Sample processing method and detection method

Abstract

The invention discloses a sample processing method and a sample detection method. According to the invention, based on the addition of the phase-splitting agent, the purifying agent and the extracting agent, extraction and oil and ester removal treatment are respectively realized, and the difficulty that enrofloxacin has great conflicts with malachite green and diazepam in the extraction and oil and ester removal processes due to great chemical property differences between the enrofloxacin and malachite green as well as between the enrofloxacin and diazepam is solved; a four-in-one unified sample pretreatment method of chloramphenicol, diazepam, malachite green and enrofloxacin is realized, and the sample pretreatment is performed once. Compared with a sample pretreatment method of a single compound, on the premise of ensuring high sensitivity, the detection time is greatly shortened, the method is subsequently suitable for on-site rapid large-scale screening detection of a colloidal gold method, the lowest detection limits of each compound are 0.1 mu g / kg, 0.5 mu g / kg, 0.5 mu g / kg and 2 mu g / kg respectively, the requirements for accuracy and stability of detection are met, and the detection time is greatly shortened. And the detection operation of grassroots personnel is facilitated.

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, respective 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 pre-treatment of the sample to be tested, and there is no unified pre-treatment detection scheme for chloramphenicol, diazepam, malachite green and enrofloxacin in the sample. The detection reagents and detection steps are also different for each compound, which greatly reduces the efficiency of rapid screening. Therefore, it is particularly important to develop a unified sample pre-treatment 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 by the following scheme:

[0008] The present invention is based on the addition of a phase separation agent, a purifier and an extractant to respectively realize extraction and oil removal treatment, wherein malachite green and diazepam are fat-soluble substances with low polarity and are easily soluble in oil and low-polarity solvents, and the method of extracting with a low-polarity solvent cannot be used to remove oil during sample processing; while chloramphenicol and enrofloxacin have relatively greater polarity and are insoluble in oil and low-polarity solvents, and can be removed by extracting with a low-polarity solvent. These two groups of compounds have opposite chemical properties, resulting in a large conflict in the extraction and oil removal process. Only by selecting a reasonable interval balance during the purification and extraction process can the maximum extraction rate of the four compounds be achieved, and at the same time, the extraction rate can be basically kept consistent for different samples during the application process, thereby solving the problem of large processing differences. 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 requirement 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, the difficulty of separating malachite green and diazepam from oily esters is solved, thereby ensuring the removal of oily esters and the extraction rate.

[0009] A method for processing a sample 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 obtained reaction product is layered, and 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 comprises 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 purifier 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 min.

[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-dicyanobenzoquinone.

[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-dicyanobenzoquinone.

[0036] More preferably, the treatment method further comprises the following steps: 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 pH=6.0.

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

[0047] The method for treating the 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 stratifying the obtained reactants, and after the reactants are separated into three layers, 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 comprises 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 purifier 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 min.

[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-dicyanobenzoquinone.

[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-dicyanobenzoquinone.

[0073] More preferably, the treatment method further comprises the following steps: 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 pH=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 the traditional single pretreatment method, the present invention realizes a four-in-one unified sample pretreatment method for chloramphenicol, diazepam, malachite green and enrofloxacin, and the sample is pretreated once. Compared with 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 suitable for on-site rapid large-scale screening and detection by colloidal gold method. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The figure is a schematic diagram of the operation flow of a sample pretreatment method for simultaneously extracting chloramphenicol, diazepam, malachite green and enrofloxacin. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0088] Example 1 A sample pretreatment method for simultaneously extracting chloramphenicol, diazepam, malachite green and enrofloxacin

[0089] This embodiment provides a process such as Figure 1 The sample pretreatment method shown in the figure 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 3 main steps. The specific operation method is 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 (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 1:1), then shake and mix at an amplitude of 120 times / min for 2 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which acts to oxidize recessive malachite green into dominant malachite green; take 4mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0094] 3. Redissolution

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

[0096] Example 2 A sample pretreatment method for simultaneously extracting chloramphenicol, diazepam, malachite green and enrofloxacin

[0097] This embodiment provides a process such as Figure 1 The sample pretreatment method shown in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0102] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0110] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0118] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0126] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 2:1), then shake and mix at an amplitude of 120 times / min for 2 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0134] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 4:1), then shake and mix at an amplitude of 120 times / min for 2 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0142] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 4:1), then shake and mix at an amplitude of 120 times / min for 2 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0150] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0158] 3. Redissolution

[0159] Same as Example 1.

[0160] Example 10 A sample pretreatment method for simultaneously extracting chloramphenicol, diazepam, malachite green and enrofloxacin

[0161] This embodiment provides a process such as Figure 1 The sample pretreatment method shown in the figure 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 3 main steps. The specific operation method is 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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0166] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0174] 3. Redissolution

[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 in the figure 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 3 main steps. The specific operation method is 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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60-70°C on a sample concentrator, and collect the residue.

[0182] 3. Redissolution

[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 (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 min, and then centrifuge at a speed of 4000 r / min for 3 min. The solution in the tube is divided 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] Fully dissolve 2,3-dichloro-5,6-dicyanobenzoquinone (CAS No. 84-58-2) in acetonitrile at a final concentration of 0.05wt% to obtain an oxidant, which is used to oxidize recessive malachite green into dominant malachite green; take 5mL of the intermediate layer clear liquid obtained in the previous step and place it in a 10mL centrifuge tube, then add 100μL of the oxidant and mix well, heat and blow dry at 60°C on a sample concentrator, and collect the residue.

[0189] 3. Redissolution

[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] It is basically the same as Example 1, except that in step "1, mixed extraction", the purifier is a n-hexane-acetic acid mixed solution 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 concentration of acetic acid is 4% (v / v).

[0201] Comparative Example 6: A sample pretreatment method

[0202] It is basically the same as Example 1, except that in step "1, mixed extraction", the purifier 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 is basically the same as Example 1, except that in step "1, mixed extraction", the phase separation agent is neutral alumina; after centrifugation, the solution in the tube is separated into two layers (the upper layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and 4.5 mL of the upper solution is collected for subsequent operations.

[0205] Comparative Example 8: A sample pretreatment method

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

[0207] Comparative Example 9: A sample pretreatment method

[0208] The method is basically the same as Example 1, except that in step "1, mixed extraction", the phase separation agent is ammonium sulfate; after centrifugation, the solution in the tube is separated into two layers (the upper layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and 4 mL of the upper solution is 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 extractant is acetonitrile.

[0211] The method is basically the same as Example 1, except that in step "1, mixed extraction", the extractant is ethyl acetate; after centrifugation, the solution in the tube is separated into two layers (the upper layer is the organic phase of the extraction layer, and the lower layer is the mixed phase of the tissue sample and water), and 7 mL of the upper solution is 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 the comparative example, and each number needs to be added in parallel with 3 concentrations and 1 negative (no concentration added) to ensure the operability of the parallel addition of concentrations.

[0219] The standards of chloramphenicol, diazepam, malachite green and enrofloxacin were added to each sample to be tested 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] According to the operation methods of Examples 1 to 13 and Comparative Examples 1 to 13, 26 numbered samples were spiked to prepare 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 for detection when conducting 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 residues of chloramphenicol, diazepam, malachite green and enrofloxacin.

[0224] The 26 numbered test samples were tested using a commercially available colloidal gold test kit (for testing chloramphenicol, diazepam, malachite chloride and enrofloxacin separately). 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 two consumables of the corresponding test compound from the aluminum foil bag, including the test card and the 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 absorb 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 blow for at least 30 seconds to completely dissolve the red substance at the bottom of the gold-labeled microwell, and let it stand horizontally for 3 minutes to react; then absorb the red solution in the gold-labeled microwell and drop it all into the sample well (S) of the corresponding compound detection card, and start timing after adding the sample;

[0228] d. The results are read within 5 to 8 minutes, and the results are judged based on the color of the control line (C line) and the test line (T line) in the test card. It should be noted that the 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 correspond to each other one by one and are listed in the corresponding numbers.

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

[0231] a. Negative (-): According to the color development results of the test card and the comparison results with the colorimetric card that comes with the kit, the color development of the T line (test line, close to one end of the sample well) is darker or the same as that of the C line (control line), indicating that the residual concentration of chloramphenicol in the sample is lower than 0.1μg / kg or there is no chloramphenicol residue; the residual concentration of diazepam is lower than 0.5μg / kg or there is no diazepam residue; the residual concentration of malachite green is lower than 0.5μg / kg or there is no malachite green residue; the residual concentration of enrofloxacin is lower than 2μg / kg or there is no enrofloxacin residue;

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

[0233] c. Invalid: The C line does not show color, indicating an incorrect operation process or the test card has deteriorated or 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 whole process of Examples 1 to 13, taking the theoretical value as an example, the time required for step 1 is: 1 (addition) + 2 (oscillation) + 3 (centrifugation) = 6 min, the time required for step 2 is: 0.5 (addition) + 8 (blow dry) = 8.5 min, the time required for step 3 is: 0.5 (addition) + 0.5 (mixing) = 1 min, the detection sample addition 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 terms of time (the total time for the detection of 4 single compounds in the prior art CN201210157423.5, CN202310993602.0, CN202120545289.0 and CN201611215776.0 is more than 80 min). The pretreatment method of the present invention can effectively shorten the pretreatment time, does not require expensive large instruments, and avoids the need to pretreat one compound at a time in the traditional pretreatment process. The entire process from pretreatment to result presentation only takes 25 to 28 minutes, effectively shortening the processing time, and having good stability and accuracy. The operational efficiency of on-site detection is greatly improved.

[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; while in Comparative Examples 1 to 13, when the purifier is a single solution without acetic acid (Comparative Examples 1 to 3), no positive results are 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 aluminum oxide, ammonium chloride or ammonium sulfate (Comparative Examples 7 to 9), no intermediate layer appears, and a large amount of grease remains after the upper layer solution is completely taken and blown dry. Only malachite green is detected to have a positive result, and the others are all negative results, indicating that the type of phase separator significantly affects the fat-soluble The extraction effects of chloramphenicol, diazepam and enrofloxacin; when the extractant is a single solution of acetonitrile (Comparative Example 10), no positive results can be detected for malachite green and enrofloxacin; when the extractant is a single solution of ethyl acetate (Comparative Example 11), no intermediate layer appears, and a large amount of oil remains after the upper solution is taken and blown dry. Only malachite green is detected to be positive, and the others are negative results; when the volume ratio of acetonitrile and ethyl acetate in the extractant is 1:2 (Comparative Example 12), there is very little intermediate layer, and only about 1 ml of intermediate layer clear liquid can be collected, and no positive results can be detected for the four compounds; when the volume ratio of acetonitrile and ethyl acetate in the extractant is 5:1 (Comparative Example 13), no positive result can be 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 separation agents, although magnesium sulfate and sodium sulfate can also meet the detection requirements, sodium sulfate needs to be shaken immediately after addition, otherwise caking will occur (Example 12), affecting the subsequent extraction and requiring manual crushing; and magnesium sulfate will release heat when in contact with water (Example 13), and obvious gas production occurs during extraction and shaking, requiring shaking while releasing gas, which increases the difficulty of operation, so sodium chloride is preferred.

[0243] In the selection of the purifier, the present invention adopts a low-polarity solvent with a certain acid content, and 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 caused by the interference of diazepam due to insufficient purification capacity (Examples 1, 4 and 7); and 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); and the selection of low-polarity solvents is relatively simple, and the selection of polarity index less than 1 can meet the requirements (Examples 5, 10 and 11), but from the analysis of solvent composition and cost, because petroleum ether is a mixture and there may be differences in different batches of reagents, the cost of n-heptane is relatively high, so n-hexane is the preferred low-polarity solvent.

[0244] In the selection of the extractant, the present invention adopts a mixed solution of acetonitrile and ethyl acetate. When the volume ratio of acetonitrile to ethyl acetate is 1:1 (Examples 1 to 3), the separation between the purification layer and the extract is slightly unclear, which slightly increases the difficulty of taking liquid from the middle layer. When the volume ratio of acetonitrile to ethyl acetate is 4:1, the detection requirements can be met, but the T line effect of enrofloxacin is slightly weak. The reason may be that the reduction in the ethyl acetate content slightly affects the extraction rate of enrofloxacin (Examples 7 to 9). In general, compared with the volume ratio of acetonitrile to ethyl acetate of 2:1, the amount of liquid in the middle layer is larger and the T line results of the four compounds are clear and obvious, and are easy to distinguish.

[0245] Based on the above analysis, the present invention unifies the pretreatment method for residual detection of diazepam, chloramphenicol, malachite green and enrofloxacin in aquatic tissue samples by the colloidal gold method through the 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 can meet the expectations in the entire pretreatment and subsequent detection process.

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A sample processing method, characterized in that: The following steps are involved: The sample to be processed is fully reacted with a phase separation agent, a purifying agent and an extracting agent, and then the obtained reaction product is layered, and 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 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); The extractant comprises 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 resulting 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 preparation method according to claim 3, characterized in that: The oxidant is acetonitrile containing 0.02wt% to 0.1wt% 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 5, 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 of claim 1 to obtain a test product, and the test product 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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