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

Abstract

The application discloses a kind of detection methods of nitrofurans metabolites in macrobrachium rosenbergii based on UPLC-MS / MS and application thereof, belong to aquatic product safety evaluation detection technical field.The application utilizes QuECHERS method to the pretreatment of macrobrachium rosenbergii sample, extracts nitrofurans metabolites, including adding acidified acetonitrile extraction, adding ammonium acetate, water removal and adding dispersed solid extraction column to reduce matrix effect and other steps.After the QuECHERS method of the application is handled, the obtained nitrofurans metabolites do not need to be subjected to derivatization procedure, can directly use HILIC hydrophilic chromatographic column, in positive ion mode liquid chromatography-mass spectrometry analysis, detect aminourea and other nitrofurans metabolites.The detection method provided by the application has the advantages of high sensitivity, strong accuracy, wide applicability, compared with traditional detection method, can avoid reagent waste, save a lot of time.

Description

Technical Field

[0001] This invention relates to the field of aquatic product safety assessment and testing technology, and in particular to a method for detecting nitrofuran metabolites in giant freshwater prawns based on UPLC-MS / MS and its application. Background Technology

[0002] Semicarbazide (SEM) is an amine hydrazine compound with moderate cumulative toxicity, particularly in reproductive toxicity and mutagenic effects. Sources of semicarbazide in food include endogenous production and metabolism from nitrofuran (NFZ). Endogenous semicarbazide is found in: (1) seasonings and flour containing azodicarbonamide; (2) foods with hypochlorite as the main disinfectant; and (3) crustaceans such as shrimp and crab. Furthermore, semicarbazide has been reported in honey and plastic gaskets in some food containers. It is important to note that the content of endogenous semicarbazide in natural foods is relatively low, while high levels of SEM are often due to the presence of NFZ. NFZ is an inexpensive and highly effective broad-spectrum redox bactericide that can effectively treat bacterial diseases in livestock and aquatic animals. However, given the toxicity of its secondary metabolites, typically SEM, the Food and Drug Administration and the European Union banned the use of NFZ as an antimicrobial treatment in livestock in 1955. Similarly, Announcement No. 250 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China explicitly states that nitrofurans are prohibited from use as drugs 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 aminourea as a marker secondary metabolite of nitrofurans and have set detection limits. Because aminourea contains amide and hydrazine bonds, it exhibits a high response value in mass spectrometry; however, its retention is poor, often eluting at the dead time of mass spectrometry analysis. In this case, the strong matrix effect can suppress the detection signal, so derivatization is often necessary. Currently, the analytical method for aminourea can generally be divided into three parts: sample pretreatment, derivatization, and detection. In the sample pretreatment process, acetic acidification with acetonitrile extraction is often required to promote the release of SEM bound to proteins. The derivatization process typically uses 2-nitrobenzaldehyde (soluble in dimethyl sulfoxide) as the derivatizing reagent to enhance the hydrophobicity of the SEM, and derivatization is performed overnight. Subsequently, after adjusting the extraction solution system to neutral, liquid-liquid extraction is performed using organic solutions such as ethyl acetate and n-hexane. After multiple centrifugations, the derivatized product is collected and subjected to defatting and other procedures. Finally, data acquisition was performed using UPLC-ESI-MS / MS combined with multi-stage reaction monitoring. It should be noted that the pretreatment process of this method remains relatively complex, requiring large amounts of reagents and involving time-consuming derivatization operations. Therefore, in SEM detection and analysis, there is an urgent need to develop a novel, efficient, highly sensitive, and highly accurate analytical method that integrates with other techniques. Summary of the Invention

[0004] The Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method enables rapid, high-throughput sample extraction. When aligned with green chemistry principles, this method is suitable for the analysis of most multi-residue pesticides in food matrices. Its high efficiency and convenience provide a reliable technical means for pesticide residue detection in food, reducing analysis time and cost while also minimizing environmental impact. Hydrophilic interaction liquid chromatography (HILIC) is a normal-phase chromatography technique with a powerful ability to separate polar compounds.

[0005] Based on the characteristics of these two methods, the purpose of this invention is to provide a method for detecting nitrofuran metabolites in giant freshwater prawns 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 objectives, the present invention provides the following solution:

[0007] This invention provides a method for detecting nitrofuran metabolites in giant freshwater prawns based on UPLC-MS / MS, comprising the following steps:

[0008] Collect samples of giant freshwater prawns;

[0009] The giant freshwater prawn sample was subjected to QuECHERS treatment to obtain an extract;

[0010] The extract was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to detect the content of the nitrofuran metabolites;

[0011] The QuECHERS process includes:

[0012] Add acidified acetonitrile to the above-mentioned giant freshwater prawn sample and shake.

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

[0014] The upper layer solution is added to the extraction column, vortexed, centrifuged, and the supernatant is collected, dried under nitrogen, and redissolved in acetonitrile to obtain the extract.

[0015] This invention incorporates acidified acetonitrile as an extraction solvent to extract nitrofuran metabolites from samples of giant freshwater prawns; utilizes sodium chloride and volatile ammonium acetate as dehydrating agents to improve recovery rates; and employs an extraction column to reduce matrix effects.

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

[0017] Preferably, the extraction column is a dSPE EMR Lipid dispersion solid-state extraction column.

[0018] Preferably, the giant freshwater prawn sample is prawn meat or prawn shell;

[0019] When the sample of giant freshwater prawn is shrimp meat, the mass-to-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 sample of giant freshwater prawn is a prawn shell, the mass-to-volume ratio of the prawn meat to the acidified acetonitrile is 1 mg:10 mL, and the mass ratio of the prawn shell, the sodium chloride, and the ammonium acetate is 2:1:4.

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

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

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

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

[0025] Preferably, the nitrofuran metabolite includes aminourea.

[0026] The present invention also provides an application of the above-mentioned method in dynamically monitoring changes in aminourea content in giant freshwater prawns under nitrofuran stress.

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

[0028] This invention utilizes the QuECHERS technique for efficient extraction of SEM from Macrobrachium rosenbergii samples, followed by hydrophilic chromatography detection and analysis to achieve dynamic analysis of SEM content changes in Macrobrachium rosenbergii under nitrofuran bath stress.

[0029] The QuECHERS method provided by this invention is simple to operate, achieves sufficient SEM extraction of samples in a short time, uses less reagent, avoids potential waste and pollution, and the reagents introduced, except for acetonitrile, have no strong toxicity, meeting the requirements of experimental safety.

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

[0031] The QuECHERS pretreatment method combined with normal phase chromatography-tandem mass spectrometry provided by this invention has high sensitivity, high accuracy, and wide applicability. It can be used to determine the residual amount of nitrofuran metabolites in giant freshwater prawns, and provides possible methodological support for the future detection of aminourea and related analyses such as endogenous aminourea. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0034] Figure 2 This is a schematic diagram of the QuECHERS preprocessing process of the present invention;

[0035] Figure 3 The following are mass spectrometry results for aminourea; where A represents the structural formulas of aminourea and its internal standard; B represents the mass spectrometry result for aminourea; and C represents the mass spectrometry result for the internal standard of aminourea.

[0036] Figure 4 The standard curve for aminourea in Macrobrachium rosenbergii samples;

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

[0038] Figure 6 This is a statistical graph showing the changes in aminourea content in giant freshwater prawns under nitrofuran stress; where A represents the aminourea content in the experimental group of giant freshwater prawns, and B represents the aminourea content in the control group of giant freshwater prawns; SEM image shows aminourea. Detailed Implementation

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

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0044] like Figure 1 As shown, this invention first provides the most suitable SEM extraction method, and then optimizes the liquid chromatography-tandem mass spectrometry method to analyze the SEM content in giant freshwater prawns, as detailed below:

[0045] Example 1

[0046] 1. Experimental Methods

[0047] 1.1 Processing of shrimp meat samples

[0048] See sample processing diagram Figure 2 Specifically, the shrimp meat was sampled from the abdominal muscle, and the shrimp shell was sampled from the abdominal exoskeleton. Both were then processed using a food processor.

[0049] For the shrimp meat, accurately weigh 3g of shrimp meat into 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, then add 4g of ammonium acetate and 1g of sodium chloride, vortex briefly, and centrifuge. After collecting the supernatant, add 2g of dSPE EMR Lipid dispersion 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 into 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, then add 4g of ammonium acetate and 1g of sodium chloride, vortex briefly, and centrifuge. After collecting the supernatant, add 2g of dSPE EMR Lipid dispersion solid extraction column, vortex for 30s, and centrifuge at low speed (1000rpm, 1min) to collect the supernatant.

[0051] 1.5UPLC-MS Analysis

[0052] The collected shrimp meat and shells were dried by nitrogen blowing, then reconstituted in 1 mL of acetonitrile and directly analyzed by UPLC-MS.

[0053] Unless otherwise specified, the techniques used in this embodiment, including nucleic acid extraction, are conventional methods well known to those skilled in the art. The mass spectrometer used in the UPLC MS method was a Waters Xevo TQ S micro mass spectrometer (Milford, USA), employing an electrospray ionization source. The liquid chromatography system was a Waters Acquity UPLC (Milford, USA). WatersAcquity was used. Compounds were separated using a BEH HILIC column, with the column temperature set to 40°C.

[0054] Mobile phases A and B are 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 for 0-3 min, decreasing to 80% B for 3-4.5 min, 80-60% B for 4.5-5.5 min, maintaining 60% B for 5.5-8 min, 60-100% B for 8-10 min, and 100% B for 10-15 min. The flow rate was set to 0.5 mL / min. Quantification was performed using the ion with the highest ion abundance under positive ion conditions (SEM, 76.1 > 30.8). 13 C- 15 N2-SEM, 79 > 33.0, cone voltage and collision voltage were 12V. Qualitative identification was performed using characteristic ion fragments (SEM, 76.1 > 58.8;). 13 C- 15 N2-SEM, 79>61.8, cone voltage and collision voltage are 12V). The mass spectrometry behavior of the SEM is shown in [reference needed]. Figure 3The capillary voltage was 0.8 kV, the ion source temperature was 300 °C, the desolvation gas flow rate was 1000 L / Hr (N2), the cone gas flow rate was 20 L / Hr (N2), the autosampler temperature was set to 4 °C, and the injection volume was 1 μL. The mass spectrometry behavior of the aminourea internal standard is shown below. Figure 3 As shown.

[0056] 1.6 Preparation of Standard Solutions

[0057] Accurately pipette 1 mg / mL of the SEM internal standard stock solution and serially dilute with acetonitrile to 10 μg / mL to prepare the working solution. Weigh 10 mg of SEM standard and dissolve it in 100 mL of acetonitrile to prepare the stock solution, then serially dilute with acetonitrile to 1 μg / mL to prepare the working solution. Store the stock solution at -20°C and prepare the working solution fresh each time.

[0058] 1.7 Establishment of Standard Curve and Determination of Sensitivity

[0059] Prepare mixed standard working solutions of SEM and 13C-15N2-SEM at 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 The mass concentration of N2-SEM was 1 μg / mL. A standard curve was plotted with the concentration ratio on the x-axis and the response ratio on the y-axis. Sensitivity was expressed by the limit of detection (LOD) and the limit of quantitation (LOQ). The LOD was defined as the concentration at which the signal / baseline ratio was 3.3, and the LOQ was defined as the concentration at which the signal / baseline ratio was 10.

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

[0061] C is defined as the SEM concentration obtained by replacing the giant freshwater prawn sample with pure solvent. 注射 The SEM concentration detected in the unspecified giant freshwater prawn sample was C. 内源性 The SEM concentration detected in the giant freshwater prawn sample with added standard before pretreatment was C. 前加标 The SEM concentration of the extract obtained after pretreatment, after adding the standard, was C. 后加标 The formulas for calculating 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 using intraday and interday precision, using the SEM described in 1.2 and 13 C- 15 A mixed standard solution with N2-SEM concentration ratios of 0.01, 0.1, and 1 was prepared, with each sample having three replicates. The solutions were injected three times on the same day at different times and three times on different days of the same week. The coefficient of variation (R0) was examined. 2 ).

[0066] 2. Results

[0067] This invention investigated the extraction efficiency, matrix effect, and recovery rate of the proposed QuECHERS method, followed by the precision, linearity, and sensitivity of the UPLC-MS / MS method. Based on the proposed QuECHERS method, SEM standard solutions with concentrations of 10 ng / mL, 100 ng / mL, and 1000 ng / mL were prepared, defined as low, medium, and high concentrations, respectively. Detection was performed according to method 1.3 (n=5). The aminourea matrix in *Macrobrachium rosenbergii* is quite complex, and purifying the high concentration of hydrophilic matrix from the co-extraction without losing the analyte is a significant challenge. With extraction efficiencies ranging from 88.2% to 96.4%, the matrix caused a response loss of 7.9% to 29.3%, and the recovery rates were between 76% and 95% (Table 1). At low concentration levels, the extract was relatively complex and the recovery rate was low, but the overall recovery rate and method precision were appropriate. For precision, based on the proposed UPLC-MS / MS method, three SEM standard solutions with concentrations of 10 ng / mL, 100 ng / mL, and 1000 ng / mL were prepared, defined as low, medium, and high concentrations, respectively. These were added to the samples for detection (n=5). The results showed that the coefficient of variation for SEM precision at the three concentration levels was less than 10%, meeting the methodological requirements (Table 2). For the linearity assessment of aminourea, a standard curve was plotted using the concentration ratio of aminourea and its internal standard as the x-axis and the peak area ratio as the y-axis. Calibration was achieved within the range of 10–50000 μg / kg. The R-value of the standard curve was [not specified]. 2 It is 0.9994 ( Figure 4 The limits of detection and limits of quantitation based on a signal-to-noise ratio of 3.3 and 10 times were 0.6 μg / kg and 1.0 μg / kg, respectively (Table 3). The formula for calculating the content of aminourea in the sample is as follows:

[0068]

[0069] In the formula, X represents the content of aminourea in the sample, expressed in micrograms per kilogram (μg / kg).

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

[0071] V - Final 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 curves and sensitivity of the UPLC-MS / MS method

[0079]

[0080] Example 2

[0081] Changes in aminourea content in giant freshwater prawns under furazolidone stress.

[0082] 1. Method

[0083] 1.1 Obtaining Giant Freshwater Prawn Samples

[0084] The giant freshwater prawns were sourced from an aquaculture base in Guangzhou. This base strictly adheres to the "Technical Specifications for Pond Culture of Giant Freshwater Prawns" during the artificial breeding process, and no nitrofuran drugs are added during the growth period. Mature individuals with intact antennae and legs, who are active and lively, are selected. After purchase, the prawns are kept in oxygenated conditions and transported to the laboratory by truck for temporary rearing in indoor recirculating aquaculture tanks.

[0085] 1.2 Conditions for Giant Freshwater Prawn Farming

[0086] The shrimp were cultured at 20℃ with continuous oxygenation using an oxygen pump, achieving the maximum dissolved oxygen level (7.8 mg / L). Disinfected plastic partitions were used within the culture tanks to prevent fighting. During the culture period, feces and food residue were removed using a siphon method, and dead shrimp were promptly removed. Before the formal experiment began, both the water and feed were tested for nitrofurans and SEM. On average, giant freshwater prawns with an individual weight of 12.0 g ± 2.0 g and a body length of 6 cm ± 1 cm were selected and placed in the culture tanks, randomly grouped, at a stocking density of 100 prawns / m². 3 .

[0087] 1.3 Nitrofuran bath stress

[0088] Giant freshwater prawns that had been fasted for two days were divided into two groups of 15 each, designated as the control group and the experimental group. The control group was not given any nitrofurans, while the experimental group was treated by soaking in a 10 mg / L nitrofuran aqueous solution for 2 hours. After that, they were transferred to clean water for normal culture. Giant freshwater prawn samples were collected at 0, 4, 12, 36, 108, 324 and 972 hours, with 3 prawns collected each time.

[0089] 1.4 Sample processing of giant freshwater prawns

[0090] The collected giant freshwater prawns were subjected to flash freezing with liquid nitrogen to induce euthanasia. The samples were then transferred to a -80°C freezer for storage, and slightly thawed before sampling to facilitate separation of the prawn meat and shell. The samples were then processed according to Example 1.

[0091] 2. Results

[0092] Extraction and chromatographic detection results of SEM using the optimized QuECHERS method are shown in the figure. Figure 5 .

[0093] Changes in the content of nitrofurans in the meat and shell of giant freshwater prawns under nitrofuran stress are shown in the figure. Figure 6 The results showed that for shrimp meat, the concentration of SEM reached a maximum of 74.14 μg / kg after 36 hours of nitrofuran bath, and then gradually decreased. SEM at 10.65 μg / kg was still detectable in the shrimp meat after 972 hours, while in the control group (without nitrofuran bath), SEM at 9.04 μg / kg was detectable. This indicates that it takes approximately 40 days for the giant freshwater prawn (Macrobrachium rosenbergii) to completely metabolize SEM after nitrofuran bath stress. In both the control and experimental groups, the SEM content in the shrimp shell increased over time, reaching its highest value at 972 hours (21.54 μg / kg and 16.11 μg / kg, respectively). This result suggests that there may be a pathway for SEM in the giant freshwater prawn meat to transfer to the shell.

[0094] Furthermore, based on the changes in SEM content in the control group of giant freshwater prawn samples, it was concluded that there is a relatively obvious endogenous SEM generation in giant freshwater prawns, and there is a possibility of it transferring from the shrimp meat to the shrimp shell.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for detecting nitrofuran metabolites in giant freshwater prawns based on UPLC-MS / MS, characterized in that, Includes the following steps: Collect samples of giant freshwater prawns; The giant freshwater prawn sample was subjected to QuECHERS treatment to obtain an extract; The extract was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to detect the content of the nitrofuran metabolites; The QuECHERS process includes: Add acidified acetonitrile to the above-mentioned giant freshwater prawn sample and shake. Add sodium chloride and ammonium acetate, shake, centrifuge, and collect the supernatant solution; The upper layer solution is added to the extraction column, vortexed, centrifuged, and the supernatant is collected, dried under nitrogen, and redissolved in acetonitrile to obtain the extract. The extraction column is a dSPE EMR Lipid dispersion solid-state extraction column; The liquid chromatography described is a hydrophilic interaction liquid chromatography, using Waters Acquity UPLC. ® BEH HILIC column, column temperature 40℃; The mobile phase A of the liquid chromatography is a 2 mM ammonium acetate aqueous solution, and the mobile phase B is formic acid-acidified acetonitrile; the volume fraction of the formic acid is 0.01%. The elution program for the liquid chromatography is as follows: 100% B for 0-3 min, gradient reduction to 80% B for 3-4.5 min, 80-60% B for 4.5-5.5 min, 60% B for 5.5-8 min, 60-100% B for 8-10 min, and 100% B for 10-15 min. The flow rate was 0.5 mL / min; The mass spectrometry conditions were: electrospray ionization; positive ion scanning mode; multiple reaction monitoring; capillary voltage of 0.8 kV; and ion source temperature of 150 °C. The nitrofuran metabolite is aminourea.

2. The method as described in claim 1, characterized in that, The acidified acetonitrile is formic acid-acidified acetonitrile; the volume fraction of the formic acid is 0.01%.

3. The method as described in claim 1, characterized in that, The sample of the giant freshwater prawn was either prawn meat or prawn shell; When the giant freshwater prawn sample is shrimp meat, the mass-to-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 sample of giant freshwater prawn is a prawn shell, the mass-to-volume ratio of the prawn meat to the acidified acetonitrile is 1 mg:10 mL, and the mass ratio of the prawn shell, the sodium chloride, and the ammonium acetate is 2:1:

4.

4. The application of the method as described in any one of claims 1-3 in the dynamic monitoring of changes in aminourea content in giant freshwater prawns under nitrofuran stress.

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