Integrated extraction, purification and filtration integrated purification column and application thereof

Abstract

The application discloses an integrated extraction, purification and filtration integrated purification column and application thereof. The integrated purification column comprises a cavity column body, a first sieve plate, a second sieve plate and a third sieve plate are sequentially arranged in the cavity column body along a fluid flow direction, purification filler is filled between the second sieve plate and the third sieve plate, and a filter membrane is arranged below the third sieve plate. The integrated purification column of the embodiment can realize that the sample after extraction does not need to be centrifuged, filtered and repeatedly transferred, directly enters the integrated purification column for purification and filtration by gravity through the sieve plate, and then direct detection can be performed, so that the sample pretreatment is controlled within 30 min from 60 min, and cost increase and environmental pollution caused by use of consumables such as centrifugal tubes, gun heads and injectors are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to an integrated extraction, purification and filtration one-body purification column and application thereof. BACKGROUND

[0002] In order to monitor the contamination of mycotoxins, pesticide residues and veterinary drug residues in grain and oil, feed and food, it is urgent to develop a simple and rapid method for detecting mycotoxins, pesticides and veterinary drugs in grain and oil, feed and food. The existing purification methods mainly include immunosorbent column and multifunctional purification column purification. The immunosorbent column and multifunctional purification column purification have the problems of complicated operation steps, complexity and time-consuming.

[0003] In summary, the existing extraction and purification methods of mycotoxins, pesticide residues and veterinary drug residues have the problems of complicated purification extraction process, low efficiency and poor effect, and a new integrated extraction, purification and filtration one-body purification column and detection method are urgently needed. SUMMARY

[0004] Therefore, the present application provides an integrated extraction, purification and filtration one-body purification column and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] The present application provides an integrated extraction, purification and filtration one-body purification column, which comprises a cavity column body, wherein a first sieve plate, a second sieve plate and a third sieve plate are sequentially arranged in the cavity column body along the fluid flow direction, and a purification filler is filled between the second sieve plate and the third sieve plate.

[0007] In an embodiment of the present application, a filter membrane is further arranged in the cavity column body, and the filter membrane is arranged close to the liquid outlet in the cavity column body.

[0008] In an embodiment of the present application, an upper cover body is arranged at the feed inlet of the cavity column body, and a lower cover body is arranged at the liquid outlet of the cavity column body.

[0009] In an embodiment of the present application, the purification filler is one or more of graphitized carbon black filler (GCB), modified carbon 18 filler (modified C18), cation exchange filler (MCX), high-nitrogen load cation mixed filler (POLY), anion exchange filler (MAX), N-propyl ethylenediamine filler (PSA), hydrophilic-lipophilic balance filler (HLB) and carbon 8 filler (C8).

[0010] The application also provides application of the integrated purification column in any one of the following: (1) as a product for extracting, purifying and filtering mycotoxins; (2) as a product for extracting, purifying and filtering pesticide residues; (3) as a product for extracting, purifying and filtering veterinary drug residues.

[0011] In one embodiment of the application, the extraction, purification and filtration process comprises the following steps:

[0012] The sample to be tested is taken and added to the integrated purification column, and an extraction solution is added.

[0013] The feed inlet and liquid outlet of the purification column are blocked, and the sample in the purification column is oscillated, homogenized and ultrasonicated; the feed inlet and liquid outlet are opened, and the purified sample liquid at the liquid outlet is collected, wherein the purified sample contains the target substance.

[0014] In one embodiment of the application, the mycotoxin is selected from one or more of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, nivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, ochratoxin A, fumonisin B1, fumonisin B2, versicolorin, T-2 toxin or HT-2 toxin, ergot alkaloid toxin, stachybotryotoxin, beauvericin or enniatin.

[0015] In one embodiment of the application, the pesticide is selected from one or more of organophosphates, organochlorines, pyrethroids or carbamates.

[0016] In one embodiment of the application, the veterinary drug residue is selected from one or more of quinolones, macrolides, sulfonamides, sex hormones, nitroimidazoles or chloramphenicol.

[0017] The application has the following advantages:

[0018] The integrated purification column purification method of the embodiment of the application can realize that after extraction, the sample does not need to be centrifuged, filtered and repeatedly transferred, but directly enters the integrated purification column for purification and filtration by gravity through the sieve plate and is collected in the sample feeding vial, so that the sample pretreatment can be controlled within 30 min from 60 min, and the cost increase and environmental pollution caused by the use of consumables such as centrifuge tubes, gun heads and syringes are reduced.

[0019] The filler used in the purification column provided by the embodiment of the present application is based on the previous filler research, and a new type of composite purification filler for detecting mycotoxins in complex substrates such as grain and oil and feed can be obtained by using a series of technical means such as core-shell growth, integral column modification, bonding and surface modification, and the purification effect of the filler on mycotoxins is good. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and other implementation drawings can be obtained according to the provided drawings without creative labor for those skilled in the art.

[0021] The structures, proportions, sizes and the like shown in the specification are only used to cooperate with the content disclosed in the specification, to be understood and read by those skilled in the art, and do not define the limiting conditions for the implementation of the present application, so they do not have technical significance. Any modification of structure, change of proportion relationship or adjustment of size, without affecting the effect and purpose that can be achieved by the present application, should still fall within the scope of the technical content disclosed by the present application.

[0022] Figure 1 The structural schematic diagram of the integrated purification column provided by the embodiment of the present application is provided.

[0023] Figure 2 The recovery rate, matrix effect and extraction time comparison chart of the integrated purification column provided by the embodiment of the present application and the existing purification column for extracting single-endotoxin mycotoxins are provided.

[0024] Figure 3 The comparison chart of the purification effect of the integrated purification column provided by the embodiment of the present application and the existing purification column on 14 kinds of mycotoxins is provided.

[0025] Figure 4 The comparison photo chart of the pesticide residue integrated purification column before and after purification provided by the embodiment of the present application is provided.

[0026] Figure 5 The mass spectrum full scan comparison chart of the veterinary drug residue integrated purification column before and after purification provided by the embodiment of the present application is provided.

[0027] In the figure: 100-cavity column body; 110-first sieve plate; 120-second sieve plate; 130-third sieve plate; 140-upper cover body; 150-lower cover body. DETAILED DESCRIPTION

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] like Figure 1 As shown, this invention provides an integrated purification column that combines extraction, purification, and filtration. The purification column includes a hollow column 100, specifically divided into an extraction column and a purification column, with the diameter of the purification column being smaller than that of the extraction column. A first sieve plate 110, a second sieve plate 120, and a third sieve plate 130 are sequentially arranged within the hollow column 100 along the liquid flow direction. Purification packing is filled between the second sieve plate 120 and the third sieve plate 130. The purification packing of the single-terminal mycotoxin integrated purification column is a mixture of 200 mg GCB, 100 mg C18-CX, 100 mg MCX, and 50 mg POLY.

[0031] The purification packing material of the multi-mycotoxin integrated purification column is a mixture of 200mg C18-CX, 100mg MCX and 100mg POLY.

[0032] In this embodiment, the first and second sieve plates have pore sizes of 20 μm, which is smaller than the particle size of the packing material. This ensures that the packing material will not overflow from the pores while also preventing small impurities from clogging the sieve plates, allowing the liquid to flow smoothly. The third sieve plate has a pore size of 5 μm, which can intercept impurity particles to the maximum extent and ensure that the lower filter membrane will not become clogged.

[0033] The first sieve plate 110 is disposed in the extraction column, and the second sieve plate 120 and the third sieve plate 130 are disposed in the purification column.

[0034] In the purification column of the present invention, a filter membrane is further provided inside the hollow column 100, and the filter membrane is disposed at the liquid outlet of the hollow column 100. Preferably, an upper cover 140 is provided at the feed inlet of the hollow column 100, and a lower cover 150 is provided at the liquid outlet of the hollow column 100. When processing the sample, the sample is also added into the purification column, the upper cover 140 and the lower cover 150 are covered, and continuous vortexing / homogenization is performed to ensure that the mycotoxins in the sample are fully dissolved in the extract.

[0035] This invention provides a method for extracting, purifying, filtering, and detecting mycotoxins using the above-mentioned purification column, comprising the following steps:

[0036] Step one, after crushing 1g of the sample to be tested, add it to the purification column, and add 4mL of extraction solution, wherein the extraction solution is acetonitrile: water: acetic acid solution, wherein the volume ratio of acetonitrile, water and acetic acid is 70:29:1.

[0037] Step two, respectively block the feed inlet and liquid outlet of the purification column with the upper cover body 140 and the lower cover body 150, and vortex / homogenize the purification column for 15-25min;

[0038] Step three, open the feed inlet and liquid outlet, and collect 1mL of the purified sample liquid from the liquid outlet;

[0039] Step four, prepare the standard curve working solution of the target fungus toxin to be detected;

[0040] Step five, detect the standard curve working solution and the purified sample liquid to be detected by liquid chromatography;

[0041] Step six, obtain the standard curve according to the detection result, and calculate the concentration of the fungus toxin in the purified sample liquid to be detected according to the standard curve.

[0042] The fungus toxin is aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, nivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, ochratoxin A, fumonisin B1, fumonisin B2, versicolor toxin, T-2 toxin or HT-2 toxin, ergot alkaloid toxin, fusariotoxin, and beauvericin.

[0043] The corn sample is extracted, purified and filtered by using the above integrated purification column, solid phase extraction column and immune affinity column.

[0044] Table 1

[0045] Item Example 1 One-Pot Purification Column Multi-Purpose Purification Column Solid Phase Extraction Cartridge Immunoaffinity Column Cost / Root (Yuan) 40 120 80 100 Operation Step / Sample (Step) 3 8 8 11 Detection Time / Sample (h) 0.5 1 1 2 Detectable Mycotoxins (Number) 9-14 4-9 4-9 1 Consumable Usage (Number) 1 4 4 4 Recovery Rate (%) 80-110 70-130 70-130 80-110

[0046] As shown in Table 1, the purification effect of the integrated purification column of Example 1 of the present application and the purification methods of the existing multifunctional purification column, solid phase extraction column and immune affinity column are compared: the recovery rate of the integrated purification column of Example 1 is better than that of the existing mainstream fungus toxin purification products, the operation steps and time, the amount of consumables used and the cost are reduced by at least one time, and from the mass spectrum full scan graph, the matrix effect graph and the purification solution graph, it can be seen that the impurity removal effect is obvious.

[0047] As Figure 2As shown, the recovery rate of the single-endospore mycotoxin special purification column of the embodiment 1 of the present application to 7 kinds of mycotoxins is 85%-100%, the matrix effect is 80%-110%, the recovery rate and the matrix effect removal effect are obviously better than those of several other commercially available products, and it can be seen from the full scan mass spectrum that the removal of four main impurity peaks (outlined by dashed lines) is obvious.

[0048] As shown, Figure 3 As shown, the recovery rate of the multi-fungus toxin integrated purification column of the embodiment 1 of the present application to 14 kinds of mycotoxins is 80%-110%, the matrix effect is 90%-130%, the recovery rate and the matrix effect removal effect are obviously better than those of several other commercially available products, and it can be seen from the full scan mass spectrum that the removal of three main impurity peaks (outlined by dashed lines) is obvious;

[0049] Embodiment 2

[0050] As shown, Figure 1 The embodiment provides an integrated purification column integrating extraction, purification and filtration. The integrated purification column comprises a cavity column body 100. Specifically, the cavity column body 100 is divided into an extraction column body and a purification column body, and the diameter of the purification column body is smaller than that of the extraction column body. A first sieve plate 110, a second sieve plate 120 and a third sieve plate 130 are sequentially arranged in the cavity column body 100 along the liquid flow direction, and a purification filler is filled between the second sieve plate 120 and the third sieve plate 130. The second sieve plate 120 and the third sieve plate 130 are arranged in the purification column body, and the purification filler is a mixture of 50 mg GCB, 200 mg C18-CX, 100 mg MCX and 100 mg POLY.

[0051] In the purification column body of the present application, a filter membrane is further arranged in the cavity column body 100, and the filter membrane is arranged at the liquid outlet of the cavity column body 100. An upper cover body 140 is arranged at the feed inlet of the cavity column body 100, and a lower cover body 150 is arranged at the liquid outlet of the cavity column body 100. When the sample is treated, the sample is also added to the purification column body, the upper cover body 140 and the lower cover body 150 are covered, and vortex oscillation / homogenization is continuously performed, so that the pesticide in the sample is fully dissolved into the extraction liquid.

[0052] The present application provides a method for extracting, purifying and detecting pesticide residues in rice by using the above-mentioned purification column, which comprises the following steps:

[0053] Step 1: After 1 g of the sample to be measured is pulverized and treated, it is added to the purification column, and 4 mL of extraction liquid is added, wherein the extraction liquid is acetonitrile: water: acetic acid solution (the volume ratio of acetonitrile, water and acetic acid is 70:29:1);

[0054] Step 2: The feed inlet and the liquid outlet of the purification column are blocked by the upper cover body 140 and the lower cover body 150 respectively, and the purification column is vortexed / homogenized for 15-25 min;

[0055] Step 3: Open the inlet and outlet, and collect 1 mL of purified sample solution from the outlet;

[0056] Step 4: Prepare the working solution for the standard curve of the target pesticide;

[0057] Step 5: Detect the standard curve working solution and the purified sample solution to be tested using liquid chromatography-mass spectrometry (LC-MS).

[0058] Step 6: Obtain the standard curve based on the test results, and calculate the concentration of pesticide residues in the purified sample solution to be tested according to the standard curve.

[0059] The pesticides included are methamidophos, imidacloprid, acetamiprid, amitraz, pirimicarb, imazalil, tebuconazole, dimethomorph, azoxystrobin, metalaxyl, chlorfenapyr, propiconazole, thiamethoxam, paclobutrazol, thiamethoxam, carbofuran, flufenoxuron, methoxyfenozide, matrine, and cyromazine.

[0060] The results of using the integrated purification column to extract, purify, and filter pesticides from rice are shown in Table 2. Figure 4 As shown in Table 2, the recovery rates of different pesticides are as follows. Table 2 indicates that the pesticide recovery rates range from 80% to 110%. Figure 4 This indicates a significant purification effect.

[0061] Table 2

[0062]

[0063] Example 3

[0064] like Figure 1 As shown, this invention provides an integrated purification column that combines extraction, purification, and filtration. The purification column includes a hollow column 100, which is divided into an extraction column and a purification column. The diameter of the purification column is smaller than that of the extraction column. A first sieve plate 110, a second sieve plate 120, and a third sieve plate 130 are sequentially arranged within the hollow column 100 along the liquid flow direction. Purification packing material is filled between the second sieve plate 120 and the third sieve plate 130. The second sieve plate 120 and the third sieve plate 130 are disposed within the purification column, and the purification packing material is a mixture of 200 mg C18-CX and 200 mg POLY.

[0065] In the purification column of the present invention, a filter membrane is also provided inside the hollow column 100, and the filter membrane is located at the liquid outlet of the hollow column 100. An upper cover 140 is provided at the feed inlet of the hollow column 100, and a lower cover 150 is provided at the liquid outlet of the hollow column 100. When processing the sample, the sample is also added into the purification column, the upper cover 140 and the lower cover 150 are covered, and the column is continuously vortexed / homogenized to ensure that the pesticides in the sample are fully dissolved in the extract.

[0066] The application provides a method for extracting and purifying veterinary drug residues by using the integrated purification column, which comprises the following steps.

[0067] Step one, 1g of the sample to be detected is crushed and then added into the purification column, and 4mL of extraction liquid is added, wherein the extraction liquid is acetonitrile: water: acetic acid solution (the volume ratio of acetonitrile, water and acetic acid is 70:29:1).

[0068] Step two, the upper cover body 140 and the lower cover body 150 are used to block the feeding port and the liquid outlet of the purification column respectively, and the purification column is vortexed / homogenized for 15-25min;

[0069] Step three, the feeding port and the liquid outlet are opened, and 1mL of the purified sample liquid at the liquid outlet is collected;

[0070] Step four, the standard curve working solution of the veterinary drug residues to be detected is prepared;

[0071] Step five, the standard curve working solution and the purified sample liquid to be detected are detected by using liquid chromatography;

[0072] Step six, the standard curve is obtained according to the detection result, and the concentration of the veterinary drug residues in the purified sample liquid to be detected is calculated according to the standard curve.

[0073] The veterinary drug residues are Trenbolone, Progesterone, Cortisone, Fluorometholone, Triamcinolone, Fluocinolone, Estradiol, Chloramphenicol, Sulfadimethoxine, Sulfachlorpyridazine, Enrofloxacin, Flumequine, Tetracycline, Oxytetracycline, Tinidazole, Ronidazole, Tilmicosin and Spiramycin.

[0074] The results of the integrated extraction, purification and filtration of the veterinary drug residues in the pork sample by using the integrated purification column are shown in Tables 3 and Figure 5 Table 3 is the standard addition recovery rate of different veterinary drugs, and Table 3 shows that the recovery rate of the veterinary drug residues is 80%-110%, Figure 5 The mass spectrum full scan diagram shows that the lipid impurity peak removal effect is obvious.

[0075] Table 3

[0076]

[0077]

[0078] Although the application has been described in detail by using general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application are within the scope of the application.

Claims

1. An integrated purification column combining extraction, purification, and filtration, characterized in that, The integrated purification column includes a hollow column body, in which a first sieve plate, a second sieve plate and a third sieve plate are arranged sequentially along the fluid flow direction, and purification filler is filled between the second sieve plate and the third sieve plate. The purification packing material is a mixture of 200 mg C18-CX, 100 mg MCX, and 100 mg POLY; or, The purification packing material is a mixture of 50 mg GCB, 200 mg C18-CX, 100 mg MCX, and 100 mg POLY; or, The purification packing material is a mixture of 200 mg C18-CX and 200 mg POLY.

2. The integrated purification column for extraction, purification, and filtration as described in claim 1, characterized in that, The hollow column is also equipped with a filter membrane, which is located in the hollow column near the liquid outlet.

3. The integrated purification column for extraction, purification, and filtration as described in claim 1, characterized in that, The hollow column is provided with an upper cover at the feed inlet and a lower cover at the liquid outlet.

4. The application of the integrated purification column according to any one of claims 1-3 in any of the following: (1) As a product for extracting, purifying, and filtering fungal toxins; (2) As a product for extracting, purifying, and filtering pesticide residues; (3) As a product for extracting, purifying and filtering veterinary drug residues.

5. The application as described in claim 4, characterized in that, The extraction, purification, and filtration process includes the following steps: Take the sample to be tested, add it into the integrated purification column, and add the extraction solution; Block the inlet and outlet of the purification column, and vibrate / homogenize / ultrasonicate the sample in the purification column; open the inlet and outlet, and collect the purified sample liquid from the outlet, wherein the purified sample contains the target analyte.

6. The application as described in claim 4, characterized in that, The mycotoxins are selected from one or more of the following: aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, ochratoxin A, fumonisin B1, fumonisin B2, variegated aflatoxin, T-2 toxin or HT-2 toxin, ergot alkaloids, Alternaria alkaloids, beauveria bassiana, or enfuracin.

7. The application as described in claim 4, characterized in that, The pesticide is selected from one or more of organophosphates, organochlorines, pyrethroids, or carbamates.

8. The application as described in claim 5, characterized in that, The veterinary drug residues are selected from one or more of quinolones, macrolides, sulfonamides, sex hormones, nitroimidazoles, or chloramphenicol.

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