Method for detecting nitrofuran metabolites in macrobrachium rosenbergii based on UPLC-MS / MS and application thereof

Abstract

The invention discloses a method for detecting nitrofuran metabolites in macrobrachium rosenbergii based on UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) and application of the method, and belongs to the technical field of safety evaluation and detection of aquatic products. According to the method, a macrobrachium rosenbergii sample is pretreated by utilizing a QuECHERS method, nitrofuran metabolites are extracted, and the method comprises the steps of adding acidified acetonitrile for extraction, adding ammonium acetate and sodium chloride for water removal, adding a dispersed solid extraction column for reducing a matrix effect and the like. After the treatment by the QuECHERS method disclosed by the invention, the obtained nitrofuran metabolite does not need to be subjected to a derivatization procedure, and an HILIC hydrophilic chromatographic column can be directly used for carrying out liquid chromatography-mass spectrometry analysis in a positive ion mode so as to detect the nitrofuran metabolite such as semicarbazide and the like. The detection method provided by the invention has the advantages of high sensitivity, strong accuracy, wide applicability and the like, and compared with the traditional detection method, the reagent waste can be avoided, and a large amount of time can be saved.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic product safety assessment and detection, and in particular to a UPLC-MS / MS-based detection method for nitrofuran metabolites in Macrobrachium rosenbergii and an application thereof. Background Art

[0002] Semicarbazide (SEM) is an amine hydrazine compound with moderate cumulative toxicity, especially in terms of reproductive toxicity and mutagenicity. The sources of semicarbazide in food include endogenous production and production from the metabolism of nitrofuran (NFZ). Endogenous semicarbazide is found in the following situations: (1) seasonings and flour added with azodicarbonamide; (2) foods with hypochlorite as the main disinfectant ingredient; (3) crustaceans such as shrimp and crab. In addition, semicarbazide has been reported to be detected in honey and plastic gaskets of some food containers. It should be emphasized that the content of endogenous semicarbazide in natural foods is at a low level, while high levels of SEM are often produced due to the presence of NFZ. NFZ is an inexpensive and highly effective broad-spectrum redox-active bactericidal drug that can effectively treat bacterial diseases in livestock and aquatic animals. However, due to the toxicity of its secondary metabolites, represented by SEM, the Food and Drug Administration and the European Union banned the use of NFZ as an antimicrobial treatment in aquaculture in 1955. Similarly, Announcement No. 250 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China clearly stated that nitrofurans are prohibited drugs for use in food animals.

[0003] SEM can bind to proteins to form stable polymers. For this reason, my country, the United States and the European Union still use semicarbazide as the iconic secondary metabolite of nitrofuran and set its detection limit. Since semicarbazide contains amide bonds and hydrazine bonds, it has a high response value in the mass spectrometry, but its retention effect is poor and it often peaks in the dead time of mass spectrometry analysis. In this case, the strong matrix effect will inhibit the detection signal, so it is often necessary to perform a derivatization operation on it. At present, the analysis method of semicarbazide can generally be divided into three parts: sample pretreatment, derivatization and detection. During the sample pretreatment process, acetic acid acidification acetonitrile extraction is often required to promote the release of SEM bound to proteins. The derivatization process usually uses 2-nitrobenzaldehyde (soluble in dimethyl sulfoxide) as a derivatization reagent to enhance the hydrophobicity of SEM and perform overnight derivatization. After the extraction solution system is adjusted to neutral, liquid-liquid extraction is performed using organic solutions such as ethyl acetate and n-hexane. After multiple centrifugation, the derivatized product is collected and degreasing operations are performed. Finally, data acquisition was performed by UPLC-ESI-MS / MS combined with multi-stage reaction monitoring mode. It should be pointed out that the pre-treatment process of this method is still relatively complicated, the amount of reagents used is large, and the derivatization operation is time-consuming. Therefore, in the detection and analysis of SEM, it is urgent to combine other technical means to develop a novel, efficient, highly sensitive and highly accurate analysis method. Summary of the invention

[0004] The Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method can achieve rapid high-throughput extraction of samples. In compliance with the principles of green chemistry, this method is suitable for most multi-residue pesticide analyses in food matrices. With its high efficiency and convenience, it provides a reliable technical means for the detection of pesticide residues in food, reducing analysis time and cost while also reducing the impact on the environment. Hydrophilic interaction liquid chromatography (HILIC) is a normal phase chromatography with a strong ability to separate polar compounds.

[0005] Based on the characteristics of these two methods, the purpose of the present invention is to provide a detection method for nitrofuran metabolites in Macrobrachium rosenbergii based on UPLC-MS / MS and its application, so as to solve the problem of complex pretreatment in the above process. This method has high sensitivity, high accuracy and wide applicability, and is suitable for rapid detection by SEM in the future.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for detecting nitrofuran metabolites in Macrobrachium rosenbergii based on UPLC-MS / MS, comprising the following steps:

[0008] Collect samples of Macrobrachium rosenbergii;

[0009] Performing QuECHERS treatment on the Macrobrachium rosenbergii sample to obtain an extract;

[0010] Performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis on the extract to detect the content of the nitrofuran metabolites;

[0011] The QuECHERS process includes:

[0012] Adding acidified acetonitrile to the Macrobrachium rosenbergii sample and shaking;

[0013] Add sodium chloride and ammonium acetate, shake, centrifuge, and collect the upper solution;

[0014] The upper layer solution is added to the extraction column, vortexed, centrifuged, the supernatant is collected, dried with nitrogen, and re-dissolved with acetonitrile to obtain the extract.

[0015] The invention adds acidified acetonitrile as an extraction solvent to extract nitrofuran metabolites from a Macrobrachium rosenbergii sample; uses sodium chloride and volatile ammonium acetate as dehydrating agents in the sample to improve the recovery rate; and uses an extraction column to reduce the matrix effect.

[0016] Preferably, the acidified acetonitrile is acetonitrile acidified with formic acid; and the volume fraction of the formic acid is 0.01%.

[0017] Preferably, the extraction column is a dSPE EMR Lipid dispersed solid extraction column.

[0018] Preferably, the Macrobrachium rosenbergii sample is shrimp meat or shrimp shell;

[0019] When the Macrobrachium rosenbergii sample is shrimp meat, the mass volume ratio of the shrimp meat to the acidified acetonitrile is 1 mg:5 mL, and the mass ratio of the shrimp meat, the sodium chloride and the ammonium acetate is 3:1:4;

[0020] When the Macrobrachium rosenbergii sample is shrimp shell, the mass volume ratio of the shrimp meat to the acidified acetonitrile is 1 mg:10 mL, and the mass ratio of the shrimp shell, the sodium chloride and the ammonium acetate is 2:1:4.

[0021] Preferably, the liquid chromatography is hydrophilic interaction liquid chromatography, and the column temperature is 40°C.

[0022] Preferably, the mobile phase A of the chromatography is 2 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile acidified with formic acid; the volume fraction of the formic acid is 0.01%;

[0023] The elution program of the chromatography was: 100% B from 0 to 3 min, gradient decreased to 80% B from 3 to 4.5 min, 80-60% B from 4.5 to 5.5 min, 60% B from 5.5 to 8 min, 60-100% B from 8 to 10 min, and 100% B from 10 to 15 min; the flow rate was 0.5 mL / min.

[0024] Preferably, the mass spectrometry conditions are electrospray ionization; positive ion scanning mode, scanning method is multiple reaction monitoring; capillary voltage is 0.8 kV, and ion source temperature is 150°C.

[0025] Preferably, the nitrofuran metabolite comprises semicarbazide.

[0026] The present invention also provides an application of the above method in dynamically monitoring the change of semicarbazide content in Macrobrachium rosenbergii under nitrofuran stress.

[0027] The present invention discloses the following technical effects:

[0028] The present invention utilizes the QuECHERS technology to efficiently extract SEM from Macrobrachium rosenbergii samples, and then combines it with hydrophilic chromatography detection and analysis technology to achieve the dynamic change analysis of SEM content in Macrobrachium rosenbergii under nitrofuran bath stress conditions.

[0029] The QuECHERS method provided by the present invention is easy to operate, can fully extract SEM in a sample in a short time, uses less reagents, avoids potential waste and pollution, and the introduced reagents have no strong toxicity except acetonitrile, which meets the requirements of experimental safety.

[0030] The normal phase chromatography-mass spectrometry method provided by the present invention avoids the derivatization operation in the conventional SEM detection method, and can achieve SEM qualitative and quantitative detection through direct sample injection, which can significantly shorten the overall analysis time.

[0031] The QuECHERS pretreatment method combined with normal phase chromatography tandem mass spectrometry analysis method provided by the present invention has high sensitivity, strong accuracy, and wide applicability, and can be used for the determination of the residual amount of nitrofuran metabolites in Macrobrachium rosenbergii, providing possible methodological support for the future detection of semicarbazide and related analysis such as endogenous semicarbazide. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 It is a schematic diagram of the technical route of the present invention;

[0034] Figure 2 Schematic diagram of the QuECHERS pretreatment process of the present invention;

[0035] Figure 3 is a mass spectrum behavior diagram of semicarbazide; wherein A is the structural formula of semicarbazide and semicarbazide internal standard; B is a mass spectrum behavior diagram of semicarbazide; C is a mass spectrum behavior diagram of semicarbazide internal standard;

[0036] Figure 4 is the standard curve of semicarbazide in Macrobrachium rosenbergii samples;

[0037] Figure 5 is the extracted ion chromatogram of semicarbazide; where A is the ion chromatogram of semicarbazide in C 18 Schematic diagram of the retention effect of chromatographic column and HILIC column; B is the retention effect of aminourea standard in C 18 Chromatography and extraction chromatogram under HILIC conditions; C is the extraction chromatogram of semicarbazide standard under HILIC conditions; D is the extraction chromatogram of Macrobrachium rosenbergii sample spiked under HILIC conditions;

[0038] Figure 6 The figure is a statistical chart showing the changes in the content of semicarbazide in Macrobrachium rosenbergii under nitrofuran stress; A is the content of semicarbazide in the Macrobrachium rosenbergii samples of the experimental group; B is the content of semicarbazide in the Macrobrachium rosenbergii samples of the control group; SEM is semicarbazide. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] like Figure 1 As shown, the present invention first provides the most suitable SEM extraction method, and then optimizes the liquid chromatography tandem mass spectrometry method to analyze the content of SEM in Macrobrachium rosenbergii, as described in detail as follows:

[0045] Example 1

[0046] 1. Experimental Methods

[0047] 1.1 Processing of shrimp meat samples

[0048] Sample processing diagram Figure 2 Specifically, the sampled part of the shrimp meat is the abdominal muscle, and the sampled part of the shrimp shell is the abdominal shell. Subsequently, they are processed separately using a blender.

[0049] For shrimp meat, accurately weigh 3g of shrimp meat in a 50mL polypropylene centrifuge tube, add 15mL of acidified acetonitrile (0.01% formic acid, v / v) and 100μL of internal standard solution (1μg / mL), vortex for 30s, add 4g of ammonium acetate and 1g of sodium chloride, vortex briefly and centrifuge. Collect the upper solution and add 2g of dSPE EMR Lipid dispersed solid extraction column, vortex for 30s and centrifuge at low speed (1000rpm, 1min) to collect the supernatant.

[0050] For shrimp shells, accurately weigh 2g of shrimp shells in a 50mL polypropylene centrifuge tube, add 20mL of acidified acetonitrile (0.01% formic acid, v / v) and 100μL of internal standard solution (1μg / mL), vortex for 30s, add 4g of ammonium acetate and 1g of sodium chloride, vortex briefly and centrifuge. Collect the upper solution and add 2g of dSPE EMR Lipid dispersed solid extraction column, vortex for 30s and centrifuge at low speed (1000rpm, 1min) to collect the supernatant.

[0051] 1.5 UPLC-MS analysis

[0052] The collected shrimp meat and shrimp shell were dried by nitrogen blowing, then redissolved in 1 mL of acetonitrile and directly subjected to UPLC-MS analysis.

[0053] Unless otherwise specified, the technical means used in this example, including the extraction of nucleic acids, are conventional means known to those skilled in the art. The mass spectrometer used in the UPLC MS method is a Waters Xevo TQ S micro massspectrometer (Milford, USA), using an electrospray ion source. The liquid phase is a Waters Acquity UPLC (Milford, USA). The compounds were separated using a BEH HILIC column, and the column temperature was set at 40 °C.

[0054] Mobile phases A and B were 2 mM ammonium acetate aqueous solution and acetonitrile (acidified with 0.01% formic acid, v / v), respectively.

[0055] The elution gradient was as follows: 100% B from 0 to 3 min, gradient decreased to 80% B from 3 to 4.5 min, 80 to 60% B from 4.5 to 5.5 min, 60% B from 5.5 to 8 min, 60 to 100% B from 8 to 10 min, and 100% B from 10 to 15 min. The flow rate was set at 0.5 mL / min, and quantification was performed under positive ion conditions using the ion with the highest ion abundance (SEM, 76.1>30.8; 13 C- 15 N 2 -SEM, 79>33.0, cone voltage and collision voltage were 12 V). Characteristic ion fragments were used for identification (SEM, 76.1>58.8; 13 C- 15 N 2 -SEM, 79>61.8, cone voltage and collision voltage are 12V). SEM mass spectrometry behavior is shown in Figure 3 The capillary voltage was 0.8 kV, the ion source temperature was 300 °C, and the desolvation gas flow was 1000 L / Hr (N2 ), cone airflow is 20L / Hr(N 2 ) The temperature of the automatic sampler was set to 4°C and the injection volume was 1 μL. The mass spectrometry behavior of the aminourea internal standard is shown in Figure 3 shown.

[0056] 1.6 Preparation of standard solution

[0057] Accurately pipette 1 mg / mL of SEM internal standard stock solution and dilute it to 10 μg / mL with acetonitrile to prepare working solution. Weigh 10 mg of SEM standard and dissolve it in 100 mL of acetonitrile to prepare stock solution, then dilute it to 1 μg / mL with acetonitrile to prepare working solution. Store the stock solution in a -20℃ refrigerator and prepare the working solution for immediate use.

[0058] 1.7 Establishment of standard curve and determination of sensitivity

[0059] Prepare mixed standard working solutions with SEM and 13C-15N2-SEM mass concentration ratios of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 (wherein, 13 C- 15 N 2 -SEM mass concentration is 1μg / mL). The standard curve is drawn using the concentration ratio as the horizontal axis and the response ratio as the vertical axis. The sensitivity is represented by the detection limit and the quantification limit. The concentration when the signal / baseline is 3.3 is defined as the detection limit, and the concentration when the signal / baseline is 10 is defined as the quantification limit.

[0060] 1.8 Determination of extraction efficiency, matrix effect, recovery and precision

[0061] The SEM concentration measured by replacing the Macrobrachium rosenbergii sample with pure solvent is defined as C 注射 , the SEM concentration detected in the unspiked Macrobrachium rosenbergii sample was C 内源性 , the SEM concentration detected in the Macrobrachium rosenbergii sample with standard added before pretreatment was C 前加标 The SEM concentration detected by adding the standard sample to the extract obtained after pretreatment is C 后加标 The calculation formulas for recovery rate and matrix effect are as follows:

[0062] Extraction efficiency (%) = [(C 前加标 -C 内源性 ) / (C 后加标 -C 内源性 )]×100;

[0063] Matrix effect (%) = [(C 后加标 -C 内源性 ) / (C 注射)]×100;

[0064] Recovery rate (%) = [(C 前加标 -C 内源性 ) / (C 注射 )]×100;

[0065] Precision is expressed as intra-day and inter-day precision, using the SEM and 13 C- 15 N 2 -SEM mass concentration ratio of 0.01, 0.1 and 1 mixed standard solution, each sample has three parallel, respectively, in the same day at different times of the injection three times and in the same week on different days of the injection three times, the coefficient of variation (R 2 ).

[0066] 2. Results

[0067] The present invention investigates the extraction efficiency, matrix effect and recovery rate of the newly proposed QuECHERS method, and then investigates the precision, linearity and sensitivity of the UPLC-MS / MS method. According to the proposed QuECHERS method, three concentrations of SEM standard solutions of 10 ng / mL, 100 ng / mL and 1000 ng / mL were prepared, which were defined as low, medium and high concentrations, respectively. The detection was carried out according to the method in 1.3 (n=5). The semicarbazide matrix of Macrobrachium rosenbergii is relatively complex, and it is a great challenge to purify the high-concentration hydrophilic matrix from the co-extraction without losing the analyte. When the extraction efficiency is 88.2-96.4%, the matrix causes a response loss of 7.9-29.3%, and the recovery rate is between 76-95% (Table 1). Under low concentration conditions, the complexity of the extract is relatively large and the recovery rate is low, but the overall recovery rate and precision of the method are appropriate. For precision, according to the proposed UPLC-MS / MS method, three concentrations of SEM standard solutions of 10 ng / mL, 100 ng / mL, and 1000 ng / mL were prepared, which were defined as low, medium, and high concentrations, respectively. They were added to the sample for detection (n=5). The results showed that the precision coefficient of variation of SEM at three concentration levels was less than 10%, which could meet the methodological requirements (Table 2). The linearity evaluation of semicarbazide used the concentration ratio of semicarbazide and semicarbazide internal standard as the horizontal axis, and the peak area ratio of semicarbazide and semicarbazide internal standard as the vertical axis to draw a standard curve, achieving calibration in the range of 10 to 50000 μg / kg, and the R of the standard curve was 0. 2 is 0.9994( Figure 4 ). The detection limit and quantification limit based on 3.3 times signal-to-noise ratio and 10 times signal-to-noise ratio were 0.6 μg / kg and 1.0 μg / kg respectively (Table 3). The calculation formula of the semicarbazide content in the sample is:

[0068]

[0069] Where X is the content of semicarbazide in the sample, in micrograms per kilogram (μg / kg);

[0070] C i -The concentration of semicarbazide in the sample preparation solution, in nanograms per milliliter (ng / mL);

[0071] V-final fixed volume, in milliliters (mL);

[0072] M-sample mass, in grams (g).

[0073] Table 1 Evaluation of the newly proposed QuECHERS method

[0074]

[0075]

[0076] Table 2 Precision evaluation of the newly proposed UPLC-MS / MS

[0077]

[0078] Table 3 Standard curve and sensitivity of UPLC-MS / MS method

[0079]

[0080] Example 2

[0081] Changes in semicarbazide content in Macrobrachium rosenbergii under furazolidone stress.

[0082] 1. Methods

[0083] 1.1 Obtaining Macrobrachium rosenbergii samples

[0084] Macrobrachium rosenbergii was collected from the Guangzhou Aquaculture Base, which strictly followed the "Technical Specifications for Macrobrachium rosenbergii Pond Culture" during the artificial breeding process and did not add nitrofuran drugs during the growth period. Mature individuals with complete tentacles and active bodies were selected, and after purchase, they were connected to oxygen throughout the process and transported to the laboratory by a transport vehicle for temporary maintenance in an indoor circulating water culture box.

[0085] 1.2 Macrobrachium rosenbergii culture conditions

[0086] The shrimps were cultured at 20°C and continuously oxygenated with an oxygen pump until the dissolved oxygen reached the maximum value (7.8 mg / L). Sterilized plastic partitions were used to isolate the shrimps in the culture boxes to prevent fighting. During the culture period, the feces and food residues were removed by siphoning, and dead shrimps were removed in time. Before the formal experiment began, nitrofurans and SEM were tested for both the water and the feed. The average individual weight of Macrobrachium rosenbergii was 12.0 g ± 2.0 g and the body length was 6 cm ± 1 cm. The shrimps were placed in the culture tanks and randomly divided into groups with a culture density of 100 / m 3 .

[0087] 1.3 Nitrofuran bath stress

[0088] The Macrobrachium rosenbergii that had been fasted for two days were evenly divided into two groups, 15 in each group, set as the control group and the experimental group. No nitrofuran was added to the control group, and the experimental group was treated by immersing them in a 10 mg / L nitrofuran aqueous solution for 2 h. They were then transferred to clean water for normal culture. Samples of Macrobrachium rosenbergii were collected at 0, 4, 12, 36, 108, 324 and 972 h, with 3 shrimp samples collected each time.

[0089] 1.4 Macrobrachium rosenbergii sample processing

[0090] The collected Macrobrachium rosenbergii were frozen to death using liquid nitrogen, and then the samples were transferred to a -80°C refrigerator for freezing and storage, and slightly thawed before sampling to facilitate separation of shrimp meat and shrimp shells. The samples were then processed according to Example 1.

[0091] 2. Results

[0092] The extraction chromatographic results of the optimized QuECHERS method by SEM are shown in Figure 5 .

[0093] The changes of nitrofuran content in the meat and shell of Macrobrachium rosenbergii under nitrofuran stress are shown in Figure 6 . The results showed that for shrimp meat, after 36 hours of drug bathing, the maximum value reached 74.14μg / kg, and then began to slowly decay. After 972 hours, 10.65μg / kg of SEM could still be detected in the shrimp meat, while 9.04μg / kg of SEM could be detected in the shrimp meat of the control group that had not been bathed in drugs. This means that it takes about 40 days for Macrobrachium rosenbergii to completely metabolize SEM after nitrofuran drug bath stress. Regardless of the control group or the experimental group, the content of SEM in the shrimp shell increased with time, reaching the highest value at 972 hours, which was 21.54μg / kg and 16.11μg / kg, respectively. This result indicates that there may be a way for the SEM in Macrobrachium rosenbergii meat to be transferred to the shrimp shell.

[0094] In addition, the changes in SEM content in the control group samples of Macrobrachium rosenbergii showed that there was obvious endogenous SEM production in Macrobrachium rosenbergii, and there was a possibility of transfer from shrimp meat to shrimp shell.

[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting nitrofuran metabolites in Macrobrachium rosenbergii based on UPLC-MS / MS, characterized in that: The following steps are involved: Collect samples of Macrobrachium rosenbergii; Performing QuECHERS treatment on the Macrobrachium rosenbergii sample to obtain an extract; Performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis on the extract to detect the content of the nitrofuran metabolites; The QuECHERS process includes: Adding acidified acetonitrile to the Macrobrachium rosenbergii sample and shaking; Add sodium chloride and ammonium acetate, shake, centrifuge, and collect the upper solution; The upper layer solution is added to the extraction column, vortexed, centrifuged, the supernatant is collected, dried with nitrogen, and re-dissolved with acetonitrile to obtain the extract.

2. The method according to claim 1, characterized in that The acidified acetonitrile is acetonitrile acidified with formic acid; the volume fraction of the formic acid is 0.01%.

3. The method according to claim 1, characterized in that The extraction column is a dSPE EMR Lipid dispersed solid extraction column.

4. The method according to claim 1, characterized in that The Macrobrachium rosenbergii sample is shrimp meat or shrimp shell; When the Macrobrachium rosenbergii sample is shrimp meat, the mass volume ratio of the shrimp meat to the acidified acetonitrile is 1 mg:5 mL, and the mass ratio of the shrimp meat, the sodium chloride and the ammonium acetate is 3:1:4; When the Macrobrachium rosenbergii sample is shrimp shell, the mass volume ratio of the shrimp meat to the acidified acetonitrile is 1 mg:10 mL, and the mass ratio of the shrimp shell, the sodium chloride and the ammonium acetate is 2:1:

4.

5. The method according to claim 1, characterized in that The liquid chromatography is hydrophilic interaction liquid chromatography, and the column temperature is 40°C.

6. The method according to claim 1, characterized in that The mobile phase A of the liquid chromatography is 2 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile acidified with formic acid; the volume fraction of the formic acid is 0.01%.

7. The method according to claim 6, characterized in that The elution program of the liquid chromatography is: 100% B in 0-3 min, gradient reduced to 80% B in 3-4.5 min, 80-60% B in 4.5-5.5 min, 60% B in 5.5-8 min, 60-100% B in 8-10 min, 100% B in 10-15 min; the flow rate is 0.5 mL / min.

8. The method according to claim 1, characterized in that The mass spectrometry conditions are electrospray ionization; positive ion scanning mode, scanning method is multiple reaction monitoring; capillary voltage is 0.8 kV, and ion source temperature is 150°C.

9. The method according to any one of claims 1 to 8, characterized in that The nitrofuran metabolites include semicarbazide.

10. Use of the method according to any one of claims 1 to 9 in dynamically monitoring the changes in semicarbazide content in Macrobrachium rosenbergii under nitrofuran stress.

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