4 mL, and the main components are low-toxicity n-heptanol, tetrahydrofuran, and methanol; while Comparative Example 1 and Comparative Example 2 use as much as 62 mL and 53 mL of solvent, respectively. The solvent consumption of this invention is reduced by more than 85%, significantly reducing environmental pollution and health hazards to operators, meeting the requirements of green analytical chemistry development.

[0059] In terms of accuracy and economy, the method of this invention achieves extremely low detection costs while ensuring high accuracy. The method of this invention (Example 1) shows spiked recoveries of 77.9% to 99.0% for four aflatoxins, fully meeting the requirements of the detection specifications. This is on par with Comparative Example 1 (86.1% to 97.3%), and far superior to the manual operation of Comparative Example 3 (70.0% to 90.0%). Regarding economic cost, the method of this invention avoids the use of expensive immunoaffinity columns or multifunctional purification columns, consuming only inexpensive conventional chemical reagents, resulting in extremely low pretreatment costs per run. In contrast, Comparative Examples 1 and 2 rely on high-value dedicated purification columns, leading to high costs per run and making them unsuitable for routine screening of large-scale samples.

[0060] The dedicated device of this invention plays a crucial role in the stability and reproducibility of the method. The outstanding advantage of the method of this invention (Example 1) lies in its excellent operational reproducibility, with a relative standard deviation (RSD) ranging from 0.6% to 5.2%, demonstrating a high level of automation and standardization. To verify the necessity of the device, a comparative example 3 (manual operation) was specifically set up, which was completely identical to Example 1 in terms of reagent types and dosages. The results showed that although the manual operation took slightly longer (approximately 15 minutes), its most critical drawback was a significant deterioration in reproducibility, with the RSD range worsening to 8.0% to 15.0%. This fully demonstrates that the salting-out extraction device of this invention, through automated quantitative liquid and salt addition and standardized stirring, significantly reduces the systematic and random errors introduced by manual operation, which is the core guarantee for achieving high precision and high reproducibility of the method—a technical effect that cannot be achieved by simply optimizing the reagent formulation.

[0061] In summary, this invention provides a complete technical solution integrating a dedicated device, core reagents, and detection methods. The core of this solution lies in the first-ever combination of supramolecular solvent extraction and salting-out assisted liquid-liquid extraction technologies, automating and standardizing the process through a self-designed salting-out extraction device. This method fully leverages the dual advantages of adjustable supramolecular solvent polarity and high salting-out extraction efficiency, achieving highly efficient detection of aflatoxins B1, B2, G1, and G2 in silkworm pupa powder. Ultimately, this solution constructs a closed-loop system from dedicated device and core reagents to detection methods. Compared to existing technologies, this complete solution, while maintaining high sensitivity and accuracy comparable to mainstream technologies, achieves a significant reduction in organic solvent consumption, a substantial decrease in detection costs, and an extreme simplification of the operation process thanks to its inherent integrated design and automation advantages. Therefore, it demonstrates extremely high practical application value and industrialization prospects in large-scale screening of aflatoxins in silkworm pupa powder.

Claims

1. A method for detecting aflatoxin in silkworm pupa powder, characterized in that, Includes the following steps: S1. Preparation of supramolecular solvent: Long-chain alkyl alcohol, tetrahydrofuran and water are mixed in volume ratio, vortexed and centrifuged, and the upper organic phase is taken as supramolecular solvent, and the lower aqueous phase is collected as saturated supramolecular solvent aqueous solution. S2. Preparation of test solution: Weigh the silkworm pupa powder sample, add the saturated supramolecular solvent aqueous solution, the supramolecular solvent and salting-out agent in sequence, vortex mix and centrifuge, take the supernatant, dilute to volume and filter to obtain the test solution; S3. Plot the matrix matching standard curve: Prepare a series of aflatoxin mixed standard working solutions using blank silkworm pupa powder matrix solution; S4. UPLC-MS / MS detection: The test solution obtained in step S2 and the series of matrix-matched standard working solutions obtained in step S3 were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. S5. Quantitative analysis: Calculate the contents of aflatoxin (AFT) B1, B2, G1, and G2 in the test solution based on the matrix-matched standard curve.

2. The method for detecting aflatoxin in silkworm pupa powder according to claim 1, characterized in that, In step S1, the long-chain alkyl alcohol is a straight-chain alkyl alcohol with 6 to 9 carbon atoms, preferably n-heptanol; the volume ratio of the long-chain alkyl alcohol, tetrahydrofuran and water is 1:(4-6):(1-3), preferably 1:5:

2.

3. The method for detecting aflatoxin in silkworm pupa powder according to claim 1, characterized in that, In step S2, the salting-out agent is ammonium sulfate.

4. A method for detecting aflatoxin in silkworm pupa powder according to claim 1 or 3, characterized in that, In step S2, the sample weight of the silkworm pupa powder is 0.5g, the amount of the saturated supramolecular solvent aqueous solution added is 0.8-1.5mL, preferably 1.0mL; the amount of the supramolecular solvent added is 0.8-1.5mL, preferably 1.2mL; and the amount of the salting-out agent added is 0.4-0.6g, preferably 0.5g.

5. The method for detecting aflatoxin in silkworm pupa powder according to claim 1, characterized in that, In step S2, the solvent used for volume adjustment is a mixture of methanol and water in a volume ratio of 1:

1.

6. The method for detecting aflatoxin in silkworm pupa powder according to claim 1, characterized in that, In step S4, the chromatographic conditions of the UPLC-MS / MS include: a C18 reversed-phase column; mobile phase A is a mixture of methanol and acetonitrile, and mobile phase B is an aqueous solution of ammonium acetate; gradient elution is used.

7. A method for detecting aflatoxin in silkworm pupa powder according to claim 1 or 6, characterized in that, In step S4, the mass spectrometry conditions of the UPLC-MS / MS include: ionization mode is electrospray positive ion mode; scanning mode is multiple reaction monitoring mode.

8. The method for detecting aflatoxin in silkworm pupa powder according to claim 1, characterized in that, The preparation of the test solution in step S2 is carried out in a salting-out extraction device, which includes a workbench (1), a support leg (2), an extraction mechanism (3), and a rotating assembly (7). The extraction mechanism (3) includes a turntable (4), which is rotatably connected to the workbench (1). A material cylinder (5) for carrying the sample is provided on the turntable (4). A stirring assembly (6) for stirring the mixture in the material cylinder (5) is provided on the workbench (1). A support frame one (8) and a support frame two (10) are also fixed on the workbench (1). A first feeder (9) is provided on the support frame one (8), and a second feeder (11) is provided on the support frame two (10).

9. The method for detecting aflatoxin in silkworm pupa powder according to claim 8, characterized in that, Both the first feeder (9) and the second feeder (11) are equipped with solenoid valves (12) for controlling the amount of feed added.

10. A method for detecting aflatoxin in silkworm pupa powder according to any one of claims 1 to 9, characterized in that, The salting-out-assisted supramolecular solvent extraction kit for implementing the method for detecting aflatoxin in silkworm pupa powder comprises: a supramolecular solvent prepared from n-heptanol, tetrahydrofuran, and water in a volume ratio of 1:(4-6):(1-3), a saturated supramolecular solvent aqueous solution, and ammonium sulfate salting-out agent, preferably in a ratio of 1:5:2.

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