A double-layer detachable purification and enrichment device and a method of using the same

Abstract

The application discloses a double-layer detachable purification and enrichment device and a use method thereof, and relates to the technical field of sample analysis. The device comprises an enrichment column, a purification column and a needle. The enrichment column and the purification column are detachably connected. According to requirements, the needle is detachably connected with the enrichment column or the purification column. The enrichment column is in the shape of a syringe. A piston, a first filter, enrichment material and a second filter are sequentially arranged in a cavity of the enrichment column from top to bottom. The piston moves up and down in the cavity. The first filter, the enrichment material and the second filter are arranged at the bottom of the cavity. The enrichment material is filled in the interlayer of the first filter and the second filter and is fixed. The purification column is provided with a third filter, purification material and a fourth filter. The purification material is filled in the interlayer of the third filter and the fourth filter and is fixed. The device integrates sampling, purification, enrichment, elution and sample loading processes, and can realize rapid purification and enrichment elution of trace target objects in a solution sample.

Description

Technical Field

[0001] This invention relates to the field of sample analysis technology, and more specifically to a double-layer detachable purification and enrichment device and its usage method. Background Technology

[0002] Antibiotics pose a serious threat to food safety and the ecological environment due to their large usage, easy accumulation, and low limit values. Rapid detection technologies such as colloidal gold immunochromatography for analyzing antibiotic residues are limited by interference from complex animal-derived food matrices and cumbersome pretreatment procedures such as nitrogen blowing enrichment, affecting the sensitivity and accuracy of the methods. Therefore, there is an urgent need to develop efficient, sensitive, and convenient sample pretreatment methods. Summary of the Invention

[0003] In view of this, the present invention provides a dual-layer detachable purification and enrichment device and its usage method. The device integrates sampling, purification, enrichment, elution, and sample loading processes, enabling rapid purification and enrichment elution of trace target substances in solution samples. It is simple, convenient, and user-friendly to operate, and can be used in conjunction with rapid detection technologies that require speed, portability, and real-time processing, solving the problems of matrix interference and low sensitivity in current rapid detection products for analyzing trace target substances.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A double-layer detachable purification and enrichment device includes: an enrichment column, a purification column, and a needle; the enrichment column and the purification column are detachably connected; the needle is detachably connected to the enrichment column or the purification column as needed.

[0006] The enrichment column is syringe-shaped, and its internal cavity contains, from top to bottom, a piston, a first filter, an enrichment material, and a second filter.

[0007] The piston moves up and down in the cavity. The first filter, the enriching material, and the second filter are disposed at the bottom of the cavity. The enriching material is filled in the interlayer between the first filter and the second filter and is fixed in place.

[0008] The purification column is equipped with a third filter, purification material, and a fourth filter; the purification material is filled in the interlayer between the third and fourth filter and is fixed in place.

[0009] Preferably, the detachable connection is a plug-in connection.

[0010] Preferably, the piston has a diameter larger than the cavity diameter, and its own elasticity allows it to adhere tightly to the cavity, forming an elastic seal. The piston moves up and down within the cavity, creating downward pressure that drives the liquid sample to flow between the purification column and the enrichment column.

[0011] Preferably, the length of the piston is greater than or equal to the length of the cavity.

[0012] Preferably, the diameters of the first and second filter elements are matched to the cavity; the diameters of the third and fourth filter elements are matched to the inner diameter of the purification column. This design allows for fixing of the packing material and control of the flow rate, enabling liquid to pass smoothly through the filter elements without perforation of the packing material.

[0013] Preferably, the first filter, the second filter, the third filter and the fourth filter are all porous structures, made of plastic, with a pore size of 10-50 μm and a thickness of ≥0.1 mm.

[0014] Preferably, the purification material is one or more fillers selected from C18, Florisil, and alumina that can adsorb grease, and the particle size of the purification material is 0.01-200μm;

[0015] The enrichment material is a functional material with specific adsorption and desorption properties, such as a metal-organic framework, a covalent organic framework, or a molecularly imprinted polymer and its modified form. The particle size of the enrichment material is 0.01-200 μm. Both the purification material and the enrichment material are solid materials that do not react with the solvent.

[0016] Preferably, the first filter, the enriching material, and the second filter constitute an enrichment assembly; the third filter, the purification material, and the fourth filter constitute a purification assembly; and the number of layers of the enrichment assembly and the purification assembly is ≥1.

[0017] Preferably, the outer diameter of the needle installed at the lower end of the purification column and enrichment column is 0.15-20mm; the needle is made of stainless steel and plastic, and the inner diameter of the sampling head is 0.1-10mm.

[0018] Another object of the present invention is to provide a method of using the above-mentioned double-layer detachable purification and enrichment device, comprising the following steps:

[0019] (1) Connect the enrichment column and the purification column, install a needle on the lower detachable purification column, keep the needle below the liquid surface of the sample solution, pull the piston upward, and drive the sample solution through the purification component and the enrichment component in sequence, and store it in the chamber.

[0020] (2) Push the piston down to drive the sample solution in the chamber through the enrichment component and the purification component again to complete a push-pull cycle. At this time, the target is retained in the enrichment material in the enrichment component and the lower purification column and sample solution are discarded.

[0021] (3) Prepare the eluent, install the needle at the lower end of the enrichment column and keep the needle below the surface of the eluent, pull the piston upward to drive the eluent through the enrichment material of the enrichment component and store it in the chamber, push the piston downward to drive the eluent through the enrichment component and the purification column again to complete one push-pull cycle.

[0022] (4) Collect the eluent. At this point, the target analyte is transferred from the enrichment material in the enrichment component to the eluent and can be directly loaded for detection.

[0023] Preferably, the volume of solution aspirated into the chamber in a single pass through the needle, purification column, and enrichment column is 0.1 mL to 10 mL.

[0024] Based on the above push-pull cycle, the test solution completes the steps of sample collection, purification, enrichment, elution and loading.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0026] In this invention, the dual-layer detachable purification and enrichment device can simultaneously complete sampling, purification, enrichment, and elution processes with only a few piston push-pull operations, and achieve the transfer of target substances between liquid / solid and organic / aqueous phases, replacing cumbersome and time-consuming steps such as vortexing, centrifugation, and nitrogen blowing. This device can effectively remove interference from animal-derived foods such as oils, reduce matrix effects, and improve the accuracy and sensitivity of antibiotic detection. It has advantages such as speed, portability, and user-friendliness, and can be coupled with rapid detection technologies such as colloidal gold immunochromatography.

[0027] The device can be reused by replacing the purification material, enrichment material, and needle, and by cleaning the filter and column chamber, thus saving testing costs. Depending on the properties of different targets and the composition of agricultural product matrices, the types and amounts of adsorption and enrichment materials can be changed to achieve simultaneous purification and enrichment of different targets in various agricultural products. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0030] Figure 2 This is a schematic diagram illustrating the usage of the double-layer detachable purification and enrichment device of the present invention;

[0031] In the diagram, 101-enrichment column; 201-purification column; 1-piston; 2-cavity; 3-first filter; 4-enrichment material; 5-second filter; 6-purification material; 7-third filter; 8-fourth filter; 9-needle. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0033] Example 1

[0034] See Figure 1 This embodiment provides a double-layer detachable purification and enrichment device, including an enrichment column 101, a purification column 201 and a needle 9; the enrichment column 101 and the purification column 201 are detachably connected; according to process requirements, the needle 9 is detachably connected to the enrichment column 101 or the purification column 201.

[0035] The enrichment column 101 is syringe-shaped, and its internal cavity 2 is provided with a piston 1, a first filter 3, an enrichment material 4, and a second filter 5 arranged from top to bottom.

[0036] The piston 1 moves up and down in the cavity 2. The first filter 3, the enriching material 4, and the second filter 5 are located at the bottom of the cavity 2. The enriching material 4 is filled in the interlayer between the first filter 3 and the second filter 5 and is fixed in place. By pushing and pulling the piston 1, the liquid is driven through the filter and material layers.

[0037] The purification column 201 is equipped with a third filter 7, a purification material 6 and a fourth filter 8; the purification material 6 is filled in the interlayer between the third filter 7 and the fourth filter 8 and is fixed in place.

[0038] In this embodiment, piston 1 is made of plastic and rubber. A handle is located at the upper end of piston 1, and rubber is installed at the lower end. The diameter of the rubber is larger than the diameter of the cavity 2. Within the cavity, the rubber adheres tightly to the cavity 2 through its own elasticity, forming an elastic seal. Piston 1 moves up and down within the cavity 2, creating downward pressure, which drives the liquid sample to flow between the purification column 201 and the enrichment column 101.

[0039] The length of piston 1 is greater than or equal to the length of cavity 2. Cavity 2 is made of plastic and can hold a liquid volume of 1-10 mL.

[0040] The diameters of the first filter element 3 and the second filter element 5 are matched with those of the cavity 2; the diameters of the third filter element 7 and the fourth filter element 8 are matched with the inner diameter of the purification column 201. This design allows for the fixing of the packing material and control of the flow rate, enabling liquid to pass smoothly through the filter elements without perforating the packing material.

[0041] In this embodiment, the first filter 3, the second filter 5, the third filter 7 and the fourth filter 8 are all porous structures made of plastic with a pore size of 20 μm and a thickness of 1 mm.

[0042] Purification material 6 is one or more of C18, Florisil, and alumina that can adsorb grease, and the particle size of the purification material is 60 μm.

[0043] Enrichment material 4 is a metal-organic framework or molecularly imprinted polymer capable of specifically enriching and eluting antibiotic fillers, with a particle size of 60 μm. Both the purification material and the enrichment material are solid materials that do not react with the solvent.

[0044] The first filter 3, the enriching material 4, and the second filter 5 constitute an enrichment module; the third filter 7, the purification material 6, and the fourth filter 8 constitute a purification module; the number of layers of the enrichment module and the purification module is 1.

[0045] The outer diameter of the needle 9 installed at the lower end of the purification column 201 and the enrichment column 101 is 0.31 mm.

[0046] The needle 9 is made of stainless steel and plastic, and has an inner diameter of 0.15 mm.

[0047] In use, connect the detachable purification column and the enrichment column. Install the needle 9 on the lower detachable purification column 201, keeping the needle 9 below the sample solution surface. Pull the piston 1 upwards, causing the sample solution to pass sequentially through the purification and enrichment components and be stored in the chamber 2. Push the piston 1 downwards, causing the sample solution in the chamber 2 to pass through the enrichment and purification components again, completing one push-pull cycle. At this point, the target analyte is retained in the enrichment material 4 in the enrichment component. Discard the lower purification column 201 and the sample solution. Prepare the eluent by installing the needle 9 at the lower end of the enrichment column 101, keeping the needle 9 below the eluent surface. Pull the piston 1 upwards, causing the eluent to pass through the enrichment component and be stored in the chamber 2. Push the piston 1 downwards, causing the eluent to pass through the enrichment component again, completing one push-pull cycle. Collect the eluent; at this point, the target analyte has transferred from the enrichment material 4 to the eluent and can be directly loaded for detection.

[0048] Example 2

[0049] The double-layer detachable purification and enrichment device described in Example 1 above was used for the purification and enrichment analysis of quinolone antibiotic extracts from fish (or pork or beef).

[0050] Pretreatment of actual fish (or pork or beef) samples: Homogenize the fish (or pork or beef) and weigh 10.0 ± 0.05 g of the homogenized fish (or pork or beef) sample into a 50 mL centrifuge tube. Add 10 mL of extraction buffer, vortex or shake vigorously for 3 minutes, and centrifuge at 5000 r / min for 5 minutes.

[0051] Take 1 ml of supernatant and install needle 9 at the lower end of the purification column 201 of the double-layer detachable purification and enrichment device, ensuring that needle 9 is below the supernatant surface. Pull piston 1 upwards, causing the supernatant to pass through the purification column 201 and enrichment component once, and be stored in chamber 2. Push piston 1 downwards, causing the sample solution in chamber 2 to pass through the enrichment component and purification column 201 again, completing one push-pull cycle. At this time, the target analyte is retained in the upper enrichment column 101, while impurities such as lipids are retained in the lower purification column 201.

[0052] Discard the lower purification column 201 and sample solution. Prepare the eluent by installing needle 9 at the lower end of the enrichment column 101, keeping needle 9 below the eluent surface. Pull piston 1 upwards to pass the eluent through the enrichment column assembly and store it in chamber 2. Push piston 1 downwards to pass the eluent through the enrichment assembly again, completing one push-pull cycle. Collect the eluent; at this point, the target analyte has moved from the enrichment material 4 into the eluent.

[0053] Take an appropriate amount of eluent and add it to the sample cell of the colloidal gold immunochromatographic card for qualitative detection, or filter it through a 0.22 μm filter membrane and then perform precise quantitative detection using liquid chromatography-tandem mass spectrometry.

[0054] Fish (or pork or beef) sample pretreatment: Homogenize the fish (or pork or beef) and weigh 10.0 ± 0.05 g of the homogenized sample into a 50 mL centrifuge tube. Add 0.01 mg / L quinolone antibiotic standard solution and let stand for 30 min. Add 10 mL of extraction buffer, vortex or shake vigorously for 3 min, and centrifuge at 5000 r / min for 5 min.

[0055] Take 1 ml of supernatant and install needle 9 at the lower end of the purification column 201 of the double-layer detachable purification and enrichment device, ensuring that needle 9 is below the supernatant surface. Pull piston 1 upwards, causing the supernatant to pass through the purification column 201 and enrichment component once, and be stored in chamber 2. Push piston 1 downwards, causing the sample solution in the chamber to pass through the enrichment component and purification column 201 again, completing one push-pull cycle. At this time, the target analyte is retained in the upper enrichment column 101, while impurities such as lipids are retained in the lower purification column 201.

[0056] Discard the lower purification column 201 and the sample solution. Prepare the eluent by installing needle 9 at the lower end of the enrichment column 101, keeping needle 9 below the eluent surface. Pull piston 1 upwards to pass the eluent through the enrichment assembly and store it in chamber 2. Push piston 1 downwards to pass the eluent through the enrichment assembly again, completing one push-pull cycle. Collect the eluent; at this point, the target analyte has been transferred from the enrichment material 4 to the eluent.

[0057] Take an appropriate amount of eluent and add it to the sample cell of the colloidal gold immunochromatographic card for qualitative detection, or filter it through a 0.22 μm filter membrane and then perform precise quantitative detection using liquid chromatography-tandem mass spectrometry.

[0058] Colloidal gold immunochromatographic assay results showed that the detection limit of quinolone antibiotics in fish, pork, and beef was as low as 0.5 μg / L after treatment with a double-layer detachable purification and enrichment device. Compared with untreated samples, the chromatography was faster and more accurate, effectively avoiding false positive and false negative results caused by matrix interference and low sensitivity.

[0059] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) results showed that the recoveries of quinolone antibiotics in fish, pork, and beef were as shown in Tables 1-3. The recoveries of quinolone antibiotics in different animal tissues ranged from 85% to 113%, with relative standard deviations ranging from 1% to 10%, exhibiting good sensitivity and accuracy. Therefore, the dual-layer detachable purification and enrichment device is practical for the detection of quinolone antibiotics in animal-derived foods.

[0060] Table 1. Recovery results of four quinolone antibiotics in fish meat.

[0061]

[0062] Table 2. Recovery results of three quinolone antibiotics in pork.

[0063]

[0064] Table 3. Recovery results of three quinolone antibiotics in beef.

[0065]

[0066] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0067] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A double-layer detachable purification and enrichment device, characterized in that, include: An enrichment column, a purification column, and a needle; the enrichment column and the purification column are detachably connected; the needle is detachably connected to either the enrichment column or the purification column as needed. The enrichment column is syringe-shaped, and its internal cavity contains, from top to bottom, a piston, a first filter, an enrichment material, and a second filter. The piston moves up and down in the cavity. The first filter, the enriching material, and the second filter are disposed at the bottom of the cavity. The enriching material is filled in the interlayer between the first filter and the second filter and is fixed in place. The purification column is equipped with a third filter, purification material, and a fourth filter; the purification material is filled in the interlayer between the third and fourth filter and is fixed in place. The first, second, third, and fourth filter sheets are all porous, made of plastic, with a pore size of 10-50 μm and a thickness of ≥0.1 mm. The purification material is one or more of C18, Florisil, and alumina, and the particle size of the purification material is 0.01-200μm; The enriched materials include: metal-organic frameworks, covalent organic frameworks or molecularly imprinted polymers and their modified materials, and the particle size of the enriched materials is 0.01-200 μm.

2. The double-layer detachable purification and enrichment device according to claim 1, characterized in that, The piston has a diameter larger than the cavity diameter, and it adheres tightly to the cavity through its own elasticity to form an elastic seal.

3. The double-layer detachable purification and enrichment device according to claim 1, characterized in that, The length of the piston is greater than or equal to the length of the cavity.

4. The double-layer detachable purification and enrichment device according to claim 1, characterized in that, The diameters of the first and second filter elements are matched with the cavity; the diameters of the third and fourth filter elements are matched with the inner diameter of the purification column.

5. The double-layer detachable purification and enrichment device according to claim 1, characterized in that, The first filter, the enriching material, and the second filter constitute an enrichment assembly; the third filter, the purification material, and the fourth filter constitute a purification assembly; the number of layers of the enrichment assembly and the purification assembly is ≥1.

6. The double-layer detachable purification and enrichment device according to claim 1, characterized in that, The outer diameter of the needles installed at the lower end of the purification column and enrichment column is 0.15-20mm; the needles are made of stainless steel and plastic, and the inner diameter of the needles is 0.1-10mm.

7. A method of using the double-layer detachable purification and enrichment device as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Install a needle on the lower removable purification column and keep the needle below the liquid surface of the sample solution. Pull the piston upward to drive the sample solution through the purification component and enrichment component in sequence and store it in the chamber. (2) Push the piston down to drive the sample solution in the chamber through the enrichment component and the purification component again to complete a push-pull cycle. At this time, the target is retained in the enrichment material in the enrichment component and the lower purification column and sample solution are discarded. (3) Prepare the eluent, install the needle at the lower end of the enrichment column and keep the needle below the surface of the eluent, pull the piston upward to drive the eluent through the enrichment assembly and store it in the chamber, push the piston downward to drive the eluent through the enrichment assembly again to complete one push-pull cycle. (4) Collect the eluent. At this point, the target analyte is transferred from the enrichment material in the enrichment component to the eluent and can be directly loaded for detection.

8. The method of using the double-layer detachable purification and enrichment device according to claim 7, characterized in that, The volume of solution aspirated into the chamber in a single pass through the needle, purification column, and enrichment column is 0.1 mL to 10 mL.

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