Integrated pretreatment tube for detecting mycotoxin in camel milk and working method thereof

Abstract

This invention discloses an integrated pretreatment tube and its working method for detecting mycotoxins in camellia milk. The pretreatment tube includes an outer tube with an outer tube cap at its top, a middle tube inside the outer tube with its top protruding from the outer tube cap and a middle tube cap at its top, and an inner tube inside the middle tube with its top protruding from the middle tube cap and its top cap. An ultrafiltration tube is located inside the inner tube, with its outer sidewall conforming to the inner sidewall of the inner tube, and its bottom forming an ultrafiltration element. At least one set of micropores is provided on the sidewall of the middle tube, and a first microporous filter membrane covers these micropores. At least one set of micropores is also provided on the sidewall of the inner tube, and a second microporous filter membrane covers these micropores. Through holes are provided on the sidewall of the ultrafiltration tube, and these through holes are directly opposite the positions of the second microporous filter membrane. A QuEChERS reagent pack is located at the bottom of the middle tube, and a salt pack is located at the bottom of the outer tube.

Description

Technical Field

[0001] This invention relates to the field of fungal toxin detection technology, and in particular to an integrated pretreatment tube and working method for detecting fungal toxins in camellia milk. Background Technology

[0002] Mycotoxins (such as aflatoxin M1) are common contaminants in dairy products, making the establishment of efficient and accurate detection methods crucial. Camel milk has a complex matrix composition, containing large amounts of proteins, fats, and carbohydrates. Detection of mycotoxins requires three key pretreatment steps: extraction, purification, and filtration. The extraction step aims to release the target mycotoxins from the protein and fat matrix and dissolve them in a solvent; the purification step removes interfering substances extracted; and filtration obtains a clear, particle-free injection solution to meet the instrument's injection requirements.

[0003] In existing technologies, completing the above series of operations typically relies on a combination of the following steps:

[0004] (1) Extraction steps: Add the sample and extraction solvent (such as acetonitrile-water solution) into a centrifuge tube, mix by vortex, and then separate by high-speed centrifugation to obtain the supernatant containing the target substance;

[0005] (2) Purification step: Transfer the above clear liquid to a solid phase extraction column or other form of purification column, and under the action of gravity, negative pressure or centrifugation, use a specific adsorbent to selectively adsorb and remove interfering substances, and collect the purified liquid;

[0006] (3) Filtration step: Transfer the purified liquid to a needle filter and perform microporous membrane filtration to remove residual particulate matter and finally obtain the sample solution.

[0007] Therefore, technical researchers have attempted to improve the above-mentioned technical solutions. For example, CN217473577U discloses an ultrafiltration centrifuge tube, including a main tube with a top-open and bottom-closed structure. A top cover is installed at the opening of the main tube, and an inner tube is installed inside the main tube. The inner tube is vertically connected, and an ultrafiltration membrane is attached to the bottom surface of the inner tube, covering the bottom of the inner tube. A support plate is attached to the bottom surface of the ultrafiltration membrane, and the ultrafiltration membrane is located between the bottom surface of the inner tube and the support plate. A bottom support is installed at the bottom of the inner tube, and the inner tube, together with the bottom support, presses the ultrafiltration membrane and the support plate together. The inner tube, ultrafiltration membrane, support plate, and bottom support are connected to the cavity space in the main tube below the bottom support, which can separate the sample solution and filtrate by centrifugation and ultrafiltration with high separation purity.

[0008] However, this technical solution still has shortcomings. It can only achieve the filtration step of the pretreatment process for detecting mycotoxins in camel milk. Significant technical defects and deficiencies still exist in the extraction and purification steps.

[0009] (1) The entire process involves at least two major liquid transfers (from centrifuge tubes to purification columns, and then from purification columns to filtration devices). Each transfer means an increase in operating steps, time consumption, and the accumulation of potential loss of the target substance. The entire pretreatment process is cumbersome and time-consuming, which seriously restricts the realization of high-throughput detection.

[0010] (2) The three steps of the experiment require the purchase of various consumables, and the material cost of a single test is added up. At the same time, the complex operation also requires technicians to have a high level of proficiency, which increases the cost of human resources training and management.

[0011] (3) This technology is difficult to automate. The discrete operation steps and non-standard consumable system make it difficult to integrate with automated liquid handling systems or online pretreatment platforms, which limits the overall intelligence of the detection process.

[0012] Therefore, to overcome the aforementioned technical bottlenecks, there is an urgent need in this field for a novel sample pretreatment device that can spatially integrate and optimize the extraction, purification, and filtration functions to achieve a complete pretreatment process with a single tube and single operation. This would not only significantly improve detection efficiency and result stability but also reduce operational difficulty and overall costs, which is of great significance for advancing dairy product safety testing technology. Summary of the Invention

[0013] The purpose of this invention is to provide an integrated pretreatment tube and working method for detecting fungal toxins in camellia milk.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] An integrated pretreatment tube for detecting mycotoxins in camellia milk includes an outer tube with an outer tube cap at the top, a middle tube inside the outer tube with the top of the middle tube protruding from the outer tube cap and a middle tube cap at the top, and an inner tube inside the middle tube with the top of the inner tube protruding from the middle tube cap and an inner tube cap at the top.

[0016] The inner tube contains an ultrafiltration tube, the outer wall of which is in contact with the inner wall of the inner tube, and the bottom of the ultrafiltration tube is an ultrafiltration element.

[0017] The middle tube has at least one set of micropores on its side wall, and the micropores of the middle tube are covered with a first microporous filter membrane. The inner tube has at least one set of micropores on its side wall, and the micropores of the inner tube are covered with a second microporous filter membrane. The ultrafiltration tube has through holes on its side wall, and the through holes are directly opposite the position of the second microporous filter membrane.

[0018] The bottom of the central tube contains a QuEChERS reagent pack.

[0019] The height of the ultrafiltration tube is less than the height of the inner tube, the height of the inner tube is less than the height of the middle tube, and the height of the middle tube is less than the height of the outer tube.

[0020] The ultrafiltration tube is a detachable structure.

[0021] The top edge of the ultrafiltration tube is folded outward at 90°, and the maximum diameter of the top edge is equal to the outer diameter of the inner tube.

[0022] The micropores on the central tube are located in the lower middle part of the side wall of the central tube.

[0023] The micropores on the inner tube are located in the lower middle part of the inner tube's side wall.

[0024] The bottom of the outer tube is equipped with a salt pack.

[0025] The outer tube cover, middle tube cover, and inner tube cover are equipped with sealing gaskets.

[0026] The working method of the integrated pretreatment tube for detecting mycotoxins in camellia milk described in this invention is as follows:

[0027] Open the outer tube cap, add the camel milk sample, then add the extraction reagent, place the vibrator, insert the middle and inner tubes into the outer tube, and tighten the outer tube cap; place the sample tube on a vortex mixer and agitate. This step completes the extraction step of the pretreatment process. Place the pretreatment tube in a centrifuge. The sample is subjected to centrifugal force, separating the extract from the extraction residue. The extract passes through the microporous membrane into the middle tube. After stopping centrifugation, agitate the sample tube on a vortex mixer. This step completes the purification step of the pretreatment process. Centrifuge the pretreatment tube again. The solution in the middle tube enters the inner tube and is filtered through an ultrafiltration cartridge. This step completes the filtration step of the pretreatment process. After stopping centrifugation, remove the pretreatment tube and aspirate the solution at the bottom of the inner tube. This solution is the test solution.

[0028] Alternatively, open the outer tube cap, add the camel milk sample, then add the extraction reagent, place the vibrator, insert the middle and inner tubes into the outer tube, and tighten the outer tube cap. Place the sample tube in a three-dimensional shaking centrifuge and perform shaking, centrifugation, shaking again, and centrifugation again. The first shaking step completes the extraction step of the pretreatment process, and the first centrifugation step separates the extract from the extraction residue. The extract passes through the microporous membrane into the middle tube. The second shaking step completes the purification step of the pretreatment process, and the second centrifugation step allows the purified solution from the middle tube to enter the inner tube and be filtered through an ultrafiltration cartridge. This step completes the filtration step of the pretreatment process. After stopping centrifugation, remove the pretreatment tube and aspirate the solution from the bottom of the inner tube; this solution is the test solution.

[0029] Compared with the prior art, the outstanding effect of the present invention is as follows:

[0030] (1) The integrated pretreatment tube for detecting mycotoxins in camellia milk of the present invention integrates multiple independent steps such as extraction, purification and filtration into a single device, avoiding the cumbersome operation of transferring samples and changing containers multiple times in the traditional method (such as centrifuging and separating the liquid first, then transferring it to the purification tube for vortex centrifugation, and finally transferring the filtrate through the membrane), greatly reducing manual intervention and improving processing efficiency. There is no need to wait or manually transfer in the middle, and the overall pretreatment time can be shortened by about 40%-50%.

[0031] (2) This invention uses only a single pretreatment tube device to physically integrate three independent steps into one tube, eliminating the need for multiple consumables such as centrifuge tubes, purification columns, ultrafiltration tubes, and pipette tips, significantly reducing experimental costs and waste, and better meeting economic and environmental protection requirements.

[0032] (3) The present invention integrates the single tube design, which facilitates standardized operation and is especially suitable for batch sample processing. It is beneficial to improve the repeatability of experiments and the reliability of results, and provides convenience for large-scale detection.

[0033] (4) This invention, through a single pretreatment tube device, greatly reduces human error caused by complex steps, making it easier to promote in the laboratory or apply to rapid on-site detection scenarios. At the same time, it avoids sample adhesion loss and possible cross-contamination caused by multiple container transfers, thus improving the accuracy and precision of the detection.

[0034] (5) This invention can be modularly adjusted to meet different detection needs (such as toxin type, sample matrix), and has good method adaptability potential, leaving room for subsequent technology upgrades. The pretreatment tube has a regular structure and standardized steps, which is easy to adapt to automated liquid handling workstations to achieve high-throughput sample pretreatment.

[0035] (6) Through ingenious structural design, this invention realizes the physical integration and optimization of the pretreatment process for camel milk fungal toxin detection, providing a new type of efficient, convenient and reliable sample pretreatment tool for the field of food safety testing.

[0036] The integrated pretreatment tube and its working method for detecting mycotoxins in camellia milk according to the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the integrated pretreatment tube for detecting mycotoxins in camellia milk.

[0038] Among them, 1-outer tube, 2-middle tube, 3-inner tube, 4-outer tube cap, 5-middle tube cap, 6-inner tube cap, 7-QuEChERS reagent pack, 8-first microporous filter membrane, 9-second microporous filter membrane, 10-through hole, 11-ultrafiltration tube, 12-ultrafiltration cartridge, 13-salt pack. Detailed Implementation

[0039] like Figure 1 As shown, an integrated pretreatment tube for detecting mycotoxins in camellia milk includes an outer tube 1, with an outer tube cap 4 at the top of the outer tube 1, a middle tube 2 inside the outer tube 1, the top of the middle tube 2 protruding from the outer tube cap 4, a middle tube cap 5 at the top of the middle tube 2, an inner tube 3 inside the middle tube 2, the top of the inner tube 3 protruding from the middle tube cap 5, and an inner tube cap 6 at the top of the inner tube 3.

[0040] The outer tube 1 is a cylindrical tube with an open top and a closed bottom, used to hold the initial sample, extractant, and for initial extraction operations. The middle tube 2 is coaxially arranged inside the outer tube 1. It is also a tube with an open top and a closed bottom, but its outer diameter is smaller than the inner diameter of the outer tube 1, and there is a certain space between its bottom and the bottom of the outer tube 1.

[0041] An ultrafiltration tube 11 is installed inside the inner tube 3. The outer wall of the ultrafiltration tube 11 is in contact with the inner wall of the inner tube 3. The bottom of the ultrafiltration tube 11 is an ultrafiltration element 12. The cross-section of the ultrafiltration element 12 is V-shaped. The pore size of the ultrafiltration element 12 can be selected according to the molecular weight of the target analyte (e.g., molecular weight cutoff of 3kDa, 10kDa, etc.) to remove large molecular impurities (such as proteins, polysaccharides, etc.) and achieve final filtration and clarification of the solution. In this embodiment, a Millipore ultrafiltration tube, 15mL, 3KD, is selected.

[0042] Two sets of micropores are provided on the sidewall of the middle tube 2, and a first microporous filter membrane 8 is covered on the micropores of the middle tube 2. Two sets of micropores are provided on the sidewall of the inner tube 3, and a second microporous filter membrane 9 is covered on the micropores of the inner tube 3. A through hole 10 is provided on the sidewall of the ultrafiltration tube 11, and the through hole 10 is directly opposite the position of the second microporous filter membrane 9. The first microporous filter membrane 8 and the second microporous filter membrane 9 are permeable to liquid but can block solid particles, ensuring that only liquid can pass through. In this embodiment, the microporous filter membrane is a built-in PTFE microporous filter membrane, 0.22 μm, brand: Anpu.

[0043] The bottom of the inner tube 2 contains a QuEChERS reagent pack 7. The QuEChERS reagent pack contains anhydrous magnesium sulfate, N-propylethylenediamine (PSA), C18, and other adsorbents to remove interfering substances in the sample matrix, such as fats, proteins, and sugars. In this embodiment, the QuEChERS reagent pack includes 450 mg of anhydrous magnesium sulfate, 150 mg of PSA, and 20 mg of C18. 18 .

[0044] The height of the ultrafiltration tube 11 is less than the height of the inner tube 3, the height of the inner tube 3 is less than the height of the middle tube 2, and the height of the middle tube 2 is less than the height of the outer tube 1.

[0045] The ultrafiltration tube 11 is a detachable structure. The top edge of the ultrafiltration tube 11 is folded outward at 90°, and the diameter of the top edge is equal to the outer diameter of the inner tube 3, that is, the top outer edge of the ultrafiltration tube 11 falls on the top of the inner tube 3.

[0046] The micropores on the middle tube 2 are located in the lower middle part of the side wall of the middle tube 2. The micropores on the inner tube 3 are located in the lower middle part of the side wall of the inner tube 3.

[0047] A salt pack 13 is located at the bottom of the outer tube 1. Sealing gaskets are installed inside the outer tube cap 4, the middle tube cap 5, and the inner tube cap 6. When the outer tube cap 4 is tightened, it compresses and secures the middle tube 2 inside the outer tube 1, ensuring the entire pretreatment tube remains sealed during vortexing and centrifugation to prevent liquid leakage or evaporation. When the middle tube cap 5 is tightened, it compresses and secures the inner tube 3 inside the middle tube 2, ensuring the entire pretreatment tube remains sealed during vortexing and centrifugation to prevent liquid leakage or evaporation. When the inner tube cap 6 is tightened, it compresses and secures the ultrafiltration tube 11 inside the inner tube 3, ensuring the entire pretreatment tube remains sealed during vortexing and centrifugation to prevent liquid leakage or evaporation. In this embodiment, the salt pack contains 3 g of sodium chloride.

[0048] Example 2: Pretreatment method for detecting aflatoxin M1 in camel milk using the pretreatment tubes from Example 1.

[0049] This embodiment takes the detection of aflatoxin M1 in camel milk as an example, and uses a vortex mixer and a high-speed centrifuge to explain in detail the usage process of the pretreatment tube of the present invention.

[0050] (1) Add sample and extraction solution

[0051] Open the outer tube cap 4. Accurately measure 6.0 mL of a homogeneous camel milk sample and add it to the outer tube 1. Then, add 12.0 mL of acetonitrile solution containing 1% formic acid to the outer tube 1 as the extraction solvent. Next, place one or more ceramic or metal vortex oscillators into the tube.

[0052] (2) Extraction

[0053] The assembled pretreatment tube is placed on a vortex mixer and vortexed for 2-3 minutes. During this process, the extractant and camel milk sample are thoroughly mixed and emulsified at the bottom of the outer tube 1, and the target mycotoxin (aflatoxin M1) is extracted from the sample matrix. At the same time, the impact of the oscillator helps to break up the emulsion and accelerate mass transfer, thus completing the extraction process.

[0054] (3) Separation and purification

[0055] The pre-treatment tube, after vortexing, is placed in a high-speed centrifuge and centrifuged at 8000 rpm for 5 minutes. Under centrifugal force, the mixture in outer tube 1 separates into layers: heavier solids such as proteins and fats deposit at the bottom of outer tube 1; the upper organic extract (acetonitrile phase) moves upward and inward within outer tube 1 and passes through the first microporous membrane 8. The first microporous membrane 8 blocks any small solid particles that may pass through, achieving coarse filtration. After entering the middle tube 2, the extract comes into contact with the QuEChERS reagent pack 7 pre-filled at the bottom.

[0056] After centrifugation stops, immediately place the pretreatment tube back onto the vortex mixer and vortex for 2-3 minutes. The purpose of this step is to ensure that the extract entering tube 2 is fully contacted and mixed with the QuEChERS reagent. The adsorbent in the reagent quickly removes polar impurities, fatty acids, and other interfering substances from the extract, completing the purification process.

[0057] (4) Filtration

[0058] The purified and shaken pretreatment tube is placed back into a refrigerated high-speed centrifuge and centrifuged at 8000 rpm for 5 minutes. Under centrifugal force, the purified extract in the middle tube 2 passes through the second microporous membrane 9, reaches the inner tube 3, and is forced to pass through the ultrafiltration cartridge 12. The ultrafiltration cartridge 12 further traps any possible small particles, large molecular colloids, and other impurities, resulting in a highly clear filtrate.

[0059] After centrifugation stops, carefully open the inner tube cap 6, remove the ultrafiltration tube 11, and use a micropipette or syringe to aspirate the solution that has undergone the integrated extraction, purification, and filtration processes from the bottom of the inner tube 3. This solution is the analyte that can be directly used for subsequent instrumental analysis (HPLC, LC-MS / MS).

[0060] (5) Measurement

[0061] The analytical conditions of liquid chromatography-tandem mass spectrometry were set, and the aflatoxin M1 standard series solutions and the test solution were injected and measured to obtain the standard curve regression equation. The content of the test solution was calculated using the regression equation.

[0062] (6) Results

[0063] The experimental results for the test solutions spiked at three different levels (low, medium, and high) are shown in the table below. The average recovery rate and relative standard deviation (RSD) were calculated. The results show that the recovery rate of aflatoxin M1 was 83.63%–86.20%, and the RSD was 3.69%–5.70%, indicating that the pretreatment tubes used in this experimental method perform well and meet the experimental requirements for the detection of mycotoxins in camel milk.

[0064]

[0065] Example 3: Pretreatment method for detecting aflatoxin M1 in camel milk using the pretreatment tubes from Example 1.

[0066] This embodiment takes the detection of aflatoxin M1 in camel milk as an example, and uses a three-dimensional oscillating centrifuge to explain in detail the usage process of the pretreatment tube of the present invention.

[0067] (1) Addition of sample and extraction reagent

[0068] Open the outer tube cap 4 and accurately measure 6.0 mL of uniform camel milk sample, adding it to the outer tube 1. Then, add 12.0 mL of acetonitrile solution containing 1% formic acid to the outer tube 1 as the extraction solvent. Next, place one or more ceramic or metal vortex oscillators into the tube.

[0069] (2) Integrated operation of extraction, purification and filtration

[0070] The assembled pretreatment tube was placed in a three-dimensional oscillating centrifuge and vortexed at 2000 rpm for 2-3 minutes. During this process, the extractant and camel milk sample were thoroughly mixed and emulsified at the bottom of the outer tube 1, and the target mycotoxin (aflatoxin M1) was extracted from the sample matrix. At the same time, the impact of the oscillator helped to break up the emulsion and accelerate mass transfer, thus completing the extraction process.

[0071] Centrifuge at 4000 rpm for 15 minutes. Under centrifugal force, the mixture in outer tube 1 separates into layers: heavier solids such as proteins and fats settle at the bottom of outer tube 1; the upper organic extract (acetonitrile phase) moves upward and inward within outer tube 1 and passes through the first microporous membrane 8. The first microporous membrane 8 blocks any small solid particles that may pass through, achieving coarse filtration. After entering the middle tube 2, the extract comes into contact with the QuEChERS reagent pack 7 pre-filled at the bottom.

[0072] After centrifugation stops, set the speed to 2000 rpm and vortex for 2-3 minutes. The purpose of this step is to ensure that the extract entering tube 2 is fully in contact with and mixed with the QuEChERS reagent. The adsorbent in the reagent quickly removes polar impurities, fatty acids and other interfering substances from the extract, thus completing the purification process.

[0073] Centrifuge at 4000 rpm for 10 minutes. Under centrifugal force, the purified extract in the middle tube 2 passes through the second microporous membrane 9, reaches the inner tube 3, and is forced to pass through the ultrafiltration cartridge 12. The ultrafiltration cartridge 12 further traps any possible small particles, large molecular colloids, and other impurities, resulting in a highly clear filtrate.

[0074] (3) Filtrate aspiration

[0075] After centrifugation stops, remove the pretreatment tube, open the inner tube cap 6, remove the ultrafiltration tube 11, and use a micropipette or syringe to aspirate the solution after the integrated extraction, purification, and filtration processes from the bottom of the inner tube 3. This solution is the analyte that can be directly used for subsequent instrumental analysis (such as HPLC, LC-MS / MS).

[0076] (4) Measurement

[0077] The analytical conditions of liquid chromatography-tandem mass spectrometry were set, and the aflatoxin M1 standard series solutions and the test solution were injected and measured to obtain the standard curve regression equation. The content of the test solution was calculated using the regression equation.

[0078] (5) Results

[0079] The experimental results for the test solutions spiked at three different levels (low, medium, and high) are shown in the table below. The average recovery rate and relative standard deviation (RSD) were calculated. The results show that the recovery rate of aflatoxin M1 was 81.50%–87.21%, and the RSD was 1.71%–2.42%, indicating that the pretreatment tubes used in this experimental method perform well and meet the experimental requirements for the detection of mycotoxins in camel milk.

[0080]

[0081] To highlight the beneficial effects of this invention, conventional experimental methods were used as comparative examples. The details are as follows:

[0082] Referring to the conventional method in the literature "Rapid Determination of 21 Mycotoxins in Milk and Dairy Products by Ultra-High Performance Liquid Chromatography-Quadrupole / Electrostatic Field Orbittrap High Resolution Mass Spectrometry" by He Zhuolin et al., the sample pretreatment steps are as follows: Accurately weigh (2.00±0.05) g of liquid or powdered milk and dairy products and place them in a 50 mL polypropylene centrifuge tube. For powdered samples, first add 2 mL of water and vortex to mix thoroughly. Then add 8 mL of acetonitrile-formic acid (98:2, v:v) solution to all samples, vortex for 1 min, ultrasonically extract at 30℃ and 50 Hz for 20 min, centrifuge at 8000 r / min for 10 min, collect the supernatant in a 10 mL polypropylene centrifuge tube, vortex for 30 s, collect 5 mL of the supernatant and purify it through a Captiva-EMR Lipid purification column under positive pressure, controlling the flow rate at 3 drops per second, so that the extract flows out of the purification column, and add 1 Elute with acetonitrile-water (80:20, v:v) eluent, dry under positive pressure, combine the eluents, place in a 40℃ water bath and purge with nitrogen until nearly dry, add 250 μL acetonitrile-water-formic acid (30:70:0.1, v:v:v) to reconstitute, vortex for 1 min, sonicate for 5 min, transfer the reconstituted solution to a 2 mL centrifuge tube, centrifuge at 12000 r / min for 10 min, collect the supernatant in a sample vial with an inner tube, and store for later analysis.

[0083] Compared with Examples 2 and 3, the above conventional method has more complex processing steps, more complicated procedures, and more experimental consumables.

[0084] Following the conventional method described in He Zhuolin et al.'s paper, "Rapid Determination of 21 Mycotoxins in Milk and Dairy Products by Ultra-High Performance Liquid Chromatography-Quadrupole / Electrostatic Field Orbital Trap High-Resolution Mass Spectrometry," the recoveries of aflatoxin M1 at low, medium, and high levels ranged from 65.1% to 87.5%, with RSDs ranging from 0.4% to 3.9%. The spike recoveries and RSDs obtained in Examples 2 and 3 were consistent with the conventional method described in the aforementioned literature.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An integrated pretreatment tube for detecting mycotoxins in camellia milk, characterized in that: Includes an outer tube (1), the top of which is fitted with an outer tube cap (4), a middle tube (2) is provided inside the outer tube (1), the top of which protrudes from the outer tube cap (4), the top of which is fitted with a middle tube cap (5), an inner tube (3) is provided inside the middle tube (2), the top of which protrudes from the middle tube cap (5), and the top of which is fitted with an inner tube cap (6). The inner tube (3) is provided with an ultrafiltration tube (11), the outer wall of the ultrafiltration tube (11) is in contact with the inner wall of the inner tube (3), and the bottom of the ultrafiltration tube (11) is an ultrafiltration element (12). The middle tube (2) has at least one set of micropores on its side wall, and the micropores of the middle tube (2) are covered with a first microporous filter membrane (8). The inner tube (3) has at least one set of micropores on its side wall, and the micropores of the inner tube (3) are covered with a second microporous filter membrane (9). The ultrafiltration tube (11) has a through hole (10) on its side wall, and the through hole (10) is directly opposite to the second microporous filter membrane (9). The bottom of the tube (2) contains a QuEChERS reagent pack (7).

2. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The height of the ultrafiltration tube (11) is less than the height of the inner tube (3), the height of the inner tube (3) is less than the height of the middle tube (2), and the height of the middle tube (2) is less than the height of the outer tube (1).

3. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The ultrafiltration tube (11) is a detachable structure.

4. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The top edge of the ultrafiltration tube (11) is folded outward at 90°, and the diameter of the top edge is equal to the outer diameter of the inner tube (3).

5. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The micropores on the middle tube (2) are located in the lower middle part of the side wall of the middle tube (2).

6. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The micropores on the inner tube (3) are located in the lower middle part of the side wall of the inner tube (3).

7. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: A salt bag (13) is provided at the bottom of the outer tube (1).

8. The integrated pretreatment tube for detecting mycotoxins in camellia milk according to claim 1, characterized in that: The outer tube cover (4), the middle tube cover (5) and the inner tube cover (6) are provided with sealing gaskets.

9. The working method of the integrated pretreatment tube for detecting mycotoxins in camellia milk according to any one of claims 1-8, characterized in that, Includes the following steps: Step 1: Add sample and extraction solution Open the outer tube cap (4) of the outer tube (1), accurately measure the camel milk sample, add it into the outer tube (1), then add acetonitrile solution containing 1% formic acid into the outer tube (1) as an extractant, and then put one or more ceramic or metal vortex oscillators into the tube. Step 2: Extraction Place the assembled pretreatment tube on a vortex oscillator and vortex for 2-3 minutes. Step 3: Separation and Purification Place the pre-treatment tube that has completed vortexing into a high-speed centrifuge and centrifuge at a speed of not less than 8000 rpm for 3-5 minutes. Under the action of centrifugal force, the mixture in the outer tube (1) will separate into layers: the solid residue with a higher specific gravity will be deposited at the bottom of the outer tube (1); the organic extract in the upper layer will move upward and inward in the outer tube (1) and pass through the first microporous filter membrane (8) of the middle tube (2). After the extract enters the middle tube (2), it will come into contact with the QuEChERS reagent package (7) that has been pre-filled at the bottom. After the centrifugation stops, immediately place the pre-treatment tube back on the vortex shaker and shake for 2-3 minutes. Step 4: Filtering The pretreatment tube that has been purified and shaken is put back into the high-speed centrifuge and centrifuged at a speed of not less than 8000 rpm for 3-5 minutes. Under the action of centrifugal force, the purified extract in the middle tube (2) moves downward, passes through the second microporous filter membrane (9) into the inner tube (3), and passes through the ultrafiltration element (12) to obtain a highly clear filtrate. After centrifugation stops, open the inner tube cap (6), remove the ultrafiltration tube (11), and use a micropipette or syringe to draw the solution after the three-step integrated treatment of extraction, purification and filtration from the bottom of the inner tube (3). The solution is the test solution that can be directly used for subsequent HPLC or LC-MS / MS analysis.

10. The working method of the integrated pretreatment tube for detecting mycotoxins in camellia milk according to any one of claims 1-8, characterized in that, Includes the following steps: Step 1: Addition of sample and extraction reagent Open the outer tube cap (4) of the outer tube (1), accurately measure the camel milk sample, add it into the outer tube (1), then add acetonitrile solution containing 1% formic acid into the outer tube (1) as an extractant, and then put one or more ceramic or metal vortex oscillators into the tube. Step 2: Integrated extraction, purification, and filtration operation Place the assembled pretreatment tube in a three-dimensional oscillating centrifuge and vortex at 2000 rpm for 2-3 minutes; Then, centrifuge at 4000 rpm for 10-15 minutes. Under centrifugal force, the mixture in the outer tube (1) separates into layers: the heavier solid residue is deposited at the bottom of the outer tube (1); the upper organic extract moves upward and inward in the outer tube (1) and passes through the first microporous filter membrane (8) of the middle tube (2). After the extract enters the middle tube (2), it comes into contact with the QuEChERS reagent pack (7) that is pre-filled at the bottom. After centrifugation stops, set the speed to 2000 rpm and vortex for 2-3 minutes; Centrifuge at 4000 rpm for 10-15 minutes. Under centrifugal force, the purified extract in the middle tube (2) moves downward, passes through the second microporous filter membrane (9) into the inner tube (3), and passes through the ultrafiltration cartridge (12) to obtain a highly clear filtrate. Step 3: Aspirate the filtrate After centrifugation stops, remove the pretreatment tube, open the top cap (6), remove the ultrafiltration tube (11), and use a micropipette or syringe to draw the solution after the three-step integrated treatment of extraction, purification and filtration from the bottom of the inner tube (3). The solution is the test solution that can be directly used for subsequent HPLC or LC-MS / MS analysis.

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