Chitosan composite filler purification and detection method for nitrofuran metabolites in aquatic products

Abstract

The invention discloses a chitosan composite filler purification and detection method for nitrofuran metabolites in aquatic products, and belongs to the technical field of physical and chemical detection of drug residues. According to the detection method, after a sample is subjected to hydrochloric acid hydrolysis, 2-nitrobenzaldehyde derivation, ethyl acetate extraction, chitosan composite filler purification and nitrogen blowing concentration redissolution, rapid separation is achieved by means of ultra-high performance liquid chromatography, determination is conducted by means of a triple quadrupole / composite linear ion trap mass spectrometer, quantitative analysis is conducted by means of an internal standard method, and the quantitative analysis result is obtained. And the residual quantity of the nitrofuran metabolites in the aquatic product can be rapidly and accurately measured. The method disclosed by the invention is economical, simple, convenient, efficient, high in sensitivity and good in repeatability, and can be used as a conventional detection method for residues of four nitrofuran metabolites in aquatic products.

Description

Technical Field

[0001] The invention relates to the technical field of physical and chemical detection of drug residues, and in particular to a rapid purification method and detection method of a chitosan composite filler for nitrofuran metabolite residues in aquatic products. Background Art

[0002] As an antibiotic that can fight most bacteria, nitrofurans mainly include four compounds: furazolidin, furatadone, nitrofurantoin and furazolidone. Studies have shown that nitrofurans have obvious toxic side effects and may cause serious consequences such as carcinogenesis, teratogenesis and mutagenesis. Therefore, the problem of their residues has attracted widespread attention at home and abroad.

[0003] Four nitrofuran drugs, namely nitrofurazone, furatadone, nitrofurantoin and furazolidone, have the characteristics of rapid metabolism in aquatic matrices such as fish and shrimp meat. Their corresponding final metabolites are semicarbazide (SEM), 3-amino-2-oxazolidone (AOZ), 1-amino-2-hydantoin (AHD) and 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ). These products are easy to bind to proteins and the final product structure is stable. Therefore, the residual levels of the four nitrofuran compounds are often reflected by detecting their corresponding four metabolites. At present, the detection methods for the residues of nitrofuran metabolites include immunochromatography, enzyme-linked immunosorbent assay and high performance liquid chromatography. Compared with ultra-high performance liquid chromatography-tandem mass spectrometry, immunochromatography is affected by the stability and sensitivity of the antigen and is easily interfered by impurities; the enzyme-linked immunosorbent assay is easily interfered by temperature during operation; and high performance liquid chromatography has the problem of low sensitivity.

[0004] The components of aquatic product matrices are relatively complex, and impure proteins and lipid components are relatively rich. Therefore, it is necessary to select appropriate sample pretreatment methods to purify the final reconstituted liquid to ensure the accuracy and stability of the determination of nitrofuran metabolite residues, such as n-hexane degreasing method, solid phase extraction method, matrix dispersed solid phase extraction and Oasis PRiMEHLB purification method. However, the operation steps of the traditional solid phase extraction purification method are cumbersome and time-consuming. GB 31656.13-2021 "National Food Safety Standard for the Determination of Multiple Residues of Nitrofuran Metabolites in Aquatic Products by Liquid Chromatography-Tandem Mass Spectrometry" requires the use of 14000r / min ultra-high speed centrifugation for 10 minutes to purify the final reconstituted liquid. Chitosan is a natural polymer extracted from the shells of crustaceans. As a natural polymer of fishery by-products, it not only solves the problem of waste disposal, but also shows broad application prospects in the field of sample pretreatment due to its unique properties. Chitosan is rich in many functional groups such as hydroxyl and amino groups, and has an outstanding ability to remove interfering substances such as fat. After mixing this material with the aquatic product extract, it can selectively adsorb and remove interfering substances such as fat and phospholipids, thereby achieving the purpose of purifying the extract. Compared with traditional sample pretreatment methods such as solid phase extraction and 14000r / min ultra-high-speed centrifugation, the use of chitosan for purification can avoid a series of steps such as activation, leaching and elution. Only one centrifugation step is required to complete the purification of the extract, which has the advantages of efficient treatment methods and convenient operation. Summary of the invention

[0005] The purpose of the present invention is to provide a chitosan composite filler purification and detection method for nitrofuran metabolites in aquatic products. The present invention uses chitosan composite filler for rapid purification and combines it with an ultra-high performance liquid chromatography-triple quadrupole / composite linear ion trap mass spectrometer (UPLC-QTRAP-MS / MS) to establish an accurate determination method for the residues of four nitrofuran metabolites in aquatic products.

[0006] The present invention first provides a chitosan composite filler rapid sample pretreatment method for detecting nitrofuran metabolite residues in aquatic products, comprising the following steps:

[0007] (1) using 2-nitrobenzaldehyde solution as a derivatization reagent to carry out a derivatization reaction on the nitrofuran metabolites in the aquatic product sample to be tested, and then using an organic solvent to extract to obtain a sample to be purified;

[0008] (2) Mixing the sample to be purified and the chitosan composite filler, drying the obtained liquid after centrifugation to obtain a solid; and re-dissolving the obtained solid with a methanol solution to obtain a sample to be tested.

[0009] In the above-mentioned sample pretreatment method, in step (1), the concentration of the 2-nitrobenzaldehyde solution is 0.05 mol / L;

[0010] The mass volume ratio of the aquatic product to be tested and the 2-nitrobenzaldehyde solution is 40:3 g / mL;

[0011] The organic solvent is ethyl acetate.

[0012] In the above-mentioned sample pretreatment method, the method in step (1) comprises the following steps: mixing the aquatic product sample to be tested, the mixed internal standard working solution, the hydrochloric acid solution and the 2-nitrobenzaldehyde solution, and performing a derivatization reaction; after the derivatization reaction is completed, adding a potassium hydrogen phosphate solution to adjust the pH to 7.0-7.5, and then adding an organic solvent for extraction, and the obtained supernatant is the sample to be purified.

[0013] In the above-mentioned sample pretreatment method, the mass volume ratio of the aquatic product sample to be detected and the mixed internal standard working solution is 40:1 g / mL;

[0014] The mixed internal standard working solution is SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 and AMOZ-D 5 Specifically, in the mixed internal standard working solution, SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 and AMOZ-D 5 The concentrations were all 100 μg / L;

[0015] The mass volume ratio of the aquatic product sample to be tested and the hydrochloric acid solution is 2:5 g / mL;

[0016] The concentration of the hydrochloric acid solution is 0.2 mol / L;

[0017] The concentration of the dipotassium hydrogen phosphate solution is 1.0 mol / L;

[0018] The mass volume ratio of the aquatic product sample to be tested and the ethyl acetate is 1:4 g / mL;

[0019] In the above sample pretreatment method, the temperature of the derivatization reaction is 37°C and the time is 16h;

[0020] The derivatization reaction is carried out under ultrasonic conditions in the dark or under shaking conditions in the dark.

[0021] In the above-mentioned sample pretreatment method, in step (2), the chitosan composite filler is a mixture of anhydrous sodium sulfate and chitosan;

[0022] The mass ratio of anhydrous sodium sulfate in the chitosan composite filler to the aquatic product sample to be tested is 0.5-0.75:1, preferably 0.6:1;

[0023] The mass ratio of chitosan in the chitosan composite filler to the aquatic product sample to be tested is 0.03-0.05:1, preferably 0.04:1;

[0024] The mass volume ratio of the aquatic product sample to be tested and the methanol solution is 2:1 g / mL;

[0025] The volume percentage concentration of the methanol solution is 5%.

[0026] In the above-mentioned sample pretreatment method, the aquatic product sample to be detected is edible tissue of fish, shrimp or crab.

[0027] The present invention also provides a method for detecting residual nitrofuran metabolites in aquatic products, comprising the following steps: treating the aquatic product sample to be detected by the above-mentioned sample pretreatment method to obtain the sample to be detected; and then detecting it by liquid chromatography-tandem mass spectrometry.

[0028] In the above detection method, in the detection of the liquid chromatography tandem mass spectrometer, the liquid chromatography detection conditions are as follows: the liquid chromatography column is a Hypersil GOLD column produced by Waters, 50mm×2.1mm, 1.7μm; the injection volume is 10.0μL; the column oven setting temperature is 40°C; the mobile phase is composed of phase A and phase B, wherein phase A is 2mmol / L ammonium acetate aqueous solution, and phase B is methanol; gradient elution, the elution program is as follows: 0-6min, 10%-90% phase B; 6-9min, 90% phase B; 9-9.1min, 90%-10% phase B; 9.1-11min, 10% phase B;

[0029] The mass spectrometry detection conditions were as follows: the analytes were ionized in the positive ion mode using an electrospray ion source, and the detection mode was the multiple reaction monitoring mode; the ion source setting parameters were: ion source temperature: 650.0°C; ionization voltage: 5.5 kV; curtain gas: 30.0 psi; nebulizer gas: 60.0 psi; auxiliary gas: 55.0 psi;

[0030] The mass spectrometry parameters of the four nitrofuran metabolites in multiple reaction monitoring mode are as follows:

[0031] The parent ions of SEM, AOZ, AHD, and AMOZ were 209.2, 236.1, 249.2, and 335.2, respectively; the quantitative ions were 166.2, 133.9, 134.1, and 291.1, respectively; the collision energies eV were 14.0, 17.0, 17.0, and 17.0, respectively; the qualitative ions were 192.1, 103.9, 104.1, and 262.2, respectively; the collision energies eV were 16.0, 31.0, 27.0, and 23.0, respectively; the isotopic internal standards SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 、AMOZ-D 5 The parent ions were 212.1, 240.1, 252.0, and 340.4, respectively; the product ions were 168.0, 134.0, 134.0, and 296.2, respectively; and the collision energies eV were 14.0, 17.0, 17.0, and 17.0, respectively.

[0032] The above detection method also includes the following steps:

[0033] (1) Drawing a standard curve: mixing standard substances, semicarbazide, 3-amino-2-oxazolidinyl ketone, 1-amino-2-hydantoin and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, and then treating them by the above-mentioned pretreatment method, and detecting them by the liquid chromatography tandem mass spectrometer in the above-mentioned detection method of the claim, with the concentration value as the horizontal axis and the area ratio of the target compound quantitative ion chromatogram peak and the corresponding isotope internal standard peak measured on the mass spectrum as the vertical axis, making a linear equation and obtaining the correlation coefficient;

[0034] (2) Substituting the quantitative ion chromatographic peak of the target compound in the aquatic product sample to be detected and the corresponding isotope internal standard peak area ratio obtained by the above detection method into the standard curve to obtain the concentration of the target compound in the aquatic product sample.

[0035] The present invention has the following beneficial effects:

[0036] (1) The method of the present invention does not require traditional high-speed centrifugation purification, and because chitosan is used to purify the ethyl acetate extract, the peak response intensity of the target object becomes higher; the residual amounts of four nitrofuran metabolites in aquatic products can be quickly and accurately determined simultaneously; this method can fully meet the detection requirements of the national food safety standard GB 31656.13-2021 for the four nitrofuran metabolites of AOZ, AMOZ, AHD and SEM;

[0037] (2) The method of the present invention is economical, simple, efficient, highly sensitive, and has good repeatability, and can be used as a routine detection method for the residues of four nitrofuran metabolites in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The following are the derivatization reaction equations for four nitrofuran metabolites.

[0039] Figure 2 This is a comparison chart of carp spiked samples treated with different masses of chitosan (10μg / kg).

[0040] Figure 3 This is a comparison chart of carp spiked samples treated with different masses of anhydrous sodium sulfate (10μg / kg).

[0041] Figure 4 The chromatograms of the purified solution after two different purification methods (10μg / kg); Figure 4 (a) is the purification method according to GB 31656.13-2021; (b) is the chitosan purification method. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0044] The quantitative tests in the following examples were performed three times unless otherwise specified, and the results were averaged.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] Materials and reagents used in the following examples:

[0047] Chitosan (CAS: 9012-76-4, Aladdin Biochemical Technology Co., Ltd.);

[0048] Methanol solution of mixed standards of four nitrofuran metabolites: SEM, AOZ, AHD, and AMOZ (100 μg / mL, Tianjin Alta Technology Co., Ltd., batch number: S155597);

[0049] SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 and AMOZ-D 5Methanol solution of 4 nitrofuran metabolite isotope internal standard mixed standards (100 μg / mL, Tianjin Alta Technology Co., Ltd., batch number: S178961);

[0050] 2-nitrobenzaldehyde (CNW Technologies, Inc.);

[0051] Ethyl acetate and methanol (chromatographic grade, JTBaker, USA);

[0052] Hydrochloric acid (analytical grade, Shanghai Xinyu Biotechnology Co., Ltd.);

[0053] Dimethyl sulfoxide, ammonium acetate (chromatographic grade);

[0054] Potassium hydrogen phosphate (analytical grade, Aladdin Biochemical Technology Co., Ltd.);

[0055] Distilled water (Guangzhou Watsons Food and Beverage Co., Ltd.).

[0056] The following instruments and equipment are used in the following examples:

[0057] QTRAP 5500HPLC-QTRAP-MS / MS (AB SCIEX, USA);

[0058] H-2050R desktop high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.);

[0059] SB-5200DTN ultrasonic water bath machine (Ningbo Xinzhi Biotechnology Co., Ltd.);

[0060] MVM-2500 multi-tube vortex mixer (Shanghai Titan Technology Co., Ltd.);

[0061] MS 3Basic vortex mixer (IKA, Germany);

[0062] ZHWY-110X 30 water bath constant temperature oscillator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.);

[0063] N-EVAPTM112 nitrogen blowdown apparatus (Organomation Aossociates, USA).

[0064] Example 1

[0065] 1. Sample preparation

[0066] The carp used for method establishment and validation was purchased from the Beijing Aquatic Product Market and transferred to the laboratory via refrigerated transportation. The fish skin and bones were removed, and the edible parts were retained. The fish were crushed into a fish paste by a homogenizer and stored frozen at -20°C for later use.

[0067] 2. Sample pretreatment

[0068] (1) Hydrolysis and derivatization

[0069] Take a 50mL centrifuge tube, accurately weigh and add 2.0g of surimi sample, add 0.05mL of mixed internal standard working solution (containing SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 and AMOZ-D 5 , concentrations of 100 μg / L; solvent is methanol) and 0.05 mL of mixed standard working solution (containing SEM, AOZ, AHD and AMOZ, concentrations of 400 μg / L, solvent is methanol), vortex mix evenly. Then add 5.0 mL of hydrochloric acid (0.2 mol / L) and 0.15 mL of 2-nitrobenzaldehyde solution (0.05 mol / L) respectively, vortex mix until evenly dispersed. Finally, place the centrifuge tube in a constant temperature water bath oscillator (water temperature 37 ° C) and oscillate in the dark for 16 hours (see the derivatization reaction equation for the derivatization reaction). Figure 1 ).

[0070] (2) Extraction and purification process

[0071] The centrifuge tube after the derivatization reaction was taken out and cooled to room temperature, 15 mL of 1.0 mol / L potassium dihydrogen phosphate was added to adjust the pH to 7.0-7.5, then 8 mL of ethyl acetate was added and vortexed for 1 min, centrifuged at 6000 rpm for 5 min, the supernatant was added to a 15 mL centrifuge tube, and chitosan composite filler (a mixture of 1.2 g of anhydrous sodium sulfate and 80 mg of chitosan) was added and vortexed for 1 min, then centrifuged at 8000 rpm for 10 min and all the liquid was transferred to a 10 mL centrifuge tube, blown dry with nitrogen at 40° C., 1 mL of 5% (V / V) methanol aqueous solution was added for redissolution, and tested on a machine through a 0.22 μm filter membrane.

[0072] 3. The samples were tested using a liquid chromatography tandem mass spectrometer (QTRAP 5500HPLC-QTRAP-MS / MS, AB SCIEX, USA). The liquid chromatography conditions were as follows:

[0073] The liquid chromatography column is a Hypersil GOLD column (50 mm × 2.1 mm, 1.7 μm) produced by Waters; injection volume: 10.0 μL; column oven set temperature: 40 °C; the mobile phase consists of phase A and phase B, wherein phase A: ammonium acetate aqueous solution (2 mmol / L), phase B: methanol; gradient elution, the elution program is detailed in Table 1 below.

[0074] Table 1 Gradient elution program

[0075] Time / min <![CDATA[Flow rate / (mL·min -1 )]]> A / ％ B / ％ 0 0.35 90.0 10.0 6.00 0.35 10.0 90.0 9.00 0.35 10.0 90.0 9.10 0.35 90.0 10.0 11.00 0.35 90.0 10.0

[0076] The mass spectrometry conditions for detection are as follows:

[0077] The analytes were ionized in positive ion mode using an electrospray ion source, and the detection mode was multiple reaction monitoring mode. Specific setting parameters of the ion source were: ion source temperature: 650.0°C; ionization voltage: 5.5 kV; curtain gas: 30.0 psi; nebulizer gas: 60.0 psi; auxiliary gas: 55.0 psi. The mass spectrometry parameters of the four nitrofuran metabolites in multiple reaction monitoring mode are shown in Table 2.

[0078] Table 2 Mass spectrometry parameters for four nitrofuran metabolites and their internal standards in multiple reaction monitoring mode

[0079] Analytes Retention time / min Quasi-molecular ions Fragment ions Collision gas energy / V SEM 2.6 209.2 <![CDATA[166.2 * ,192.1]]> 14,16 AOZ 2.5 236.1 <![CDATA[133.9 * ,103.9]]> 17,31 AHD 2.3 249.2 <![CDATA[134.1 * ,104.1]]> 17,27 AMOZ 3.3 335.2 <![CDATA[291.1 * ,262.2]]> 17,23 <![CDATA[SEM- 13 C- 15 N 2 ]]> - 212.1 168.0 14 <![CDATA[AOZ-D 4 ]]> - 240.1 134.0 17 <![CDATA[AHD- 13 C 3 ]]> - 252.0 134.0 17 <![CDATA[AMOZ-D 5 ]]> - 340.4 296.2 17

[0080] Note: *Quantitative ion.

[0081] 4. Selection of purification method

[0082] After a series of pre-treatments such as hydrolysis, derivatization, extraction, and concentration, the reconstituted solution of nitrofuran metabolites still contains interfering substances, which affect the quantitative detection results. In this study, carp samples were used as experimental objects, and chitosan was used to remove impurities and purify the ethyl acetate extract. Carp spiked with a concentration of 10 μg / kg was used as the experimental object, and chitosan was used to remove impurities and purify the ethyl acetate extract.

[0083] (1) Effect of chitosan dosage

[0084] The specific sample pretreatment and detection are as follows: 2.0 g of blank carp surimi sample was weighed into a 50 mL centrifuge tube, and then the sample was pretreated using the above methods (1) hydrolysis and derivatization and (2) extraction and purification, except that 1 g of anhydrous sodium sulfate was used in combination with different masses of chitosan (0, 10, 20, 50, 60, 80, 100 mg) for purification. Then, liquid chromatography-tandem mass spectrometry was used to detect the sample, and the peak area of ​​the quantitative ion measured on the mass spectrum was obtained. The results are shown in FIG. Figure 2 As shown. Figure 2It can be seen that the response intensity of the target compound increases with the increase in the amount of chitosan used. The peak response intensity of the target compound is the highest when 80 mg of chitosan is used to purify the ethyl acetate extract. When the amount of chitosan is less than 80 mg, the instrument signal of nitrofuran metabolites may be suppressed by the matrix effect, resulting in low instrument response; when the amount of chitosan is higher than 80 mg, the effect of nitrofuran metabolites being adsorbed by chitosan is significant, resulting in a decrease in the instrument response of nitrofuran metabolites. Therefore, this experiment uses 80 mg of chitosan to remove impurities and purify the ethyl acetate extract.

[0085] (2) Effect of the dosage of anhydrous sodium sulfate

[0086] The specific sample pretreatment and detection are as follows: 2.0 g of blank carp surimi sample was weighed into a 50 mL centrifuge tube, and then the sample was pretreated using the above methods (1) hydrolysis and derivatization and (2) extraction and purification, except that 80 mg of chitosan was combined with different masses of anhydrous sodium sulfate (200, 500, 700, 1000, 1200, 1500 mg) for purification. Then, liquid chromatography-tandem mass spectrometry was used to detect it, and the peak area of ​​the quantitative ion measured on the mass spectrum was obtained. The results are shown in FIG. Figure 3 As shown. Figure 3 The results showed that the chromatographic peaks of the four target substances had the highest intensities when 80 mg chitosan and 1200 mg anhydrous sodium sulfate were used to purify the reconstituted solution. Therefore, 80 mg chitosan and 1200 mg anhydrous sodium sulfate were finally selected as the best purification filler combination in this study.

[0087] Example 2: Comparative study on purification effect with GB 31656.13-2021

[0088] In GB 31656.13-2021, the reconstituted solution is purified by high-speed centrifugation at 14000r / min for 10min. The specific method is as follows:

[0089] The hydrolysis and derivatization treatment was completed by (1) in the sample pretreatment of Example 1, and then the centrifuge tube was taken out after the reaction was completed and cooled to room temperature. 15 mL of 1.0 mol / L potassium dihydrogen phosphate solution was used to adjust the pH to 7.0-7.5, and then 8 mL of ethyl acetate was added and vortexed for 1 min, centrifuged at 6000 rpm for 5 min, and the supernatant was taken to a 10 mL centrifuge tube and blown to dryness with nitrogen at 40° C. 1 mL of 5% volume fraction methanol aqueous solution was added, and then the liquid was transferred to a 5 mL centrifuge tube, centrifuged at 14000 rpm for 10 min, and passed through a 0.22 μm filter membrane for detection.

[0090] The method of the present invention adopts 2. Sample pretreatment in Example 1 to pretreat the sample.

[0091] The detection conditions were the same as those of the liquid chromatography in Example 1.

[0092] In this study, carp samples were used as experimental objects, and the chromatograms of the purified liquid after being treated by the two different purification methods were compared (see Figure 4 ). The results show that the one-step purification of chitosan composite filler (i.e., the method of the present invention) significantly improves the intensity of the chromatographic peaks of the four targets; when compared with the experimental process of the national standard method, the one-step purification method using chitosan is simpler in experimental steps, and the additional high-speed centrifugation to separate impurities before the final machine detection is omitted, and the overall experimental process is safer and more convenient; in addition, the addition of chitosan can adsorb and remove lipid impurities in the ethyl acetate extract, making the peak response value of the target stronger. After comprehensive consideration, the chitosan composite filler was selected to purify the sample.

[0093] Example 3: Methodological Verification

[0094] 1. Preparation of standard solution

[0095] Take appropriate amount of methanol solution of four nitrofuran metabolite mixed standard substances (100 μg / mL, Tianjin Alta Technology Co., Ltd.) and methanol solution of four nitrofuran metabolite isotope internal standard mixed standard substances (100 μg / mL, Tianjin Alta Technology Co., Ltd.) and dilute them with methanol to prepare a series of mixed standard intermediate solutions (SEM, AOZ, AHD and AMOZ concentrations were 100.0, 10.0, and 1.0 μg / mL, respectively) and mixed internal standard intermediate solutions (SEM- 13 C- 15 N 2 、AOZ-D 4 、AHD- 13 C 3 and AMOZ-D 5 The concentrations were all 100.0, 10.0, and 1.0 μg / mL), and stored at -20°C in a dark place for future use. Immediately before use, the mixed standard working solutions (100.0, 10.0 ng / mL) and the mixed internal standard working solutions (50.0 ng / mL) were prepared by diluting them step by step with water.

[0096] 2. Preparation of standard curve

[0097] Four nitrofuran metabolite mixed standard working solutions were diluted with methanol to obtain 10.0 ng / mL and 100.0 ng / mL mixed standard working solutions, respectively, to prepare a series of standard solutions with concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 ng / mL. Except for not adding actual samples and not performing purification treatment, the remaining experimental steps were performed according to 2. Sample pretreatment in Example 1, and the conditions of 3. Liquid chromatography tandem mass spectrometer were used for detection, and the standard curve was prepared by the internal standard method. The concentration value was used as the horizontal coordinate, and the area ratio of the target compound quantitative ion chromatographic peak measured on the mass spectrometer and the corresponding isotope internal standard peak was used as the vertical coordinate to prepare a linear equation and obtain the correlation coefficient.

[0098] 3. Linear equation and limit of quantitation

[0099] The linear regression equations, correlation coefficients and quantification limits of the four target compounds are shown in Table 3. The four nitrofuran metabolites showed good linear relationships in the concentration range of 0.5-10.0 μg / L, and the correlation coefficients (r) were all greater than 0.998. The spiked concentration at ten times the signal-to-noise ratio was selected as the quantification limit, i.e., 0.5 μg / kg, indicating that this method has high sensitivity.

[0100] Table 3 Linear regression equations and quantification limits of four nitrofuran metabolites

[0101] Compound Linear range / (μg / L) Regression equation Correlation coefficient (r) Limit of quantification / (μg / kg) SEM 0.5～10.0 Y=0.29683X+0.01936 0.99964 0.5 AOZ 0.5～10.0 Y=0.23747X+0.00523 0.99875 0.5 AHD 0.5～10.0 Y=0.32279X-0.00140 0.99977 0.5 AMOZ 0.5～10.0 Y=0.38553X+0.01646 0.99937 0.5

[0102] 4. Accuracy, correctness and precision

[0103] Carp, turbot and white shrimp were selected as blank samples respectively, and the machine detection liquid was obtained according to the sample pretreatment method in Example 1 2. The unspiked matrix was selected as the blank sample. According to the requirements of GB 2704-2008, spiked samples (0.5, 2.0, 10.0 μg / kg) were prepared corresponding to three different concentration levels of one, four and twenty times the quantitative limit for experiment, so as to ensure that the different changes in the concentration of the sample within a certain degree during the detection can be correctly determined by the designed method, and the authenticity and reliability of the experimental results under different concentration ranges are ensured. The relative standard deviation (RSDs) was selected as the measure of the repeatability of the method. Six parallel samples were used for each spiked concentration, and the recovery rate and relative standard deviation were calculated. The results are shown in Table 4. At the three added concentration levels, the average recovery rate of the four nitrofuran metabolites was 96.5% to 116.5%, the intra-day RSDs were 2.2% to 9.3%, and the inter-day RSDs were 2.7% to 9.7%. The results showed that the detection method established in this study has high accuracy and good repeatability, and can meet the detection needs of four nitrofuran metabolites in common aquatic products.

[0104] Table 4 Recoveries and RSDs of four nitrofuran metabolites in aquatic products (n=6)

[0105]

[0106] As can be seen from the above, the present invention uses chitosan composite filler for rapid purification, combined with UPLC-QTRAP-MS / MS, to establish a method for accurately determining the residues of 4 nitrofuran metabolites in aquatic products. The experimental results show that this method does not require traditional high-speed centrifugation purification, and because chitosan is used to purify the ethyl acetate extract, the peak response intensity of the target object is also higher. The residues of 4 nitrofuran metabolites in aquatic products can be quickly and accurately determined at the same time. This method can fully meet the national food safety standard GB 31656.13-2021 for the detection requirements of AOZ, AMOZ, AHD and SEM 4 nitrofuran metabolites.

Claims

1. A sample pretreatment method for detecting nitrofuran metabolite residues in aquatic products, comprising the following steps: (1) using 2-nitrobenzaldehyde solution as a derivatization reagent to carry out a derivatization reaction on the nitrofuran metabolites in the aquatic product sample to be tested, and then using an organic solvent to extract to obtain a sample to be purified; (2) Mixing the sample to be purified and the chitosan composite filler, drying the obtained liquid after centrifugation to obtain a solid; and re-dissolving the obtained solid with a methanol solution to obtain a sample to be tested.

2. The sample pretreatment method according to claim 1, characterized in that: In step (1), the concentration of the 2-nitrobenzaldehyde solution is 0.05 mol / L; The mass volume ratio of the aquatic product to be tested and the 2-nitrobenzaldehyde solution is 40:3 g / mL; The organic solvent is ethyl acetate.

3. The sample pretreatment method according to claim 1 or 2, characterized in that: The method in step (1) comprises the following steps: mixing the aquatic product sample to be tested, the mixed internal standard working solution, the hydrochloric acid solution and the 2-nitrobenzaldehyde solution, and performing a derivatization reaction; after the derivatization reaction is completed, adding a potassium hydrogen phosphate solution to adjust the pH to 7.0-7.5, and then adding an organic solvent for extraction, and the obtained supernatant is the sample to be purified.

4. The sample pretreatment method according to claim 3, characterized in that: The mass volume ratio of the aquatic product sample to be tested and the mixed internal standard working solution is 40:1 g / mL; The mixed internal standard working solution is SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 C3 and AMOZ-D5 mixed solution; Specifically, in the mixed internal standard working solution, SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 The concentrations of C3 and AMOZ-D5 were both 100 μg / L; The mass volume ratio of the aquatic product sample to be tested and the hydrochloric acid solution is 2:5 g / mL; The concentration of the hydrochloric acid solution is 0.2 mol / L; The concentration of the dipotassium hydrogen phosphate solution is 1.0 mol / L; The mass volume ratio of the aquatic product sample to be tested and the ethyl acetate is 1:4 g / mL.

5. The sample pretreatment method according to any one of claims 1 to 4, characterized in that: The derivatization reaction temperature is 37°C and the reaction time is 16h; The derivatization reaction is carried out under ultrasonic conditions in the dark or under shaking conditions in the dark.

6. The sample pretreatment method according to any one of claims 1 to 5, characterized in that: In step (2), the chitosan composite filler is a mixture of anhydrous sodium sulfate and chitosan; The mass ratio of anhydrous sodium sulfate in the chitosan composite filler to the aquatic product sample to be tested is 0.5-0.75:1, preferably 0.6:1; The mass ratio of chitosan in the chitosan composite filler to the aquatic product sample to be tested is 0.03-0.05:1, preferably 0.04:1; The mass volume ratio of the aquatic product sample to be tested and the methanol solution is 2:1 g / mL; The volume percentage concentration of the methanol solution is 5%.

7. The sample pretreatment method according to any one of claims 1 to 6, characterized in that: The aquatic product sample to be tested is edible tissue of fish, shrimp or crab.

8. A method for detecting residual nitrofuran metabolites in aquatic products, comprising the following steps: treating aquatic product samples to be detected by the sample pretreatment method described in any one of claims 1 to 7 to obtain the samples to be detected; and then detecting the samples by liquid chromatography tandem mass spectrometry.

9. The detection method according to claim 8, characterized in that: In the detection of the liquid chromatography tandem mass spectrometer, the liquid chromatography detection conditions are as follows: the liquid chromatography column is a Hypersil GOLD column produced by Waters, 50mm×2.1mm, 1.7μm; the injection volume is 10.0μL; the column oven setting temperature is 40°C; the mobile phase is composed of two phases, A phase and B phase, wherein A phase: 2mmol / L ammonium acetate aqueous solution, B phase: methanol; gradient elution, the elution program is as follows: 0-6min, 10%-90% B phase; 6-9min, 90% B phase; 9-9.1min, 90%-10% B phase; 9.1-11min, 10% B phase; The mass spectrometry detection conditions were as follows: the analytes were ionized in the positive ion mode using an electrospray ion source, and the detection mode was the multiple reaction monitoring mode; the ion source setting parameters were: ion source temperature: 650.0°C; ionization voltage: 5.5 kV; Air curtain gas: 30.0psi; Atomizing gas: 60.0psi; Auxiliary gas: 55.0psi; The mass spectrometry parameters of the four nitrofuran metabolites in multiple reaction monitoring mode are as follows: The parent ions of SEM, AOZ, AHD, and AMOZ were 209.2, 236.1, 249.2, and 335.2, respectively; the quantitative ions were 166.2, 133.9, 134.1, and 291.1, respectively; the collision energies eV were 14.0, 17.0, 17.0, and 17.0, respectively; the qualitative ions were 192.1, 103.9, 104.1, and 262.2, respectively; the collision energies eV were 16.0, 31.0, 27.0, and 23.0, respectively; the isotopic internal standards SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 The parent ions of C3 and AMOZ-D5 were 212.1, 240.1, 252.0, and 340.4, respectively; the product ions were 168.0, 134.0, 134.0, and 296.2, respectively; and the collision energies eV were 14.0, 17.0, 17.0, and 17.0, respectively.

10. The detection method according to claim 8 or 9, characterized in that: The method further comprises the steps of: (1) Drawing a standard curve: mixing standard substances, semicarbazide, 3-amino-2-oxazolidinyl ketone, 1-amino-2-hydantoin and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, and then treating them by the pretreatment method described in any one of claims 1 to 7, and detecting them by the liquid chromatography tandem mass spectrometer in the detection method described in claim 8 or 9, with the concentration value as the horizontal coordinate and the area ratio of the target compound quantitative ion chromatogram peak and the corresponding isotope internal standard peak measured on the mass spectrum as the vertical coordinate, making a linear equation and obtaining a correlation coefficient; (2) Substitute the quantitative ion chromatographic peak of the target compound in the aquatic product sample to be detected and the corresponding isotope internal standard peak area ratio obtained by the detection method described in claim 8 or 9 into the standard curve to obtain the concentration of the target compound in the aquatic product sample.

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