CN115014890B - Sample processing method and nitrofuran metabolite determination method - Google Patents
Sample processing method and nitrofuran metabolite determination method Download PDFInfo
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Abstract
The application relates to the technical field of biochemical analysis, in particular to a sample processing method and a nitrofuran metabolite determination method. The sample processing method of the application comprises the following steps: mixing an initial sample for nitrofuran metabolite determination with a derivatization reagent, and performing derivatization treatment under an acidic condition to obtain a derivatized sample; mixing the derived sample with polyethylene glycol alkylphenyl ether, and performing cloud point extraction treatment under alkaline conditions to obtain micelle phase solution; and diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected. The sample treatment method is simple in process, and can effectively separate nitrofuran metabolites, so that a good sample solution to be measured is provided for measuring the content of the nitrofuran metabolites, and the process does not use toxic solvents, so that the method has the characteristics of economy, safety, high efficiency, simplicity, convenience, time saving and environmental protection, and has a good application prospect.
Description
Technical Field
The application belongs to the technical field of biochemical analysis, and particularly relates to a sample processing method and a nitrofuran metabolite determination method.
Background
Nitrofurans (Nitrofurans) are a class of synthetic broad spectrum antibiotics having the basic structure 5-Nitrofurans, including furazolidone (furazolidone), furaltadone (furaltadone), nitrofurantoin (nitrofurantoin), nitrofurazone (nitrofurazone), and the like. Nitrofurans are commonly used to treat and prevent infections of the digestive system of swine, fish and poultry caused by salmonella and escherichia coli infections, and these drugs have a short half-life and are rapidly metabolized in animals and the metabolites associated with the proteins produce stable residues. The corresponding metabolites of furazolidone, furaltadone, nitrofurantoin and furacilin are respectively: 3-Amino-2-Oxazolidinone (3-Amino-2-Oxazolidinone, AOZ), 5-methylmorpholine-3-Amino-2-Oxazolidinone (5-morpholine-3-Amino-2-Oxazolidinone, AMOZ), 1-Amino-2-hydantoin (1-Aminohydantoin hydrochloride, AHD), semicarbazide (SEM).
At present, the cloud point extraction technology is widely applied to researches such as analytical chemistry, food, trace metal element analysis and the like, so that wide attention of scientific researchers at home and abroad is obtained. The cloud point extraction has mild conditions and can process a plurality of organic macromolecular substances and volatile organic compounds which are unstable to heat. Cloud point extraction was developed from aqueous two-phase extraction, which is mainly based on two important functions of surfactant: solubilization and cloud point phenomena, when the temperature is lowered, the surfactant is easily dissolved in water to form a clear solution, and when the temperature is raised to a certain extent, the solubility is reduced instead, and phenomena of turbidity, precipitation and delamination occur in the aqueous solution, so that the extraction method using this phenomenon is called cloud point extraction (CPE method).
The method for measuring nitrofuran metabolite residues comprises high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, enzyme-linked immunosorbent assay and the like. These methods have long detection times and large instrument volumes, and are not suitable for on-site detection. In addition, the immune colloidal gold rapid detection kit meets the rapid detection requirement, and has the advantages of short detection time, high sensitivity, convenience in carrying and the like. The nitrofuran metabolite determination mainly aims at fresh and aquatic products, the pretreatment of a sample to be detected is needed, and for food fishes and the like rich in lipid matrixes, the fat is removed by utilizing normal hexane. However, the current sample processing method has complex steps and poor separation effect, so that the determination effect of nitrofurans metabolites needs to be improved.
Disclosure of Invention
The present application aims to provide a sample processing method and a nitrofuran metabolite measurement method, which aim to solve the technical problem of detecting nitrofuran metabolites by simply and effectively separating the nitrofuran metabolites from a sample.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a sample processing method comprising the steps of:
mixing an initial sample for nitrofuran metabolite determination with a derivatization reagent, and performing derivatization treatment under an acidic condition to obtain a derivatized sample; wherein the derivatization reagent is selected from 2, 3-dichloro-5, 6-dicyano-p-benzoquinone, hydrochloric acid is used for regulating acidity in the derivatization treatment, and the temperature of the derivatization treatment is 58-62 ℃ and the time is 50-70 min;
mixing the derived sample with Triton X-114 with the mass concentration of 2.8-3.2%, and performing cloud point extraction treatment under an alkaline condition with pH=7.0-7.5 to obtain micelle phase solution; the temperature of the cloud point extraction treatment is 38-42 ℃ and the time is 5-15 min;
and diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected.
Further, sodium chloride was also added to the step of mixing the derivatized sample with Triton X-114.
Further, the filtration treatment comprises 0.22 μm membrane filtration.
Further, the buffer is selected from the group consisting of tris solutions; and/or the number of the groups of groups,
the initial sample is selected from the group consisting of edible muscles of an aquatic sample.
In a second aspect, the present application provides a method for determining nitrofurans metabolites, comprising the steps of:
according to the sample treatment method, a sample solution to be detected is obtained;
detecting nitrofuran metabolites in the sample solution to be detected by adopting a colloidal immune gold test strip.
Further, the step of detecting the sample solution to be detected by using a colloidal immune gold test strip comprises the following steps: and (3) dripping 70-80 mu L of the sample solution to be detected into the sample hole of the colloidal immune gold test strip, and judging the result after reacting for 8-12 min.
Further, the nitrofuran metabolites include at least one of AOZ, AMOZ, AHD and SEM.
The sample processing method provided by the first aspect of the application is a sample processing method for rapidly determining nitrofuran metabolites, which comprises the steps of firstly mixing an initial sample for determining the nitrofuran metabolites with a derivatization reagent, performing derivatization treatment under an acidic condition to obtain a derivatized sample, then mixing the derivatized sample with polyethylene glycol alkylphenyl ether, performing cloud point extraction treatment under an alkaline condition, enabling the derivatized nitrofuran metabolites to be better separated in micelle phase solution through the action of the polyethylene glycol alkylphenyl ether, and finally diluting and filtering the micelle phase solution, so that a sample solution to be detected suitable for rapidly determining the nitrofuran metabolites can be obtained. The sample treatment method is simple in process, and can effectively separate nitrofuran metabolites, so that a good sample solution to be measured is provided for measuring the content of the nitrofuran metabolites, and the process does not use toxic solvents, and has the characteristics of economy, safety, high efficiency, simplicity, time saving and environmental protection, so that the method has a good application prospect.
According to the method for determining the nitrofuran metabolites, which is provided by the second aspect of the application, a sample solution to be detected, which is obtained by a special sample treatment method of the application, is used, and then a colloidal immune gold test strip is adopted to detect the nitrofuran metabolites derived from the sample solution to be detected; the sample treatment method has the advantages of simple process, effective separation of nitrofuran metabolites, good sample solution to be detected for measuring the content of the nitrofuran metabolites, short detection time, high sensitivity, convenient carrying and the like based on the detection of the immune colloidal gold test strip, and can better measure the nitrofuran metabolites in fresh samples, aquatic products and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of experimental data for determining an optimal concentration of Triton X-114 provided in the examples of the present application;
FIG. 2 is a graph showing the judgment of the result of the reagent plate for rapid detection of immune colloidal gold according to the embodiment of the present application;
FIG. 3 is a graph of blank measurement results for two extractants provided in the examples of the present application;
FIG. 4 is a graph of SEM measurements corresponding to two extractants provided in examples herein;
FIG. 5 is a graph of AMOZ assay results provided in an embodiment of the present application;
fig. 6 is a graph of AOZ assay results provided in the examples of the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the embodiments of the present application may refer not only to specific contents of the components, but also to the proportional relationship between the weights of the components, and thus, any ratio of the contents of the relevant components according to the embodiments of the present application may be enlarged or reduced within the scope disclosed in the embodiments of the present application. Specifically, the mass described in the specification of the examples of the present application may be a mass unit known in the chemical industry such as μ g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
An embodiment of the present application provides a sample processing method, including the following steps:
s01: mixing an initial sample for nitrofuran metabolite determination with a derivatization reagent, and performing derivatization treatment under an acidic condition to obtain a derivatized sample;
s02: mixing the derived sample with polyethylene glycol alkylphenyl ether, and performing cloud point extraction treatment under alkaline conditions to obtain micelle phase solution;
s03: and diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected.
The sample processing method provided by the embodiment of the application is a sample processing method for rapidly determining nitrofuran metabolites, which comprises the steps of firstly mixing an initial sample containing the nitrofuran metabolites with a derivatization reagent, performing derivatization treatment under an acidic condition to obtain a derivatized sample, then mixing the derivatized sample with polyethylene glycol alkylphenyl ether, performing cloud point extraction treatment under an alkaline condition, enabling the derivatized nitrofuran metabolites to be better separated in micelle phase solution through the action of the polyethylene glycol alkylphenyl ether, and finally diluting and filtering the micelle phase solution, so that a sample solution to be detected suitable for rapidly determining the nitrofuran metabolites can be obtained. The sample treatment method is simple in process, and can effectively separate nitrofuran metabolites, so that a good sample solution to be measured is provided for measuring the content of the nitrofuran metabolites, and the process does not use toxic solvents, and has the characteristics of economy, safety, high efficiency, simplicity, time saving and environmental protection, so that the method has a good application prospect.
Specifically, in step S01, the initial sample may be a raw sample to be used for the measurement of nitrofurans metabolites, for example, a fresh sample, an aquatic sample, or the like. Specifically, the initial sample may be obtained by homogenizing the edible muscle portion of an aquatic sample such as fish with a homogenizer. Of course, the initial sample may be other sample solutions containing nitrofurans metabolites.
In order to better improve the detection sensitivity of the nitrofuran metabolites, the initial sample and the derivatization reagent are mixed under the acidic condition for derivatization treatment, so that a derivatized sample is obtained, and thus the nitrofuran metabolites can be better determined by detecting the nitrofuran metabolite derivatives. Further, the derivatizing reagent may be 2, 3-dichloro-5, 6-dicyano-p-benzoquinone (DDQ), and the 2, 3-dichloro-5, 6-dicyano-p-benzoquinone may be reacted with nitrofurans metabolites to be derivatized to better detect nitrofurans metabolites. During this derivatization treatment, acidic conditions may be provided by a hydrochloric acid solution. For example, the acid is adjusted by using a 0.2mol/L hydrochloric acid solution, specifically, the acid is adjusted, so that 4 nitrofurans metabolites are hydrolyzed under weak acidic conditions for derivatization.
Further, the temperature of the derivatization treatment performed by mixing the initial sample and the derivatization reagent under the acidic condition is 58-62 ℃ and the time is 50-70 min. The derivatization efficiency gradually increases along with the lengthening of the derivatization time, and the generation amount of the derivatization product is relatively stable after the derivatization time exceeds a certain time; the derivatization efficiency is gradually improved along with the increase of the derivatization temperature, and when the derivatization temperature reaches a certain degree, the generation amount of the derivatization product is relatively stable, so that the derivatization temperature is selected to be 58-62 ℃ for 50-70 min under the condition of better derivatization effect in consideration of the requirement of the reagent plate as a rapid detection method; for example, the incubation may be carried out for 50 to 70 minutes under the condition of a constant temperature water bath at 58 to 62 ℃.
Specifically, in step S02, the structural formula of the polyethylene glycol alkylphenyl ether is as follows:
wherein R is an alkyl group having 6 to 10 carbon atoms, and n is an integer of 5 to 10.
Polyethylene glycol alkyl phenyl ether is used as a nonionic surfactant, is an environment-friendly surfactant, corresponds to the cloud point temperature, and can be mutually dissolved with water when the temperature is lower than the cloud point temperature, so that the polyethylene glycol alkyl phenyl ether can be used for cloud point extraction treatment; further, the polyethylene glycol alkylphenyl ether is Triton X-114 (polyethylene glycol tert-octylphenyl ether) and has the following structural formula:
because of the relatively low cloud point temperature (23 ℃) of Triton X-114, triton X-114 is miscible with water below this temperature; above this temperature, then separates into an aqueous phase and a micellar phase; tritonX-114 has a good solubilization effect on biomolecules, so that it can be used for separating aqueous phase proteins and lipoproteins.
Further, the polyethylene glycol alkyl phenyl ether is used as a surfactant, the addition amount has a certain influence on the extraction efficiency of nitrofurans metabolites, and in the embodiment of the application, 4ml of aqueous solution of Triton X-114 with mass concentration of 1%,2%,3% and 4% is respectively added by taking 2.0g of edible muscle part of a homogenized aquatic product sample as an example. As a result, as shown in FIG. 1, the extraction efficiency of the target analytes (AOZ, AMOZ, AHD and SEM) tended to decrease with increasing Triton X-114 concentration. This is because when the concentration of the polyethylene glycol alkylphenyl ether is too low, the surfactant-rich phase is too small to be phase-separated during extraction and delamination, and as the concentration increases, more and more micelles are formed, the stronger the solubilization becomes, which is more advantageous for extraction of the target analyte. However, after the concentration of TritonX-114 is higher than 3%, the extraction efficiency tends to decrease, because the concentration of the surfactant is increased, the viscosity of the solution is increased, the diffusion capability is weakened, the micelle is difficult to penetrate into the matrix, and the internal structure of the micelle is changed, so that the extraction rate is reduced. Therefore, the optimal concentration of Triton X-114 is about 3%, such as 2.8-3.2%, and in the embodiment of the application, a Triton X-114 solution with concentration of 2.8-3.2% can be selected for cloud point extraction treatment.
Further, the temperature of the cloud point extraction treatment is 38-42 ℃ and the time is 5-15 min. The temperature of the cloud point extraction treatment may be higher than the cloud point temperature of the polyethylene glycol alkylphenyl ether, thereby facilitating better separation of the nitrofurans metabolite in the micelle phase solution. Because the equilibrium temperature is lower than the cloud point temperature of the polyethylene glycol alkylphenyl ether, the two phases are in a mutual-soluble state, when the equilibrium temperature rises, H bonds in the polyethylene glycol alkylphenyl ether micelle are broken and dehydrated in a hydrated form in an aqueous solution, so that the polyethylene glycol alkylphenyl ether enrichment phase becomes a volume reduction, the polyethylene glycol alkylphenyl ether is easier to settle, and better separation of the two aqueous phases is facilitated.
Further, the alkaline condition that the sample after the derivatization is mixed with polyethylene glycol alkylphenyl ether to carry out cloud point extraction treatment under the alkaline condition is that the pH is 7.0-7.5; in cloud point extraction, the target analyte and polyethylene glycol alkyl phenyl ether micelle are mainly distributed in a hydrophobic mode, the pH value of a sample solution influences the effect of two-phase distribution, and experiments show that the extraction rate is maximum by adjusting the pH value to 7-7.5. When the pH of the extract is too high, the polyethylene glycol alkylphenyl ether enrichment phase is transferred from the lower phase to the upper layer of the solution, which is inconvenient for the separation of the two phases. Thus, the alkaline condition is selected to have a pH of 7 to 7.5.
The cloud point temperature of TritonX-114 is 23 ℃, so that the effective equilibrium temperature is 15-25 ℃ higher than the cloud point temperature. However, the high temperature may cause decomposition of the drug and the extraction rate may be lowered. In addition, the extension of the equilibration temperature is beneficial to the distribution of target analytes in the cloud point, the increase of the equilibration time can improve the extraction rate, but the overlong equilibration time has no obvious influence on the extraction rate, the treatment time of the sample is increased, and the experimental period can be influenced. The optimal conditions of the cloud point extraction of nitrofurans metabolites by Triton X-114 are finally obtained by examining the equilibrium temperature, the equilibrium time and the target analyte temperature response value range of Triton X-114 through experiments: 2.8-3.2% Triton X-114, pH value of 7-7.5, balancing temperature of 35 ℃ and balancing time of 10min.
Further, sodium chloride may be added in the step of mixing the derivatized sample with polyethylene glycol alkylphenyl ether. NaCl belongs to electrolyte, and the addition of NaCl can lead to the increase of the aggregation number of polyethylene glycol alkyl phenyl ether micelle, so that the ionic strength of the solution can be further increased, the density of an aqueous phase is increased, the separation of two phases is accelerated, the cloud point temperature is lowered, and the solubilization of a target analyte solution is improved, so that the addition of NaCl is beneficial to cloud point extraction.
Specifically, in step S03, the micelle phase solution is diluted with a buffer solution and then filtered to obtain a sample solution to be measured. Wherein the buffer is selected from the group consisting of tris solutions, e.g., 8-12 mmol/L tris solutions, which allows for better dilution of the micelle phase solution. Further, the filtration treatment comprises 0.22 μm membrane filtration. And filtering to obtain a uniformly dispersed sample solution to be measured so as to facilitate the subsequent determination of nitrofurans metabolites.
In summary, in the cloud point extraction, polyethylene glycol alkyl phenyl ether is used as a surfactant, critical Micelle Concentration (CMC) is achieved in a solution, vortex shaking is performed fully, the solution is heated to the cloud point in a water bath to keep the temperature of the solution for a period of time, water phase is removed after centrifugal separation, so that micelle phase solution containing derivative nitrofuran metabolites is obtained, and the micelle phase is diluted appropriately, so that a sample solution to be detected which can be directly used for colloidal immune gold test strip measurement is obtained.
In a second aspect, embodiments of the present application provide a method for determining nitrofurans metabolites, comprising the steps of:
t01: according to the sample processing method, a sample solution to be detected is obtained;
t02: detecting nitrofuran metabolites in the sample solution to be detected by adopting a colloidal immune gold test strip.
According to the method for measuring the nitrofuran metabolites, which is provided by the embodiment of the application, a sample solution to be measured, which is obtained by a special sample treatment method, is used, and then a colloidal immune gold test strip is adopted to detect the nitrofuran metabolites in the sample solution to be measured; the sample treatment method has the advantages of simple process, effective separation of nitrofuran metabolites, good sample solution to be detected for measuring the content of the nitrofuran metabolites, short detection time, high sensitivity, convenient carrying and the like based on the detection of the immune colloidal gold test strip, so that the nitrofuran metabolites in fresh samples, aquatic products and the like can be better measured.
According to the embodiment of the application, the principle of competitive inhibition immunochromatography is applied to the colloidal immune gold test strip, nitrofurans metabolites in a sample are combined with specific antibodies marked by colloidal gold in the flowing process, and the combination of the antibodies and an antigen-BSA conjugate on an NC membrane detection line (T line) is inhibited, so that the color depth of the detection line is changed; whether the sample contains the substance to be detected or not, the quality control line (C line) can develop color to indicate that the detection is effective. Specifically, the step of detecting the sample solution to be detected by using the colloidal immune gold test strip comprises the following steps: and (3) dripping 70-80 mu L of sample solution to be detected into the sample hole of the colloidal immune gold test strip, and judging the result after reacting for 8-12 min.
In the embodiment of the application, triton X-114 is used as an extractant for extracting nitrofuran metabolites, the conditions of optimal concentration, pH value, equilibrium time, equilibrium temperature and the like during low-cloud-point extraction of Triton X-114 are explored, and a sample treatment method is established by using the conditions of cloud-point extraction. Meanwhile, based on the sample treatment method, a determination method of nitrofuran metabolites is determined. According to the method, samples respectively containing about 20ppb of nitrofurans metabolites (AOZ/SEM/AMOZ) are subjected to hydrolysis derivatization, then Triton X-114 is added to extract a target solution, after extraction is completed, a slow-release solution is added to filter, then a sample solution to be detected is obtained, and then the sample solution is titrated into a colloidal immune gold test strip, and manual interpretation is performed to determine the detection result of the target. Therefore, triton X-114 can effectively extract nitrofuran metabolites, breaks through the traditional extraction of nitrofuran metabolites by using ethyl acetate as an organic extractant, and the method does not use toxic solvents, and has the advantages of economy, safety, high efficiency, simplicity, time saving, environmental protection and the like.
The following description is made with reference to specific embodiments.
Example 1
1.1 Experimental materials and instruments
AOZ, AMOZ, AHD, SEM standard (purity > 97%, LGC Labar GmbH), nitrofurans metabolite standard derivatizing reagent (DDQ) (purity > 98%, tianjin Alta technologies Co., ltd.), hydrochloric acid (analytical purity, margaritite Cheng Chemie Co., ltd.), dipotassium hydrogen phosphate (analytical purity, west Long chemical Co., ltd.), triton X-114 (molecular biology grade, YEASWN), 2-nitrobenzaldehyde (analytical purity, jiangsu Ning kang chemical Co., ltd.), TBS buffer (Solaro Co., ltd.), and laboratory water is ultrapure water. The main instruments and equipment used for the experiments are shown in table 1 below:
TABLE 1
1.2 preparation of reagents
(1) AOZ and AMOZ standard stock: 1.0mg/ml. Accurately weighing 10mg of standard substance, dissolving with methanol, and keeping constant volume in a 10ml volumetric flask at 4deg.C.
(2) SEM standard stock: 1.0mg/ml, the standard was dissolved in methanol and fixed to a 10ml volumetric flask, stored at 4 ℃.
(3) ADH standard stock: 1.0mg/ml, the standard was dissolved in methanol and fixed to a 10ml volumetric flask, stored at 4 ℃.
(4) 10mM Tris,150mM NaCl,pH7.4: 1L of the packed TBS buffer salt powder (1 xTBS) was taken and dissolved in 1000ml of pure water.
(5) Triton x-114 (15%, w/w): and (3) adding 16.68ml or less of triton TritonX-114 into 100ml of TBS solution, gradually diluting to 1%,2%,3%,4% and 5% and refrigerating for later use.
(6) 0.2mol/L hydrochloric acid solution: concentrated hydrochloric acid 0.6ml was measured and diluted to 100ml with water.
(7) 0.05mol/L o-nitrobenzaldehyde solution: 0.0378g was weighed out and dissolved in 5ml of methanol.
(8) 1mol/L dipotassium hydrogen phosphate solution: 17.42g of dipotassium hydrogen phosphate are weighed out and dissolved in 100ml of water.
(9) 10mmol/L tris solution: 1.211g of the extract was weighed, dissolved in 80ml of water, adjusted to pH 8.0 by adding appropriate hydrochloric acid, and then fixed to volume of 1L by water.
1.3 sample processing methods
Homogenizing edible muscle parts of fish aquatic samples by using a homogenizer, weighing 2.0g of homogenized samples, obtaining initial samples in a 50mL centrifuge tube, then adding 5mL of 0.2mol/L hydrochloric acid solution and 0.15mL of derivatization reagent (DDQ) of the samples, and fully mixing for 3min; incubating for 60min under the condition of constant temperature water bath at 60 ℃ to obtain a derived sample.
Taking out the derived sample, adding 3-5 ml of 1.0mol/L dipotassium hydrogen phosphate solution and 0.4ml of 1.0mol/L sodium hydroxide solution, and adjusting the pH to 7.5; adding 4ml of 3% triton X-114 vortex to oscillate for 2min, carrying out constant-temperature water bath at 40 ℃ for 5min to obtain a turbid solution, immersing a test tube into an ice bath for 5min, centrifuging for 5min at 4000r/min to promote separation of a surfactant-rich phase, precipitating the surfactant phase at the bottom of a conical tube, collecting a bottom concentrate by using a micro-injector, placing recovered liquid drops into a sample injection vial, adding proper 10mmol/L tris solution, fully mixing, and filtering with a 0.22 mu m filter membrane to obtain the solution to be tested.
Example 2
Detection of nitrofurans metabolites
2.1 colloidal immune gold test strip detection
The test strip applies the principle of competitive inhibition immunochromatography, and the derivatized nitrofurans metabolites in the sample are combined with specific antibodies marked by colloidal gold in the flowing process, so that the combination of the antibodies and antigen-BSA conjugate on an NC membrane detection line (T line) is inhibited, thereby causing the change of the color depth of the detection line; whether the sample contains the substance to be detected or not, the quality control line (C line) can develop color to indicate that the detection is effective.
The colloidal immune gold test strip comprises the following operation steps: (1) the test strip and the sample to be tested are used at room temperature. (2) Taking out the test strip from the original packaging bag, and using the test strip within 60 minutes after opening. (3) About 75. Mu.L of the sample solution to be measured is sucked up by a micropipette and vertically dropped into a sample well (S well). (4) And (3) starting timing the flow of the liquid, reacting for 10min, judging the result according to the detection requirement of the colloidal immune gold test strip, and invalidating the other time.
The colloidal immune gold results are shown in fig. 2: a is invalid: the C line does not appear, which indicates that the operation process is incorrect or the test strip is failed; in this case, the instructions should be read again carefully and retested with a new test strip. b is negative: the color development of the T line is stronger than that of the C line or five obvious differences from that of the C line, which indicates that the sample does not contain the to-be-detected object or is lower than the detection limit. c is positive: the T line color development is obviously weaker than that of the C line or the T line does not develop, which indicates that the concentration of the to-be-detected object in the sample is equal to or higher than the detection limit.
2.2SEM assay
Blank samples are respectively prepared, 0.2ml of 100ng/ml of SEM standard solution is accurately removed and added into a negative standard sample, the negative standard sample is placed into a 50ml polyethylene centrifuge tube, the sample is treated by taking the SEM standard solution as an initial sample according to the step of the sample treatment method of 1.3 of the embodiment 1, the sample solution to be detected is obtained (meanwhile, the target analyte is extracted by taking ethyl acetate announced by the Ministry of agriculture 783 as an extractant, after centrifugal separation, the solution of non-target analyte is discarded, then 1ml of 10mmol/L of tris solution is added for diluting the target object, the sample solution is filtered by a 0.22 mu m filter membrane and is taken as a comparison), the sample solution is dripped onto a colloidal immune gold test strip for observation, and the two extractants (triton X-114 of the application and ethyl acetate of the comparison) are subjected to blank experiments, so that the detection result of the colloidal immune gold test strip is effective, as shown in figure 3, and the detection result of the two extractants can be detected by the SEM, as shown in figure 4.
2.3AMOZ assay
Respectively preparing a blank sample and accurately transferring 0.2ml of 100ng/ml of AMOZ standard solution, adding the sample into a negative standard sample, placing the negative standard sample into a 50ml polyethylene centrifuge tube, performing sample treatment by taking the AMOZ standard solution as an initial sample according to the step of the sample treatment method 1.3 of the embodiment 1 to obtain a sample solution to be detected, and then dripping the sample solution to the colloidal immune gold test strip for observing the result. A blank experiment of the Triton X-114 extractant proves that the detection result of the AMOZ colloidal immune gold test strip is effective (left in figure 5), and the Triton X-114 extractant can be detected for extracting the labeled AMOZ target (right in figure 5).
2.4AOZ assay
Respectively preparing a blank sample and accurately transferring 0.2ml of 100ng/ml AOZ standard solution, adding the sample into a negative standard sample, placing the negative standard sample into a 50ml polyethylene centrifuge tube, performing sample treatment by taking the AOZ standard solution as an initial sample according to the step of the sample treatment method 1.3 of the embodiment 1 to obtain a sample solution to be detected, and then dripping the sample solution to the colloidal immune gold test strip for observation. A blank experiment of Triton X-114 extractant proves that the detection result of the AOZ colloidal immune gold test strip is effective (left in figure 6), and the Triton X-114 extractant can be detected for extracting the labeled AOZ target (right in figure 6).
In summary, according to the embodiment of the application, according to the T, C line color development difference when the AOZ, AMOZ, SEM immune colloidal gold rapid detection reagent plate is used for detection, the manually judged measurement result is positive. According to the ethyl acetate organic solvent extraction method in the 785 publication of the Ministry of agriculture, the detection result is positive by the requirement of manual judgment through T, C line color development difference when the AOZ, AMOZ, SEM immune colloidal gold rapid detection reagent plate is used for detection. The color development effect of the fluorescent dye is better, the detection time is short, the sensitivity is high, the fluorescent dye is convenient to carry, and the fluorescent dye has the advantages of economy, safety, high efficiency, simplicity, convenience, time saving and environmental protection.
The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.
Claims (7)
1. A method of sample processing comprising the steps of:
mixing an initial sample for nitrofuran metabolite determination with a derivatization reagent, and performing derivatization treatment under an acidic condition to obtain a derivatized sample; wherein the derivatization reagent is selected from 2, 3-dichloro-5, 6-dicyano-p-benzoquinone, hydrochloric acid is used for regulating acidity in the derivatization treatment, and the temperature of the derivatization treatment is 58-62 ℃ and the time is 50-70 min;
mixing the derived sample with Triton X-114 with the mass concentration of 2.8-3.2%, and performing cloud point extraction treatment under an alkaline condition with pH=7.0-7.5 to obtain micelle phase solution; the temperature of the cloud point extraction treatment is 38-42 ℃ and the time is 5-15 min;
and diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected.
2. The method of sample processing according to claim 1, wherein sodium chloride is further added to the step of mixing the derivatized sample with Triton X-114.
3. The method of sample processing according to any one of claims 1-2, wherein the filtration treatment comprises 0.22 μm membrane filtration.
4. The method of sample processing according to any one of claims 1-2, wherein the buffer is selected from the group consisting of tris solutions; and/or the number of the groups of groups,
the initial sample is selected from the group consisting of edible muscles of an aquatic sample.
5. A method for determining nitrofurans metabolites, comprising the steps of:
the sample processing method according to any one of claims 1 to 4, obtaining a sample solution to be measured;
detecting nitrofuran metabolites in the sample solution to be detected by adopting a colloidal immune gold test strip.
6. The assay of claim 5, wherein the step of detecting the sample solution using a colloidal immune gold test strip comprises: and (3) dripping 70-80 mu L of the sample solution to be detected into the sample hole of the colloidal immune gold test strip, and judging the result after reacting for 8-12 min.
7. The assay of claim 5, wherein said nitrofurans metabolite comprises at least one of AOZ, AMOZ, AHD and SEM.
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