A sample processing method and detection method

Abstract

The present invention discloses a method for processing and detecting a sample. The present invention is based on the addition of a phase separator, a purifier and an extractant to respectively realize extraction and oil removal treatment, solves the difficulty of large conflicts in the extraction and oil removal process due to the large difference in the chemical properties of enrofloxacin, malachite green and diazepam, realizes a four-in-one unified sample pretreatment method of chloramphenicol, diazepam, malachite green and enrofloxacin, and the sample is pretreated once. Relative to the sample pretreatment method of a single compound, under the premise of ensuring high sensitivity, the detection time is greatly shortened, and it is subsequently applicable to the on-site rapid large-scale screening test of the colloidal gold method, and the minimum detection limit of each compound is 0.1μg / kg, 0.5μg / kg, 0.5μg / kg and 2μg / kg, respectively, which meets the accuracy and stability requirements of the detection and is convenient for the detection operation of grassroots personnel.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a sample processing method and a detection method. Background Art

[0002] Chloramphenicol and enrofloxacin are broad-spectrum antibiotics, diazepam is a commonly used sedative and anti-anxiety drug, and malachite green was once a commonly used antibacterial agent in aquaculture. Although these four chemicals have certain anti-disease effects, they have now been banned or restricted in order to ensure the safety of animal-derived food.

[0003] In the prior art, corresponding detection methods have been established for chloramphenicol, diazepam, malachite green, and enrofloxacin, mainly including high-performance liquid chromatography, high-performance liquid chromatography-tandem mass spectrometry, enzyme-linked immunosorbent assay, and colloidal gold immunochromatography. These detection methods all require pretreatment of the sample to be tested, and there is currently no unified pretreatment detection scheme for chloramphenicol, diazepam, malachite green, and enrofloxacin in the sample. The detection reagents and detection steps are also different for each compound, greatly reducing the efficiency of rapid screening. Therefore, it is particularly important to develop a unified sample pretreatment method that can meet the detection needs of multiple compounds and shorten the detection time. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a sample processing method and a detection method.

[0005] An object of the present invention is to provide a method for processing a sample.

[0006] Another object of the present invention is to provide a method for detecting a sample.

[0007] In order to achieve the above object, the present invention is implemented through the following scheme:

[0008] The present invention utilizes a phase separator, a purifier, and an extractant to achieve extraction and oil removal, respectively. Malachite green and diazepam are fat-soluble substances with low polarity, making them easily soluble in oils and low-polarity solvents. Low-polarity solvent extraction methods cannot be used for oil removal during sample processing. Chloramphenicol and enrofloxacin, on the other hand, have relatively high polarity and are insoluble in oils and low-polarity solvents, making them suitable for oil removal. These two groups of compounds exhibit opposing chemical properties, leading to significant conflicts during the extraction and oil removal processes. Only by selecting a reasonable balance between the purification and extraction processes can the maximum extraction yield of these four compounds be achieved. Furthermore, the extraction yield can be kept essentially consistent across different samples during application, thereby addressing the issue of significant processing variability. Among them, the selection of extractant and purifier is particularly critical. The extractant selected in the present invention not only ensures the extraction rate of multiple compounds, but also satisfies the requirements of being able to separate from the water layer and the purifier layer under the action of a phase separator. At the same time, when removing oily esters, it solves the difficulty of separating malachite green and diazepam from oily esters, thereby ensuring the removal of oily esters and the extraction rate.

[0009] A sample processing method comprises the following steps:

[0010] The sample to be processed is fully reacted with a phase separation agent, a purifying agent and an extracting agent, and then the resulting reaction product is layered. After the reaction product is separated into three layers, the middle layer is collected;

[0011] The phase separation agent includes any one of sodium sulfate, magnesium sulfate or sodium chloride;

[0012] The purifier comprises a low-polarity solvent containing acetic acid, wherein the low-polarity solvent is any one of n-hexane, petroleum ether or n-heptane, and the content of acetic acid in the low-polarity solvent is 0.5% (v / v) to 2% (v / v);

[0013] The extractant includes a mixture of acetonitrile and ethyl acetate, and the volume ratio of the acetonitrile to the ethyl acetate is (1-4):1.

[0014] Preferably, the sample to be processed includes a tissue sample.

[0015] More preferably, the sample to be processed is a tissue sample of an aquatic animal.

[0016] Preferably, the sample to be processed is first homogenized and then fully reacted with the phase separation agent, the purifying agent and the extracting agent.

[0017] In some specific embodiments, the mass volume ratio of the sample to be processed to the phase separation agent, the cleanup agent and the extractant is 4 g:2 g:2 mL:6 mL.

[0018] Preferably, the phase separation agent is any one of sodium sulfate, magnesium sulfate or sodium chloride.

[0019] More preferably, the phase separation agent is sodium chloride.

[0020] Preferably, the low polarity solvent is n-hexane.

[0021] Preferably, the purifier is a low-polarity solvent containing acetic acid.

[0022] Preferably, the content of acetic acid in the low-polarity solvent is 1% (v / v) to 2% (v / v).

[0023] More preferably, the content of acetic acid in the low-polarity solvent is 1% (v / v).

[0024] Preferably, the extractant is a mixture of acetonitrile and ethyl acetate.

[0025] More preferably, the volume ratio of the acetonitrile to the ethyl acetate is (2-4):1.

[0026] Further preferably, the volume ratio of the acetonitrile to the ethyl acetate is 2:1.

[0027] Preferably, the method of fully reacting comprises mixing.

[0028] More preferably, the mixing method comprises shaking.

[0029] In some specific embodiments, the shaking conditions include: shaking and mixing at an amplitude of 120 times / min for 2 minutes.

[0030] Preferably, the method of separating the resulting reaction products comprises centrifugation.

[0031] In some specific embodiments, the centrifugal conditions include: centrifugation at a rotation speed of 4000 r / min for 3 minutes.

[0032] Preferably, the treatment method further comprises the following step: fully reacting the obtained intermediate layer with an oxidant.

[0033] More preferably, the oxidizing agent comprises 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0034] More preferably, the oxidant is acetonitrile containing 0.02 wt% to 0.1 wt% of 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0035] More preferably, the oxidant is acetonitrile containing 0.05 wt% of 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0036] More preferably, the treatment method further comprises the following step: collecting the obtained reaction product and drying it.

[0037] Further preferably, the drying method includes heating and blowing.

[0038] More preferably, the heating temperature is 50°C to 70°C.

[0039] Still more preferably, the heating temperature is 60°C.

[0040] Further preferably, the steps of fully reacting the obtained intermediate layer with the oxidant and then drying are both performed in a sample concentrator.

[0041] Further preferably, the treatment method further comprises the following step: re-dissolving the dried residue.

[0042] More preferably, the dried residue is re-dissolved with a weakly acidic buffer.

[0043] Still further preferably, the weakly acidic buffer solution is a weakly acidic sodium citrate buffer solution.

[0044] Still more preferably, the concentration of the sodium citrate buffer solution is 0.05 mol / L to 0.2 mol / L, and the pH is 5.5 to 6.2.

[0045] Most preferably, the concentration of the sodium citrate buffer solution is 0.1 mol / L, and the pH is 6.0.

[0046] A method for detecting a sample, processing a sample to be tested to obtain a test sample, and detecting the test sample by immunochromatography;

[0047] The method for treating a sample to be tested comprises the following steps: fully reacting the sample to be treated with a phase separation agent, a purifying agent and an extracting agent, then separating the resulting reactants into three layers, and collecting the middle layer;

[0048] The phase separation agent includes any one of sodium sulfate, magnesium sulfate or sodium chloride;

[0049] The purifier comprises a low-polarity solvent containing acetic acid, wherein the low-polarity solvent is any one of n-hexane, petroleum ether or n-heptane, and the content of acetic acid in the low-polarity solvent is 0.5% (v / v) to 2% (v / v);

[0050] The extractant includes a mixture of acetonitrile and ethyl acetate, and the volume ratio of the acetonitrile to the ethyl acetate is (1-4):1.

[0051] Preferably, the sample to be processed includes a tissue sample.

[0052] More preferably, the sample to be processed is a tissue sample of an aquatic animal.

[0053] Preferably, the sample to be processed is first homogenized and then fully reacted with the phase separation agent, the purifying agent and the extracting agent.

[0054] In some specific embodiments, the mass volume ratio of the sample to be processed to the phase separation agent, the cleanup agent and the extractant is 4 g:2 g:2 mL:6 mL.

[0055] Preferably, the phase separation agent is any one of sodium sulfate, magnesium sulfate or sodium chloride.

[0056] More preferably, the phase separation agent is sodium chloride.

[0057] Preferably, the low polarity solvent is n-hexane.

[0058] Preferably, the purifier is a low-polarity solvent containing acetic acid.

[0059] Preferably, the content of acetic acid in the low-polarity solvent is 1% (v / v) to 2% (v / v).

[0060] More preferably, the content of acetic acid in the low-polarity solvent is 1% (v / v).

[0061] Preferably, the extractant is a mixture of acetonitrile and ethyl acetate.

[0062] More preferably, the volume ratio of the acetonitrile to the ethyl acetate is (2-4):1.

[0063] Further preferably, the volume ratio of the acetonitrile to the ethyl acetate is 2:1.

[0064] Preferably, the method of fully reacting comprises mixing.

[0065] More preferably, the mixing method comprises shaking.

[0066] In some specific embodiments, the shaking conditions include: shaking and mixing at an amplitude of 120 times / min for 2 minutes.

[0067] Preferably, the method of separating the resulting reaction products comprises centrifugation.

[0068] In some specific embodiments, the centrifugal conditions include: centrifugation at a rotation speed of 4000 r / min for 3 minutes.

[0069] Preferably, the treatment method further comprises the following step: fully reacting the obtained intermediate layer with an oxidant.

[0070] More preferably, the oxidizing agent comprises 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0071] More preferably, the oxidant is acetonitrile containing 0.02 wt% to 0.1 wt% of 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0072] More preferably, the oxidant is acetonitrile containing 0.05 wt% of 2,3-dichloro-5,6-dicyano-p-benzoquinone.

[0073] More preferably, the treatment method further comprises the following step: collecting the obtained reaction product and drying it.

[0074] Further preferably, the drying method includes heating and blowing.

[0075] More preferably, the heating temperature is 50°C to 70°C.

[0076] Still more preferably, the heating temperature is 60°C.

[0077] Further preferably, the steps of fully reacting the obtained intermediate layer with the oxidant and then drying are both performed in a sample concentrator.

[0078] Further preferably, the treatment method further comprises the following step: re-dissolving the dried residue.

[0079] More preferably, the dried residue is re-dissolved with a weakly acidic buffer.

[0080] Still further preferably, the weakly acidic buffer solution is a weakly acidic sodium citrate buffer solution.

[0081] Still more preferably, the concentration of the sodium citrate buffer solution is 0.05 mol / L to 0.2 mol / L, and the pH is 5.5 to 6.2.

[0082] Most preferably, the concentration of the sodium citrate buffer solution is 0.1 mol / L, and the pH is 6.0.

[0083] Preferably, the immunochromatography method comprises colloidal gold immunochromatography.

[0084] Compared with the prior art, the present invention has the following beneficial effects:

[0085] Compared with traditional single pretreatment methods, the present invention implements a four-in-one unified sample pretreatment method for chloramphenicol, diazepam, malachite green, and enrofloxacin, requiring only a single sample pretreatment. Compared with sample pretreatment methods for individual compounds, this method significantly shortens detection time while maintaining high sensitivity. It is subsequently suitable for rapid, large-scale, on-site screening using colloidal gold methods, with minimum detection limits of 0.1 μg / kg, 0.5 μg / kg, 0.5 μg / kg, and 2 μg / kg for each compound, respectively. This method meets the accuracy and stability requirements of the test and facilitates detection operations by grassroots personnel. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The figure shows the operating flow of a sample pretreatment method for the simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0088] Example 1 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0089] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0090] 1. Mixed extraction

[0091] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99.5:0.5, wherein the acetic acid concentration is 0.5% (v / v)) and 6 mL of extractant (an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 1:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0092] 2. Oxidation drying

[0093] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 4 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0094] 3. Redissolve

[0095] Use acidic sodium citrate buffer solution (0.1 mol / L, pH=6.0) as a resolvent and use 400 μL of the resolvent to fully dissolve the residue obtained in the previous step to obtain the test sample.

[0096] Example 2 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0097] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0098] 1. Mixed extraction

[0099] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 1:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0100] 2. Oxidation drying

[0101] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0102] 3. Redissolve

[0103] Same as Example 1.

[0104] Example 3 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0105] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0106] 1. Mixed extraction

[0107] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 98:2, wherein the acetic acid concentration is 2% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 1:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0108] 2. Oxidation drying

[0109] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0110] 3. Redissolve

[0111] Same as Example 1.

[0112] Example 4 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0113] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0114] 1. Mixed extraction

[0115] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99.5:0.5, wherein the acetic acid concentration is 0.5% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0116] 2. Oxidation drying

[0117] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0118] 3. Redissolve

[0119] Same as Example 1.

[0120] Example 5 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0121] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0122] 1. Mixed extraction

[0123] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0124] 2. Oxidation drying

[0125] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0126] 3. Redissolve

[0127] Same as Example 1.

[0128] Example 6 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0129] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0130] 1. Mixed extraction

[0131] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 98:2, wherein the acetic acid concentration is 2% (v / v)) and 6 mL of extractant (an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0132] 2. Oxidation drying

[0133] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0134] 3. Redissolve

[0135] Same as Example 1.

[0136] Example 7 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0137] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0138] 1. Mixed extraction

[0139] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99.5:0.5, wherein the acetic acid concentration is 0.5% (v / v)) and 6 mL of extractant (an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 4:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0140] 2. Oxidation drying

[0141] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0142] 3. Redissolve

[0143] Same as Example 1.

[0144] Example 8 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0145] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0146] 1. Mixed extraction

[0147] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 4:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0148] 2. Oxidation drying

[0149] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0150] 3. Redissolve

[0151] Same as Example 1.

[0152] Example 9 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0153] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0154] 1. Mixed extraction

[0155] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 98:2, wherein the acetic acid concentration is 2% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 4:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0156] 2. Oxidation drying

[0157] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0158] 3. Redissolve

[0159] Same as Example 1.

[0160] Example 10 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0161] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0162] 1. Mixed extraction

[0163] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (petroleum ether-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0164] 2. Oxidation drying

[0165] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0166] 3. Redissolve

[0167] Same as Example 1.

[0168] Example 11 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0169] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0170] 1. Mixed extraction

[0171] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium chloride), 2 mL of purifier (n-heptane-acetic acid mixture obtained by mixing n-heptane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0172] 2. Oxidation drying

[0173] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0174] 3. Redissolve

[0175] Same as Example 1.

[0176] Example 12 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0177] This embodiment provides a process such as Figure 1 The sample pretreatment method shown here is to simultaneously extract chloramphenicol, diazepam, malachite green, and enrofloxacin from the sample to be tested to prepare the test sample for subsequent testing. It includes three main steps, and the specific operation methods are as follows:

[0178] 1. Mixed extraction

[0179] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (sodium sulfate), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0180] 2. Oxidation drying

[0181] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green. 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed. The mixture was then heated and dried at 60-70°C on a sample concentrator to collect the residue.

[0182] 3. Redissolve

[0183] Same as Example 1.

[0184] Example 13 A sample pretreatment method for simultaneous extraction of chloramphenicol, diazepam, malachite green and enrofloxacin

[0185] 1. Mixed extraction

[0186] Weigh 4 g of homogenized tissue sample and place it in a 15 mL centrifuge tube. Then add 2 g of phase separator (magnesium sulfate), 2 mL of purifier (a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 99:1, wherein the acetic acid concentration is 1% (v / v)) and 6 mL of extractant (acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 2:1). Then, shake and mix at an amplitude of 120 times / min for 2 minutes, and then centrifuge at a speed of 4000 r / min for 3 minutes. The solution in the tube is separated into three layers (the upper layer is the organic phase of the oil-removing layer, the middle layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and the clear liquid in the middle layer is collected.

[0187] 2. Oxidation drying

[0188] 2,3-Dichloro-5,6-dicyano-p-benzoquinone (CAS No. 84-58-2) was fully dissolved in acetonitrile at a final concentration of 0.05 wt% to obtain an oxidizing agent, which oxidized recessive malachite green to dominant malachite green; 5 mL of the intermediate layer supernatant obtained in the previous step was placed in a 10 mL centrifuge tube, and 100 μL of the oxidizing agent was added and mixed, and then heated and dried at 60°C on a sample concentrator to collect the residue.

[0189] 3. Redissolve

[0190] Same as Example 1.

[0191] Comparative Example 1: A sample pretreatment method

[0192] The method is basically the same as Example 1, except that in step “1, mixed extraction”, the purifying agent is n-hexane.

[0193] Comparative Example 2: A sample pretreatment method

[0194] The method is basically the same as Example 1, except that in step “1, mixed extraction”, the purifying agent is petroleum ether.

[0195] Comparative Example 3: A sample pretreatment method

[0196] The method is basically the same as Example 1, except that in step “1, mixed extraction”, the purifying agent is n-heptane.

[0197] Comparative Example 4: A sample pretreatment method

[0198] The method is basically the same as Example 1, except that in step "1, mixed extraction", the purifying agent is a n-hexane-acetic acid mixture obtained by mixing n-hexane and acetic acid in a volume ratio of 96:4, wherein the acetic acid concentration is 4% (v / v).

[0199] Comparative Example 5: A sample pretreatment method

[0200] The method is basically the same as Example 1, except that in step "1, mixed extraction", the purifier is a petroleum ether-acetic acid mixture obtained by mixing petroleum ether and acetic acid in a volume ratio of 96:4, wherein the acetic acid concentration is 4% (v / v).

[0201] Comparative Example 6: A sample pretreatment method

[0202] The method is basically the same as Example 1, except that in step "1, mixed extraction", the purifying agent is a n-heptane-acetic acid mixture obtained by mixing n-heptane and acetic acid in a volume ratio of 96:4, wherein the acetic acid concentration is 4% (v / v).

[0203] Comparative Example 7: A sample pretreatment method

[0204] The method was basically the same as Example 1, except that in step "1. Mixed extraction", the phase separator was neutral alumina; after centrifugation, the solution in the tube separated into two layers (the upper layer was the organic phase of the extraction layer, and the lower layer was the mixed phase of the tissue sample and water), and 4.5 mL of the upper layer solution was collected for subsequent operations.

[0205] Comparative Example 8: A sample pretreatment method

[0206] The method was basically the same as Example 1, except that in step "1. Mixed extraction", ammonium chloride was used as the phase separator; after centrifugation, the solution in the tube separated into two layers (the upper layer was the organic phase of the extraction layer, and the lower layer was the mixed phase of the tissue sample and water), and 4 mL of the upper layer solution was collected for subsequent operations.

[0207] Comparative Example 9: A sample pretreatment method

[0208] The method was basically the same as Example 1, except that in step "1. Mixed extraction", ammonium sulfate was used as the phase separator; after centrifugation, the solution in the tube separated into two layers (the upper layer was the organic phase of the extraction layer, and the lower layer was the mixed phase of the tissue sample and water), and 4 mL of the upper layer solution was collected for subsequent operations.

[0209] Comparative Example 10: A sample pretreatment method

[0210] The method is basically the same as Example 1, except that in step "1, mixed extraction", the extraction agent is acetonitrile.

[0211] The method was basically the same as Example 1, except that in step "1. Mixed extraction", ethyl acetate was used as the extractant; after centrifugation, the solution in the tube separated into two layers (the upper layer was the organic phase of the extraction layer, and the lower layer was the mixed phase of the tissue sample and water), and 7 mL of the upper layer solution was collected for subsequent operations.

[0212] Comparative Example 12: A sample pretreatment method

[0213] The method is basically the same as Example 1, except that in step “1. mixed extraction”, the extractant is an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 1:2.

[0214] Comparative Example 13: A sample pretreatment method

[0215] The method is basically the same as Example 1, except that in step “1. mixed extraction”, the extractant is an acetonitrile-ethyl acetate solution obtained by mixing acetonitrile and ethyl acetate in a volume ratio of 5:1.

[0216] Application example: Detection of chloramphenicol, diazepam, malachite green and enrofloxacin in aquatic products

[0217] 1. Sample spike

[0218] The collected aquatic animal tissue samples (from grass carp) were homogenized by conventional methods and randomly divided into 26 samples of equal mass to be tested (numbered: 1 to 26); each number represents the serial number of the operating method of the embodiment and comparative example, and each number requires parallel addition of 3 concentrations and 1 negative portion (no concentration) to ensure the operability of the parallel addition of concentrations.

[0219] Chloramphenicol, diazepam, malachite green and enrofloxacin standards were added to each sample at a final concentration of 0.1 μg / mL, 0.5 μg / mL, 0.5 μg / mL and 2 μg / mL, respectively, to obtain 26 numbered spiked samples.

[0220] 2. Sample pretreatment

[0221] The 26 numbered samples were spiked according to the operating methods of Examples 1 to 13 and Comparative Examples 1 to 13 to prepare the corresponding test samples. Unspiked aquatic tissue samples were used as negative controls.

[0222] 3. Colloidal gold detection

[0223] In actual application scenarios, technical personnel in this field generally use the colloidal gold method to conduct random inspections on various aquatic tissues. Therefore, they need to prepare the solution to be tested and add it to the sample well of the test card. By observing the color development of the "C line" and the "T line", they can judge the residual conditions of chloramphenicol, diazepam, malachite green and enrofloxacin.

[0224] The 26 numbered test samples were tested using a commercially available colloidal gold test kit (for separate detection of chloramphenicol, diazepam, malachite chloride, and enrofloxacin). The specific steps are as follows:

[0225] a. Before use, restore the test card and the sample solution packaged in the aluminum bag to room temperature (20-30°C);

[0226] b. Take out the two consumables corresponding to the test compound from the aluminum foil bag, including the test card and gold-labeled microwell, and place them horizontally in front of the observer; the test card has a sample well (S), a control line (C line), and a test line (T line);

[0227] c. Use the dropper in the aluminum foil bag to draw 100 μL of the sample solution prepared in the above pretreatment step and drop it into the gold-labeled microwell of the corresponding compound. Use the matching pipette to gently pipette for at least 30 seconds to completely dissolve the red substance at the bottom of the gold-labeled microwell. Let it stand horizontally and wait for the reaction for 3 minutes; then draw the red solution in the gold-labeled microwell and drop it all into the sample well (S) of the corresponding compound detection card. Start timing after adding the sample;

[0228] d. The results are read within 5 to 8 minutes and are determined based on the color of the control line (C line) and the test line (T line) on the test card. It should be noted that results read at other times are invalid.

[0229] The homogenized tissue samples corresponding to each of the above numbers correspond to the prepared test products and test cards in the subsequent processing process, that is, the homogenized tissue samples, test products and test cards are all in one-to-one correspondence and are listed in the corresponding numbers.

[0230] 4. Interpretation of the test card display results

[0231] a. Negative (-): Based on the color development results of the test card and its comparison with the colorimetric card provided with the kit, if the color of the T line (test line, near one end of the sample well) is darker or as dark as that of the C line (control line), it indicates that the residual concentration of chloramphenicol in the sample is less than 0.1 μg / kg or no chloramphenicol residue is present; the residual concentration of diazepam is less than 0.5 μg / kg or no diazepam residue is present; the residual concentration of malachite green is less than 0.5 μg / kg or no malachite green residue is present; and the residual concentration of enrofloxacin is less than 2 μg / kg or no enrofloxacin residue is present.

[0232] b. Positive (+): According to the color development results of the test card and the comparison results with the colorimetric card provided with the test kit, when the T line is lighter than the C line, it indicates that the residual concentration of chloramphenicol in the sample is higher than 0.1 μg / kg, the residual concentration of diazepam is higher than 0.5 μg / kg, the residual concentration of malachite green is higher than 0.5 μg / kg, and the residual concentration of enrofloxacin is higher than 2 μg / kg; wherein, 0.1 μg / kg is used as the sensitivity of chloramphenicol detection to define the negative and positive results of chloramphenicol detection; 0.5 μg / kg is used as the sensitivity of diazepam detection to define the negative and positive results of diazepam detection; 0.5 μg / kg is used as the sensitivity of malachite green detection to define the negative and positive results of malachite green detection; 2 μg / kg is used as the sensitivity of enrofloxacin detection to define the negative and positive results of enrofloxacin detection;

[0233] c. Invalid: Line C does not show color, indicating incorrect operation process or the test card has deteriorated and damaged. In this case, the measured result is invalid and needs to be retested.

[0234] 5. Test results

[0235] (1) Detection time

[0236] In the entire treatment process of Examples 1 to 13, taking theoretical values ​​as an example, the time required for step 1 is: 1 (addition) + 2 (concussion) + 3 (centrifugation) = 6 min, the time required for step 2 is: 0.5 (addition) + 8 (drying) = 8.5 min, the time required for step 3 is: 0.5 (addition) + 0.5 (mixing) = 1 min, the detection time is: 0.5 (droplet blowing) + 3 (waiting) = 3.5 min, and the result waiting time is 6 min. The total time required for the above process is 25 min, which is significantly better than the traditional single pre-treatment detection method in time (the total time for the 4 single compound detections of prior art CN201210157423.5, CN202310993602.0, CN202120545289.0 and CN201611215776.0 is more than 80 min). The pretreatment method of the present invention effectively shortens pretreatment time, eliminates the need for expensive, large-scale equipment, and avoids the traditional compound-by-compound pretreatment process. The entire process from pretreatment to result presentation takes only 25-28 minutes, effectively shortening processing time and providing excellent stability and accuracy. This significantly improves the operational efficiency of on-site testing.

[0237] (2) Test card interpretation results

[0238] Table 1 Colloidal gold test results

[0239]

[0240] Note: Since comparative examples 7, 8, 9, and 11 have no middle layer, only the top layer can be used to process the obtained results; * indicates a false negative result; prior arts 1 to 4 correspond to Chinese patent applications with application numbers 201210157423.5, 202310993602.0, 202120545289.0, and 201611215776.0, respectively.

[0241] As shown in Table 1, in Examples 1 to 13, the spiked experiments all gave positive results, indicating that the pretreatment method of the present invention can meet the detection limit requirements of each test compound; in Comparative Examples 1 to 13, when the purifier is a single solution without acetic acid (Comparative Examples 1 to 3), no positive result is detected for diazepam, and when the acetic acid content in the mixed solution is 4% (v / v) (Comparative Examples 4 to 6), no positive results are detected for diazepam and malachite green, indicating that the acetic acid content in the purifier significantly affects the extraction effect of diazepam and malachite green; when the phase separator is neutral alumina, ammonium chloride or ammonium sulfate (Comparative Examples 7 to 9), no intermediate layer appears, and a large amount of oil remains after the upper layer solution is completely taken and blown dry. Only malachite green is detected as positive, while the others are all negative, indicating that the type of phase separator significantly affects the fat-soluble The extraction effects of chloramphenicol, diazepam and enrofloxacin; when the extractant was a single solution of acetonitrile (Comparative Example 10), neither malachite green nor enrofloxacin tested positive; when the extractant was a single solution of ethyl acetate (Comparative Example 11), no intermediate layer appeared, and a large amount of oil remained after the upper solution was taken and blown dry. Only malachite green tested positive, and the others were negative; when the volume ratio of acetonitrile to ethyl acetate in the extractant was 1:2 (Comparative Example 12), there was very little intermediate layer, and only about 1 ml of the intermediate layer clear liquid could be collected, and no positive results were detected for the four compounds; when the volume ratio of acetonitrile to ethyl acetate in the extractant was 5:1 (Comparative Example 13), no positive result was detected for enrofloxacin, indicating that the content ratio of acetonitrile and acetic acid in the extractant significantly affects the effect of simultaneous extraction of the four compounds.

[0242] In terms of the selection of phase separators, although magnesium sulfate and sodium sulfate can also meet the detection requirements, sodium sulfate needs to be shaken immediately after addition, otherwise it will clump (Example 12), affecting the subsequent extraction and requiring manual crushing; and magnesium sulfate will release heat when it comes into contact with water (Example 13), and will produce obvious gas during extraction and shaking, requiring shaking while releasing gas, which increases the difficulty of operation. Therefore, sodium chloride is preferred.

[0243] In terms of the selection of purifiers, the present invention uses a low-polarity solvent with a certain acid content. The acetic acid content is preferably 1% (v / v), and 0.5% (v / v) can also meet the detection requirements. However, the T-line effect of diazepam is slightly weak, which may be due to the interference of diazepam caused by insufficient purification capacity (Examples 1, 4 and 7); when the acetic acid content is 2% (v / v), the increase in acetic acid leads to a longer drying time and also increases the cost (Examples 3, 6 and 9); The selection of a low-polarity solvent is relatively simple, and any solvent with a polarity index less than 1 can meet the requirements (Examples 5, 10 and 11). However, from the perspective of solvent composition and cost analysis, because petroleum ether is a mixture and there may be differences between different batches of reagents, the cost of n-heptane is relatively high, so n-hexane is the preferred low-polarity solvent.

[0244] In terms of the selection of the extraction agent, the present invention uses a mixture of acetonitrile and ethyl acetate. When the volume ratio of acetonitrile to ethyl acetate is 1:1 (Examples 1-3), the separation between the purification layer and the extract is slightly unclear, which slightly increases the difficulty of extracting the liquid from the intermediate layer. When the volume ratio of acetonitrile to ethyl acetate is 4:1, the detection requirements can be met. However, the T line effect of enrofloxacin is slightly weaker. The reason may be that the reduction in ethyl acetate content slightly affects the extraction rate of enrofloxacin (Examples 7-9). Overall, compared with the case where the volume ratio of acetonitrile to ethyl acetate is 2:1, the amount of liquid in the intermediate layer is larger and the T line results of the four compounds are clear and obvious, making them easy to distinguish.

[0245] Based on the above analysis, the present invention unifies the pretreatment methods for the detection of diazepam, chloramphenicol, malachite green, and enrofloxacin residues in aquatic tissue samples using the colloidal gold method by performing three key steps of phase separation, purification, and extraction during the pretreatment of homogenized tissue samples of aquatic products. Based on the feedback of the test results, the stability and accuracy of the entire pretreatment and subsequent detection process can meet the expectations.

[0246] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A sample processing method, characterized in that: It is used to simultaneously extract chloramphenicol, diazepam, malachite green and enrofloxacin, comprising the following steps: The sample to be processed is fully reacted with a phase separation agent, a purifying agent and an extracting agent, and then the resulting reaction product is layered. After the reaction product is separated into three layers, the middle layer is collected; The phase separation agent includes any one of sodium sulfate, magnesium sulfate or sodium chloride; The purifier includes a low-polarity solvent containing acetic acid, wherein the low-polarity solvent is any one of n-hexane, petroleum ether or n-heptane, and the volume percentage of acetic acid in the low-polarity solvent is 0.5% to 2%; The extractant includes a mixture of acetonitrile and ethyl acetate, and the volume ratio of the acetonitrile to the ethyl acetate is (1-4):

1.

2. The processing method according to claim 1, characterized in that The following steps are also included: The obtained intermediate layer is fully reacted with an oxidizing agent.

3. The processing method according to claim 2, characterized in that The oxidizing agent includes 2,3-dichloro-5,6-dicyano-p-benzoquinone.

4. The processing method according to claim 3, characterized in that The oxidant is acetonitrile containing 0.02 wt% to 0.1 wt% of 2,3-dichloro-5,6-dicyano-p-benzoquinone.

5. The processing method according to claim 2, characterized in that: The following steps are also included: The resulting reaction product was collected and dried.

6. The processing method according to claim 5, characterized in that: The following steps are also included: Reconstitute the dried residue.

7. The processing method according to claim 6, characterized in that The dried residue was reconstituted with a weakly acidic buffer.

8. The processing method according to claim 7, characterized in that: The weakly acidic buffer solution is a weakly acidic sodium citrate buffer solution.

9. A method for detecting a sample, characterized in that: The sample to be tested is treated with the treatment method according to claim 1 to obtain a test sample, and the test sample is detected by immunochromatography.

10. The method according to claim 9, characterized in that The immunochromatography method includes colloidal gold immunochromatography.

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