CN110836938A - Detection method of furan metabolites in aquatic products and preparation method of solid-phase extraction column - Google Patents
Detection method of furan metabolites in aquatic products and preparation method of solid-phase extraction column Download PDFInfo
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Abstract
The method comprises the steps of hydrolyzing, derivatizing, purifying and purifying the residual furan metabolites in muscle tissues of a sample under an acidic condition, dissolving an obtained test solution in gold-labeled micropores after adsorption and elution of the solid-phase extraction column prepared by the method, and then dropwise adding a colloidal gold card for detection. The reagent used in the method is a common chemical reagent, the safety is high, a large-scale detection instrument is not needed, the detection time is greatly shortened, the method is simple and convenient to operate, the detection cost is low, the sensitivity is high, the detection accuracy is high, the detection limit of four furan metabolites is 0.3ppb, the requirement on the experimental environment is low, and the method is suitable for rapid detection of aquatic products.
Description
Technical Field
The disclosure relates to the field of detection, in particular to a detection method of furan metabolites in aquatic products and a preparation method of a solid-phase extraction column.
Background
Furan drugs are broad-spectrum antibiotics and have been widely applied to livestock and poultry and aquaculture industry, and furan drugs and metabolites thereof have carcinogenic and teratogenic side effects on human bodies, so that furan drugs furazolidone, furaltadone, nitrofurantoin and nitrofural are listed in a black list of inedible substances which may be illegally added by 3-22 months in 2010 by the ministry of health in China. Furan drugs are rapidly metabolized in a living body, the metabolites thereof are AOZ (3-amino-2-oxazolidinone), SEM (semicarbazide), AMOZ (5-methylmorpholine-3-amino-2-oxazolidone) and AHD (N-amino-2-hydantoin), and these metabolites are combined with proteins to be quite stable, so that the detection of metabolites is often used to reflect the residual status of furan drugs.
Disclosure of Invention
According to one aspect of the present disclosure, there is provided a method of preparing a solid phase extraction column, comprising:
preparing a filler: under the protection of inert gas, mixing N-vinyl pyrrolidone and divinylbenzene, adding a diluent to obtain a mixture, adding an initiator to the mixture, uniformly mixing, and carrying out polymerization reaction to obtain a filler, wherein the mass ratio of the diluent to the divinylbenzene to the N-vinyl pyrrolidone in the mixture is 1:1:1, the diluent comprises diethylene glycol butyl ether, ethylene glycol methyl acetate and diphenyl ether, and the mass ratio of the diethylene glycol butyl ether to the ethylene glycol methyl acetate to the diphenyl ether in the diluent is 3:2: 1;
preparing a solid phase extraction column: a fixed extraction column is prepared and obtained by using the packing.
In some embodiments, the solid phase extraction column is used to detect a furan metabolite selected from at least one of 3-amino-2-oxazolidinone, 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, 1-amino-2 hydantoin, and semicarbazide. The metabolite of furazolidone in a living body is 3-amino-2-oxazolidinone, the metabolite of furaltadone in the living body is 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, the metabolite of nitrofurantoin in the living body is 1-amino-2 hydantoin, and the metabolite of nitrofural in the living body is semicarbazide.
The beneficial effect of this disclosure: the solid phase extraction column prepared by the preparation method disclosed by the invention can be used for concentrating and enriching furan metabolites and detecting the furan metabolites in a sample, so that the time can be saved, the cross contamination in the concentration process is avoided, and the detection accuracy is improved. The filler used by the solid phase extraction column disclosed by the invention is diethylene glycol monobutyl ether: ethylene glycol methyl ether acetate: the diphenyl ether is obtained by polymerization reaction of a mixed solution of 3:2:1 serving as a diluent and N-vinyl pyrrolidone and divinylbenzene serving as monomers, has good adsorption effect on furan metabolites, and is different from the conventional solid-phase extraction column packing on the market. The furan metabolite is polar substance, and the N-vinyl pyrrolidone has hydrophilicity and has the function of retaining the furan metabolite. The filler used in the method improves the relative content of the N-vinyl pyrrolidone, further increases the adsorption capacity of the filler on the furan metabolites in the sample, and improves the detection accuracy of the furan metabolites.
In some embodiments, the solid phase extraction column obtained by the preparation method of the present disclosure has a packing made of N-vinyl pyrrolidone: divinylbenzene: diluent 1:1:1 is prepared.
In some embodiments, a method for detecting a furan metabolite in an aquatic product, comprising:
step a, homogenizing a sample, adding hydrochloric acid and an aldehyde group-containing compound, and performing derivatization;
step b, extracting the derivatized sample to obtain a first sample solution;
step c, adding the first sample solution into the solid phase extraction column obtained by the preparation method disclosed by the invention to obtain an eluent;
step d, mixing the eluent and the buffer solution to obtain a mixed solution, and dropwise adding the mixed solution into the gold-labeled micropores to obtain a second sample solution;
and e, dropwise adding the second sample solution into a colloidal gold card for determination. The colloidal gold card can utilize the colloidal gold technology to carry out rapid detection on the furan metabolites in the second sample liquid, and has high detection sensitivity and high accuracy. By using the solid-phase extraction column obtained by the preparation method disclosed by the invention, the operation procedures are reduced, the pretreatment time is saved, the detection time is greatly shortened, the detection efficiency is improved, the cross contamination is avoided, the reliability of the detection result is improved, a large instrument is not required, the cost is reduced, and the requirement for immediately obtaining the detection result can be met, so that the quality supervision of aquatic products is carried out.
In some embodiments, step a comprises adding a hydrochloric acid solution and an aldehyde group-containing compound to the sample, performing vortex oscillation, and then performing water bath incubation on the sample;
step b, adding an extracting agent into the sample incubated in the water bath, fully mixing, then centrifuging to obtain a first supernatant, taking the first supernatant and adding a purifying agent, uniformly mixing, centrifuging, standing to obtain a second supernatant, adding diethylene glycol into the second supernatant, and uniformly mixing to obtain a first sample solution; said step c comprises a first sample enrichment:
step c1, activating the solid phase extraction column by methanol and water in sequence before use;
step c2, adding the first sample solution into the activated solid phase extraction column, leaching the solid phase extraction column with water after the first sample solution completely passes through the solid phase extraction column, and vacuumizing to dry the solid phase extraction column; and eluting the solid phase extraction column by using methanol to obtain an eluent. The diglycol can improve the extraction efficiency of the furan metabolites, has good defoaming effect, can eliminate interference and is beneficial to result detection.
In some embodiments, the solid phase extraction column has a size of 60mg/3ml, an average particle size of 50-60 μm, and an average pore size of 60 μm
Under the condition, the solid phase extraction column can fully adsorb the furan metabolites in the sample, improve the content of the furan metabolites in the eluent and avoid the loss of the furan metabolites in the sample.
In some embodiments, the concentration of the hydrochloric acid solution in step a is 0.1 to 0.2mol/L, the aldehyde group-containing compound is one of 2-nitrobenzaldehyde or 2-chlorobenzaldehyde, and the temperature of the water bath in step a is 70 to 80 ℃. Under the conditions of 0.1-0.2mol/L hydrochloric acid solution and 70-80 ℃ water bath, the step a can simultaneously carry out the hydrolysis of the sample and the derivatization of the furan metabolites, thereby reducing the operation steps and saving the processing time.
In some embodiments, the extractant comprises 0.1mol/L dipotassium hydrogen phosphate, 1.0mol/L sodium hydroxide and n-hexane, the volume ratio of 0.1mol/L dipotassium hydrogen phosphate, 1.0mol/L sodium hydroxide and n-hexane in the extractant is 46:4:100, and the purifying agent is a mixture of 1:1, the rotating speed of the first centrifugation is 4000 revolutions per minute, and the rotating speed of the second centrifugation is 4000 revolutions per minute. In the step b, the volume ratio is 1: the mixed solution of methanol and water of 1 is a purifying agent, and can be used for purifying and removing impurities in the sample solution by matching with the rotation speed centrifugation of 4000 revolutions per minute. And b, adopting a composite extracting agent, wherein the extracting agent prepared from dipotassium hydrogen phosphate, sodium hydroxide and normal hexane according to corresponding concentration and volume ratio has the strongest extraction capability on furan metabolites, and saving the extraction time.
In some embodiments, the volume ratio of methanol to water in step c1 is 1:4 and the volume ratio of eluent to buffer in step d is 1: 1. The volume ratio of methanol to water in step c1 is 1:4, which can activate the solid phase extraction column to maximize the adsorption capacity of the solid phase extraction column on furan metabolites. The volume ratio of the eluent to the buffer solution is 1:1, which is beneficial to the combination of furan metabolites in the eluent and the gold-labeled antibody in the gold-labeled micropores, is beneficial to the color development of the gold-labeled card dripped after redissolution, improves the detection accuracy rate, and is beneficial to the judgment of the detection result.
In some embodiments, the method for preparing a buffer solution comprises the following operations: accurately weighing 0.96g of disodium hydrogen phosphate monohydrate, 0.17g of potassium dihydrogen phosphate, 9.0g of sodium chloride and 10.0g of trehalose, dissolving with water, diluting to 1.0L, adding 0.15ml of tween-20, and mixing uniformly for later use. Trehalose is added into the buffer solution, which is helpful for combination of furan metabolites and the gold-labeled antibody in the gold-labeled micropores and helps to maintain stability of the gold-labeled antibody.
Detailed Description
The present disclosure will be described in further detail with reference to examples.
Example 1
In this embodiment, the concentrated hydrochloric acid is analytically pure hydrochloric acid supplied by Nanjing chemical reagent GmbH, the methanol is analytically pure methanol supplied by Kande science and technology GmbH of Tianjin city, the 2-nitrobenzaldehyde is analytically pure 2-nitrobenzaldehyde supplied by Shanghai Biotechnology GmbH, the ethyl acetate is analytically pure ethyl acetate supplied by Fuyu Fine chemical technology GmbH of Tianjin city, the n-hexane is analytically pure hexane supplied by Majon chemical reagent factory of Tianjin city, the dipotassium hydrogen phosphate is analytically pure dipotassium hydrogen phosphate supplied by Xiong science GmbH, the sodium hydroxide is analytically pure sodium hydroxide supplied by Kunshan south chemical material GmbH, the disodium hydrogen phosphate monohydrate is analytically pure disodium hydrogen phosphate supplied by Wuxi Crystal Co., the Nykungco chemical Co., the Potassium dihydrogen phosphate is analytically pure potassium dihydrogen phosphate supplied by Xiong science GmbH, the sodium chloride is analytically pure sodium chloride supplied by Guangdong Guanghua science and technology GmbH, the trehalose is D-trehalose supplied by Nanjing Dulai biotechnology GmbH, the Tween-20 is analytically pure Tween-20 supplied by Dajingmeng chemical reagent GmbH, the N-vinylpyrrolidone is analytically pure N-vinylpyrrolidone supplied by Guangdong Dulai chemical reagent GmbH, the divinylbenzene is 98% pure divinylbenzene supplied by Zhengzhou Jiekang chemical products GmbH, the diethylene glycol monobutyl ether is analytically pure diethylene glycol monobutyl ether supplied by Guangdong Ningjiang chemical reagent GmbH, the azodiisobutyronitrile is analytically pure azobisisobutyronitrile supplied by Guangdong Guangxiang chemical reagent GmbH, and the diethylene glycol is analytically pure diethylene glycol supplied by Dajingmeng chemical reagent GmbH.
The vortex oscillator in this embodiment selects MIT135C multi-tube vortex oscillator supplied by shangham institute limited, the constant temperature water bath selects HH-4A digital display constant temperature water bath supplied by huacheng huahua laboratory instruments factory in the gold altar area, the tissue triturator selects SJ-1 high speed tissue triturator supplied by shanghai shi instruments limited, the liquid-moving gun selects the anbend single-channel adjustable micro-range pipettor supplied by anbend china limited, the centrifuge selects TG16-WS desktop high speed centrifuge supplied by hunan xiang instruments limited, the automatic SPE column assembler selects the full-automatic SPE column assembler supplied by kazu bio technology limited, the colloidal gold card selects the furans metabolite colloidal gold card supplied by guangzhou Zhihui bio technology limited, the colloidal gold selects the furans metabolite colloidal gold test paper supplied by guangzhou Zhihui bio technology limited, the centrifugal tube is selected from commercial products, and the hollow column tube of the solid phase extraction column is selected from commercial products.
Reagents required for formulation in this example:
0.1mol/L hydrochloric acid solution: measuring 8.5ml of concentrated hydrochloric acid, and adding water to a constant volume of 1L;
0.2mol/L hydrochloric acid solution: measuring 17ml of concentrated hydrochloric acid, and adding water to a constant volume of 1L;
0.1mol/L dipotassium hydrogenphosphate: weighing 17.42g of dipotassium hydrogen phosphate, dissolving with water and fixing the volume to 1L;
1.0mol/L sodium hydroxide: weighing 40g of sodium hydroxide, dissolving with water and fixing the volume to 1L;
purifying agent: mixing methanol and water according to the volume of 1: 1;
buffer solution: weighing 0.96g of disodium hydrogen phosphate monohydrate, 0.17g of potassium dihydrogen phosphate, 9g of sodium chloride and 10g of D-trehalose, dissolving with water to a constant volume of 1L, adding 0.5ml of Tween-20, and mixing uniformly.
Preparation of the filler in this example:
under the protection of nitrogen, 50g of N-vinyl pyrrolidone and 50g of divinylbenzene are taken, the N-vinyl pyrrolidone and the divinylbenzene are mixed according to the mass ratio of 1:1, 50g of diluent is added, the mixture is uniformly stirred to obtain a mixture, the mass ratio of the diluent to the divinylbenzene to the N-vinyl pyrrolidone in the mixture is 1:1:1, the diluent comprises diethylene glycol butyl ether, ethylene glycol methyl ether acetate and diphenyl ether, and the mass ratio of the diethylene glycol butyl ether, the ethylene glycol methyl ether acetate and the diphenyl ether in the diluent is 3:2: 1; the mass ratio of the diethylene glycol butyl ether to the ethylene glycol methyl ether acetate to the diphenyl ether is 3:2:1, and the diethylene glycol butyl ether, the ethylene glycol methyl ether acetate and the diphenyl ether are used as diluents, so that the conversion rate of polymerization reaction can be improved, the molecular weight of the filler obtained by reaction can be improved, and the adsorbability of the filler to furan metabolites can be improved.
Adding 15g of azodiisobutyronitrile into 50g of diethylene glycol butyl ether, and uniformly stirring to obtain an initiator, wherein the mass ratio of the azodiisobutyronitrile to the diethylene glycol butyl ether in the initiator is 3: 10.
Adding an initiator into the mixture at the temperature of 65 ℃, uniformly stirring, keeping the temperature at 100 ℃, and carrying out polymerization reaction for 8 hours to obtain the filler for later use. The divinylbenzene is used as a cross-linking agent, the diethylene glycol butyl ether solution of azodiisobutyronitrile is used as an initiator, the N-vinyl pyrrolidone is subjected to polymerization reaction at the temperature of 100 ℃, so that a polymer of the N-vinyl pyrrolidone and the divinylbenzene is obtained, and the polymer is used as a filler of a solid phase extraction column and has high adsorption rate on furan metabolites.
Preparation of solid phase extraction column in this example:
selecting an empty column tube with the specification of 3ml, placing the empty column tube on an automatic SPE column assembling machine, adding the filler prepared in the embodiment on the automatic SPE column assembling machine, and assembling the column by using the automatic SPE column assembling machine to obtain 60mg/3ml, wherein the average particle size is 50-60 mu m, and the average pore size is
The solid phase extraction column for standby.
The reagents, apparatus, prepared solution and solid phase extraction column of example 1 were used in examples 2 to 7 below.
Example 2
The detection method of the furan metabolites in the aquatic products comprises the following steps:
step a, sample extraction and derivatization:
mashing fresh fish meat in an SJ-1 high-speed tissue mashing machine to ensure that the fresh fish meat is in a homogenized state, weighing 6.0g of homogenized sample, putting the sample into a 50ml centrifuge tube, adding 4.0ml of 0.1mol/L hydrochloric acid solution and 0.6ml of 2-nitrobenzaldehyde into the 50ml centrifuge tube, putting the 50ml centrifuge tube into an MIT135C multi-tube vortex oscillator to carry out vortex oscillation for 20s, putting the 50ml centrifuge tube into an HH-4A digital display constant temperature water bath kettle to carry out water bath incubation for 8min, wherein the water bath temperature is 80 ℃;
step b, extraction and purification:
adding 15.0mL of an extracting agent into the incubated sample, wherein the extracting agent consists of 4.6mL of 0.1mol/L dipotassium hydrogen phosphate solution, 0.4mL of 1mol/L sodium hydroxide solution and 10mL of ethyl acetate, fully mixing for 10s, then putting into a TG16-WS table type high-speed centrifuge, centrifuging for 1min at the centrifugal speed of 4000 revolutions/min to obtain a first supernatant, taking a centrifuge tube, taking 2.0mL of the first supernatant, adding into the centrifuge tube, adding 8.0mL of 1 +/-1 methanol aqueous solution, inverting for 4-5 times, centrifuging for 0.8min after uniformly mixing, standing for 0.8min to obtain a second supernatant, adding 80.0 mu L of diethylene glycol into the second supernatant, inverting for 0.6min, and uniformly mixing to obtain a first sample solution;
step c, enriching the first sample solution:
step c1, taking a solid phase extraction column, preparing a filler of the solid phase extraction column from N-vinyl pyrrolidone and divinyl benzene in a mass ratio of 1:1, and sequentially activating the solid phase extraction column by using 2.0ml of methanol and 8.0ml of water;
and c2, adding the first sample solution into the activated solid phase extraction column, rinsing the solid phase extraction column by using 5.0ml of water after the first sample solution completely passes through the solid phase extraction column, and vacuumizing to dry the solid phase extraction column. Eluting the solid phase extraction column with 400.0 μ l methanol, and collecting the eluate;
step d, preparing a second sample solution:
putting 3 drops of eluent and 3 drops of buffer solution into a 1.5ml centrifuge tube for mixing to obtain a mixed solution, sucking the mixed solution, dropwise adding the mixed solution into the gold-labeled micropores, and waiting for 1.5min to obtain a second sample solution;
step e, determination:
and (3) sucking the second sample liquid, dropwise adding the second sample liquid into the sample adding hole of the colloidal gold card for detection, judging the detection result, and judging the detection result to obtain the content of the furan metabolites in the sample.
Example 3
The detection method of the furan metabolites in the aquatic products comprises the following steps:
step a, sample extraction and derivatization:
putting fresh shrimp meat into an SJ-1 high-speed tissue triturator for triturating to enable the fresh shrimp meat to be in a homogeneous state, weighing 6.0g of homogenized sample, putting the sample into a 50ml centrifuge tube, adding 5.0ml of 0.1mol/L hydrochloric acid solution and 0.8ml of 2-chlorobenzaldehyde into the 50ml centrifuge tube, putting the 50ml centrifuge tube into an MIT135C multi-tube vortex oscillator for vortex oscillation for 30s, putting the 50ml centrifuge tube into an HH-4A digital display constant temperature water bath kettle for water bath incubation for 10min, wherein the water bath temperature is 75 ℃;
step b, extraction and purification:
adding 15.0mL of an extracting agent into the incubated sample, wherein the extracting agent consists of 4.6mL of 0.1mol/L dipotassium hydrogen phosphate solution, 0.4mL of 1mol/L sodium hydroxide solution and 10mL of ethyl acetate, fully mixing for 15s, then putting into a TG16-WS table type high-speed centrifuge, centrifuging for 2min at the centrifugal speed of 4000 revolutions/min to obtain a first supernatant, taking a centrifuge tube, taking 2.0mL of the first supernatant, adding into the centrifuge tube, adding 10.0mL of a purifying agent, inverting for 5 times, centrifuging for 1min after uniform mixing, standing for 1min to obtain a second supernatant, adding 100.0 mu L of diethylene glycol into the second supernatant, inverting for 1min, and obtaining a first sample after uniform mixing;
step c, enriching the first sample solution:
step c1, taking a solid phase extraction column, preparing a filler of the solid phase extraction column from N-vinyl pyrrolidone and divinyl benzene in a mass ratio of 1:1, and sequentially activating the solid phase extraction column by using 2.0ml of methanol and 8.0ml of water;
and c2, adding the first sample solution into the activated solid phase extraction column, rinsing the solid phase extraction column by using 5.0ml of water after the first sample solution completely passes through the solid phase extraction column, and vacuumizing to dry the solid phase extraction column. Eluting the solid phase extraction column with 400.0 μ l methanol, and collecting the eluate;
step d, preparing a second sample solution:
putting 3 drops of eluent and 3 drops of buffer solution into a 1.5ml centrifuge tube for mixing to obtain a mixed solution, sucking the mixed solution, dropwise adding the mixed solution into the gold-labeled micropores, and waiting for 2min to obtain a second sample solution;
step e, determination:
and (4) sucking the second sample liquid, dropwise adding the second sample liquid into the sample adding hole of the colloidal gold card for detection, and judging the detection result to obtain the content of the furan metabolites in the sample.
Example 4
The detection method of the furan metabolites in the aquatic products comprises the following steps:
step a, sample extraction and derivatization:
putting fresh shellfish meat into an SJ-1 high-speed tissue triturator for triturating to enable the fresh shellfish to be in a homogeneous state, weighing 6.0g of the homogenized sample, putting the sample into a 50ml centrifuge tube, then adding 6.0ml of 0.2mol/L hydrochloric acid solution and 1.0ml of 2-nitrobenzaldehyde into the 50ml centrifuge tube, putting the 50ml centrifuge tube into an MIT135C multi-tube vortex oscillator for vortex oscillation for 40s, then putting the 50ml centrifuge tube into an HH-4A digital display constant temperature water bath kettle for water bath incubation for 12min, wherein the water bath temperature is 70 ℃;
step b, extraction and purification:
adding 15.0mL of an extracting agent into the incubated sample, wherein the extracting agent consists of 4.6mL of 0.1mol/L dipotassium hydrogen phosphate solution, 0.4mL of 1mol/L sodium hydroxide solution and 10mL of n-hexane, fully mixing for 20s, then putting into a TG16-WS table type high-speed centrifuge, centrifuging for 3min at the centrifugal speed of 4000 revolutions/min to obtain a first supernatant, taking a centrifuge tube, taking 2.0mL of the first supernatant into the centrifuge tube, adding 12.0mL of 1 +/-1 methanol aqueous solution, turning upside down for 4 times, centrifuging for 1.2min after uniformly mixing, standing for 1.2min to obtain a second supernatant, adding 120.0 mu L of diglycol into the second supernatant, turning upside down for 1.4min, and obtaining a first sample liquid after uniformly mixing;
step c, enriching the first sample solution:
step c1, taking a solid phase extraction column, preparing a filler of the solid phase extraction column from N-vinyl pyrrolidone and divinyl benzene according to the mass ratio of 1:1, and sequentially activating the solid phase extraction column by using 3.0ml of methanol and 12.0ml of water;
and c2, adding the first sample solution into the activated solid phase extraction column, rinsing the solid phase extraction column by using 5.0ml of water after the first sample solution completely passes through the solid phase extraction column, and vacuumizing to dry the solid phase extraction column. Eluting the solid phase extraction column with 400.0 μ l methanol, and collecting the eluate;
step d, preparing a second sample solution:
taking 4 drops of eluent and 4 drops of buffer solution, putting the eluent and the buffer solution into a 1.5ml centrifugal tube for mixing to obtain a mixed solution, sucking the mixed solution, dropwise adding the mixed solution into the gold-labeled micropores, and waiting for 2.5min to obtain a second sample solution;
step e, determination:
and (3) sucking the second sample liquid and dropwise adding the second sample liquid to the sample adding region of the colloidal gold card for detection, and judging the detection result to obtain the content of the furan metabolites in the sample.
Example 5
And (3) respectively taking each furan metabolite as a separate internal standard substance to be added into the negative sample for detection by adopting an internal standard method. Four furan metabolites were AOZ (3-amino-2-oxazolidinone), SEM (semicarbazide), AMOZ (5-methylmorpholin-3-amino-2-oxazolidone), AHD (N-amino-2-hydantoin), and each furan metabolite was provided with a concentration gradient of 0, 0.1, 02, 0.3, 0.8, 1.2, 1.6, 3.2, 6.4, 10, 100ppb (μ g/kg), and was added to the negative sample according to this concentration gradient. The added samples were tested as in example 3 and the following table was obtained:
note: + represents that the detection line and the control line are both colored, and + -represents that only the quality control line is colored; the detection results in the table above are statistical results after 100 times of repetition under the same detection conditions.
As can be seen from the above table, the detection limit of this example 4 is 0.3ppb, i.e. the lowest amount of furan metabolites in the sample that can be detected in this example 5 is 0.3ppb, and the detection limit of the existing similar detection method is 0.5ppb, so the accuracy of the detection method of the present disclosure is better than that of the existing similar method. Compared with the existing similar method, the detection limit of the method is reduced by 40%, and the detection accuracy is greatly improved.
Example 6
The method (refer to the method of example 3), the high performance liquid chromatography (refer to the high performance liquid chromatography-tandem mass spectrometry method for detecting the residual quantity of nitrofurans drug metabolites in GB/T21311-2007 animal-derived food) and the enzyme-linked immunosorbent assay (refer to the furazolidone residual marker residue detection-enzyme-linked immunosorbent assay in agricultural division 1025 bulletin-17-2008 animal-derived food) of the invention are used for detecting the furans metabolites of the same aquatic product samples, and the following table is obtained:
| categories | The method of the invention | High performance liquid chromatography | Enzyme linked immunosorbent assay |
| Hydrolysis and derivatization time/h | 0.25 | 17.00 | 15.00 |
| Extraction, purification and enrichment time/h | 0.25 | 1.50 | 0.50 |
| Internal standard liquid preparation time/h | — | 2.00 | 2.50 |
| Measurement time/h | 0.10 | 4.00 | 2.50 |
| Total time/h | 0.60 | 24.50 | 20.50 |
| Detection time saving rate/%) | — | 97.55 | 97.07 |
From the above table, the total time for detecting the furan metabolites in the sample by using the method disclosed by the invention is 0.60 hour, which is reduced by 97.55 percent compared with the time for detecting by using the high performance liquid chromatography and 97.07 percent compared with the time for detecting by using the enzyme linked immunosorbent assay.
Example 7
The packing for the solid phase extraction column used in this example 7 was prepared using butyl diglycol: ethylene glycol methyl ether acetate: the mixed solution of phenyl ether ═ 3:2:1 was used as a diluent, acetonitrile was used as a diluent in the preparation of the filler of comparative example 1, toluene was used as a diluent in the preparation of the filler of comparative example 2, and N-vinylpyrrolidone: divinylbenzene: diluent 1:1: 1. Referring to the detection procedure of the present disclosure (referring to the detection procedure of example 3), the same fish meat samples were subjected to detection of furan metabolites (exemplified by semicarbazide) using the above different solid phase extraction columns, resulting in the following table:
as can be seen from the above table, the solid phase extraction column of this example 7 uses diethylene glycol butyl ether as the filler in the preparation process: ethylene glycol methyl ether acetate: the mixed solution of diphenyl ether-3: 2:1 is used as a diluent, so that the conversion rate of polymerization reaction can be improved, the molecular weight of a polymer filler obtained by polymerization reaction is improved, the adsorbability of the filler to semicarbazide (furacilin metabolite) is improved, the adsorbability of the filler to furan metabolites is enhanced, the standard recovery rate reaches 87.4 percent, namely, the content of the furan metabolite in a sample can be detected to be 0.343ppb at the lowest under the condition of 0.3ppb detection limit. The detection limits of the solid phase extraction columns of the comparative example 1 and the comparative example 2 are 0.455ppb and 0.789ppb respectively, so the recovery rate of the furan metabolites by using the filler prepared in the disclosure is better than that of the filler prepared in the comparative example 1 and the comparative example 2, and the detection accuracy is greatly improved.
The solid phase extraction column of the filler prepared by the method disclosed by the invention can achieve the effect of 100% elution by using a small amount of eluent, does not need to carry out additional concentration operation, shortens the elution time and the concentration time, controls the elution concentration time within 15s, greatly reduces the detection time, and is more suitable for market batch rapid detection of aquatic products.
In summary, according to the detection method for the furan metabolites in the aquatic products disclosed by the present disclosure, the furan metabolites remaining in the muscle tissue of the sample are hydrolyzed under an acidic condition, derivatized with 2-nitrobenzaldehyde or 2-chlorobenzaldehyde, purified and purified, adsorbed and eluted by the solid phase extraction column to obtain the test solution, the obtained test solution is dissolved in the gold-labeled micropores, and then the colloidal gold card is added dropwise for detection. Used reagent is ordinary chemical reagent, and the security is high, need not use large-scale detecting instrument, and the solid-phase extraction post that uses in this disclosure adopts with diethylene glycol butyl ether: ethylene glycol methyl ether acetate: the filler prepared by using the mixed solution of diphenyl ether-3: 2:1 as a diluent greatly shortens the detection time, is simple and convenient to operate, has low detection cost, high sensitivity and high detection accuracy, has detection limits of 0.3ppb for four furan metabolites, has low requirements on experimental environment, and is suitable for rapid detection of aquatic products.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (10)
1. The preparation method of the solid phase extraction column comprises the following steps:
preparing a filler: under the protection of inert gas, mixing N-vinyl pyrrolidone and divinylbenzene, adding a diluent to obtain a mixture, adding an initiator to the mixture, uniformly mixing, and carrying out polymerization reaction to obtain a filler, wherein the mass ratio of the diluent to the divinylbenzene to the N-vinyl pyrrolidone in the mixture is 1:1:1, the diluent comprises diethylene glycol butyl ether, ethylene glycol methyl acetate and diphenyl ether, and the mass ratio of the diethylene glycol butyl ether to the ethylene glycol methyl acetate to the diphenyl ether in the diluent is 3:2: 1;
preparing a solid phase extraction column: a fixed extraction column is prepared and obtained by using the packing.
2. The method of claim 1, wherein the solid phase extraction column is used for measuring furan metabolites selected from at least one of 3-amino-2-oxazolidinone, 5-methylmorpholine-3-amino-2-oxazolidone, 1-amino-2 hydantoin, and semicarbazide.
3. The solid phase extraction column obtained by the preparation method of claim 1, wherein the packing of the solid phase extraction column is prepared from N-vinyl pyrrolidone: divinylbenzene: diluent 1:1:1 is prepared.
4. The method for detecting the furan metabolites in the aquatic products comprises the following steps:
step a, homogenizing a sample, adding hydrochloric acid and an aldehyde group-containing compound, and performing derivatization;
step b, extracting the derivatized sample to obtain a first sample solution;
step c, adding the first sample solution into the solid phase extraction column of any one of claim 1 or claim 3 to obtain an eluent;
step d, mixing the eluent and the buffer solution to obtain a mixed solution, and dropwise adding the mixed solution into the gold-labeled micropores to obtain a second sample solution;
and e, dropwise adding the second sample solution into a colloidal gold card for determination.
5. The method for detecting furans metabolites in aquatic product according to claim 4, wherein the step a comprises
Adding a hydrochloric acid solution and an aldehyde group-containing compound into the sample, carrying out vortex oscillation, and then carrying out water bath incubation on the sample;
said step b comprises
Adding an extracting agent into the sample incubated in the water bath, fully mixing, then carrying out first centrifugation to obtain a first supernatant, taking the first supernatant and adding a purifying agent, uniformly mixing, carrying out second centrifugation, standing to obtain a second supernatant, adding diglycol into the second supernatant, and uniformly mixing to obtain a first sample solution;
said step c comprises a first sample enrichment:
step c1, activating the solid phase extraction column by methanol and water in sequence before use;
step c2, adding the first sample solution into the activated solid phase extraction column, leaching the solid phase extraction column with water after the first sample solution completely passes through the solid phase extraction column, and vacuumizing to dry the solid phase extraction column; and eluting the solid phase extraction column by using methanol to obtain an eluent.
6. The method for detecting furan metabolites in aquatic products of claim 5, wherein the specification of the solid phase extraction column is 60mg/3ml, the average particle size of the solid phase extraction column is 50-60 μm, and the average pore size of the solid phase extraction column is
7. The method for detecting furan metabolites in aquatic products according to claim 5, wherein the concentration of the hydrochloric acid solution is 0.1-0.2mol/L, the aldehyde group-containing compound is one of 2-nitrobenzaldehyde or 2-chlorobenzaldehyde, and the water bath temperature in the step a is 70-80 ℃.
8. The method for detecting the furan metabolites in aquatic products of claim 5, wherein the extractant comprises 0.1mol/L dipotassium hydrogen phosphate solution, 1.0mol/L sodium hydroxide solution and n-hexane, the volume ratio of the 0.1mol/L dipotassium hydrogen phosphate solution, the 1.0mol/L sodium hydroxide solution and the n-hexane in the extractant is 46:4:100, and the purifying agent is a mixture of the following components in the volume ratio of 1:1, the rotating speed of the first centrifugation is 4000 revolutions per minute, and the rotating speed of the second centrifugation is 4000 revolutions per minute.
9. The method for detecting furans metabolites in aquatic product according to claim 5, wherein the volume ratio of methanol to water in step c1 is 1:4, and the volume ratio of eluent to buffer solution in step d is 1: 1.
10. The method for detecting the furan metabolites in the aquatic product according to claim 5, wherein the method for preparing the buffer solution comprises the following operations: accurately weighing 0.96g of disodium hydrogen phosphate monohydrate, 0.17g of potassium dihydrogen phosphate, 9.0g of sodium chloride and 10.0g of trehalose, dissolving with water, diluting to 1.0L, adding 0.15ml of tween-20, and mixing uniformly for later use.
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