A method for detecting four nitrofuran metabolites in aquatic products

Abstract

The present invention discloses a detection method for 4 kinds of nitrofuran metabolites in aquatic products, and belongs to the technical field of physical and chemical detection of drug residues. The detection method provided by the present invention comprises the following steps: (1) using 2-pyrrole carboxaldehyde solution as a derivatization reagent to carry out a derivatization reaction on 4 kinds of nitrofuran metabolites in aquatic product samples, and then extracting with an organic solvent to obtain a sample to be purified; (2) purifying the sample to be purified obtained in step (1), and detecting it with a liquid chromatography tandem mass spectrometer after purification. The method of the present invention uses 2-pyrrole carboxaldehyde for derivatization reaction, ethyl acetate extraction, push rod filter type purification column purification, and UPLC-MS / MS analysis and determination of drug residues in aquatic products after purification, the method has less interference, simple and fast pre-treatment, and the recovery rate, precision and sensitivity indexes all meet the requirements of drug residue analysis.

Description

Technical Field

[0001] The invention relates to the technical field of physical and chemical detection of drug residues, and specifically to a method for detecting four nitrofuran metabolites in aquatic products. Background Art

[0002] my country is a major fishing country, and the total output of aquatic products has ranked first in the world for many years, which greatly meets people's demand for aquatic product consumption. However, high-density and large-scale aquaculture has also brought some negative effects, such as water environment pollution and excessive residues of various drugs in aquatic products. Drugs in aquatic products will accumulate in organisms through the food chain and eventually pass to the human body, posing potential hazards to human consumption safety.

[0003] Nitrofuran compounds are a class of synthetic antibacterial drugs. Nitrofuran compounds can be rapidly metabolized in aquatic products, and their metabolites are semicarbazide (SEM), 3-amino-2-oxazolidinyl ketone (AOZ), 1-amino-2-hydantoin (AHD), and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone (AMOZ). Therefore, the residual status of nitrofuran compounds is often reflected by detecting the residual level of metabolites. Nitrofuran drugs and metabolites are toxic substances with toxic side effects such as carcinogenicity, teratogenicity, and mutagenicity. The European Union and the United States banned the use of nitrofurans in food and animal production in 1995 and 2002, respectively, and the EU stipulated that the minimum required performance level (MRPL) of each nitrofuran metabolite is 1 μg / kg; my country's Ministry of Agriculture and Rural Affairs also issued Announcement No. 193 and Announcement No. 235 in 2002 and Announcement No. 560 in 2005, listing nitrofurans as banned veterinary drugs, which are strictly prohibited from use in all food animals.

[0004] Although my country has issued many method standards for the determination of residual nitrofuran metabolites in aquatic products, such as the national testing standard GB 31656.13-2021 (Determination of residual nitrofuran metabolites in aquatic products by liquid chromatography-tandem mass spectrometry), the Ministry of Agriculture Announcement No. 1077-2-2008 (Determination of residual nitrofuran metabolites in aquatic products by high performance liquid chromatography) and the agricultural industry testing standard Ministry of Agriculture Announcement No. 783-1-2006 (Determination of residual nitrofuran metabolites in aquatic products by liquid chromatography-tandem mass spectrometry), etc. However, due to the small molecular weight of nitrofuran metabolites, they all need to be chemically derivatized with 2-nitrobenzaldehyde at 37°C in the dark for 16 hours before detection. Aquatic samples have high levels of interfering substances such as fat and protein, and need to be purified and enriched by solvent extraction, solid phase extraction, nitrogen drying, rotary evaporation concentration and other pre-treatment technologies. It can be seen that the detection of nitrofuran metabolites in the prior art has the disadvantages of complex sample pretreatment steps and long time consumption. Summary of the invention

[0005] In view of the shortcomings of the detection of nitrofuran metabolites in the prior art, such as complex sample pretreatment steps and long time consumption, the present invention provides a method for detecting four nitrofuran metabolites in aquatic products. The sample pretreatment of the method of the present invention adopts 2-pyrrolecarboxaldehyde for derivatization reaction, which overcomes the shortcomings of the existing methods of long derivatization time and complex pretreatment; the method of the present invention has a short derivatization time, one-step purification, and less interference.

[0006] The present invention first provides a method for detecting four nitrofuran metabolites in aquatic products, comprising the following steps:

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

[0008] (2) Purifying the sample to be purified obtained in step (1), and detecting it by liquid chromatography-tandem mass spectrometry after purification.

[0009] In the above detection method, in step (1), the concentration of the 2-pyrrolecarboxaldehyde solution is 0.5 to 6.0 mol / L, preferably 1.0 mol / L;

[0010] The mass volume ratio of the aquatic product sample to the 2-pyrrole carboxaldehyde solution is 40:3 g / mL;

[0011] The organic solvent is ethyl acetate.

[0012] The above detection method, the method in step (1) comprises the following steps: mixing the aquatic product sample with a mixed internal standard working solution, a hydrochloric acid solution, and a 2-pyrrole carboxaldehyde solution and then performing a derivatization reaction; after the derivatization reaction is completed, adding neutral alumina and a potassium dihydrogen phosphate solution to adjust the pH to 7.0-7.5; centrifuging to obtain a supernatant; adding ethyl acetate to extract, centrifuging, drying the supernatant, and then adding a methanol-water solution to re-dissolve to obtain a sample to be purified.

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

[0014] The mixed internal standard working solution is SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 C3 and AMOZ-D5 mixed solution; 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;

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

[0016] The concentration of the hydrochloric acid is 0.15-0.6 mol / L, preferably 0.2 mol / L;

[0017] The mass ratio of the aquatic product sample to the neutral alumina is 1:1;

[0018] The concentration of the dipotassium hydrogen phosphate solution is 1.0 mol / L;

[0019] The mass volume ratio of the aquatic product sample to the ethyl acetate is 1:4 g / mL;

[0020] The mass volume ratio of the aquatic product sample to the methanol-water solution is 2:1 g / mL;

[0021] In the methanol-water solution, the volume ratio of methanol to water is 5:95.

[0022] In the above detection method, the temperature of the derivatization reaction is 20-50°C, preferably room temperature (25±3°C); the time is 5-60 min, preferably 5-30 min, more preferably 5 min;

[0023] The derivatization reaction is carried out under ultrasonic conditions in the dark or under shaking conditions in the dark.

[0024] In the above detection method, in step (2), purification is performed using a push rod filter type purification column;

[0025] The filler in the push rod filter type purification column is 100 mg C 18 .

[0026] In the above detection method, in step (2), in the detection of the liquid chromatography tandem mass spectrometer, the liquid chromatography detection conditions are as follows: chromatographic column: BEH C18 chromatographic column (2.1mm×100mm, 1.7μm); liquid automatic sampler: injection volume 5μL; mobile phase flow rate: 0.30mL / min; binary mobile phase: phase A is an aqueous solution (containing 0.1% formic acid, V / V), phase B is methanol (containing 0.1% formic acid, V / V); gradient elution conditions are: 0-1min, phase B volume concentration: 5%-5%; 1-8min, phase B volume concentration: 5%-75%; 8-10min, phase B volume concentration: 75%-75%; 10-10.1min, phase B volume concentration: 75%-5%; 10.1-12min, phase B volume concentration: 5%-5%;

[0027] The mass spectrometry detection conditions are as follows: ion source: electrospray ion source (ESI); scanning mode: positive ion scanning; detection mode: multiple reaction monitoring (MRM); ion source temperature: 650°C; nebulizer gas (Gas1, zero-order air): 60.0psi; nebulizer auxiliary heating gas (Gas2, zero-order air): 55.0psi; curtain gas (CUR, nitrogen): 30.0psi; spray voltage 5500V; scanning time: 50.0msec; selected ion detection: select a quantitative ion and a qualitative ion for each compound;

[0028] The parent ions of the four target compounds SEM, AOZ, AHD, and AMOZ were 153.0, 180.1, 193.0, and 279.1, respectively; the quantitative ions (collision energy V) were 136.1 (15.0), 93.0 (24.0), 92.9 (31.0), and 235.1 (14.0), respectively; the qualitative ions (collision energy V) were 110.1 (14.0), 119.1 (23.0), 122.3 (30.0), and 125.0 (16.0), respectively;

[0029] Isotope internal standard SEM of four target compounds 13 C- 15 N2, AOZ-D4, AHD- 13The parent ions of C3 and AMOZ-D5 were 156.1, 184.0, 196.1, and 284.2, respectively; the product ions (collision energy V) were 139.1 (15.0), 93.0 (24.0), 93.0 (31.0), and 240.1 (14.0), respectively.

[0030] The above detection method also includes the following steps:

[0031] (1) Steps for drawing a standard curve

[0032] In the step of drawing the standard curve, standard substances, semicarbazide hydrochloride, 3-amino-2-oxazolidinyl ketone, 1-amino-2-hydantoin hydrochloride and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, are weighed, and then treated by the derivatization and extraction methods in the detection method, and detected by liquid chromatography-tandem mass spectrometry, 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, to prepare a linear equation and obtain the correlation coefficient;

[0033] (2) Substituting the measured quantitative ion chromatographic peak of the target compound in the aquatic product sample and the corresponding isotope internal standard peak area ratio into the standard curve to obtain the concentration of the target compound in the aquatic product sample.

[0034] In the above detection method, the aquatic product sample is edible tissue of fish, shrimp or crab.

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

[0036] The method of the present invention uses 2-pyrrole carboxaldehyde for derivatization reaction, ethyl acetate extraction, push rod filter type purification column purification, and after purification, UPLC-MS / MS analysis is used to determine the drug residue in aquatic products. The method has less interference, simple and fast pretreatment, and the recovery rate, precision and sensitivity indicators all meet the requirements of drug residue analysis. The method is suitable for the detection of nitrofuran metabolite residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the derivatization process of four nitrofuran metabolites.

[0038] Figure 2 The peak area of ​​the derivatives of four nitrofuran metabolites varies with temperature under oscillating conditions (n=3).

[0039] Figure 3 The graph shows the trend of the peak areas of the derivatives of four nitrofuran metabolites changing with the concentration of hydrochloric acid under room temperature oscillation conditions (n=3).

[0040] Figure 4The graph shows the trend of the peak areas of the derivatives of four nitrofuran metabolites changing with the concentration of the derivatizing agent under room temperature oscillation conditions (n=3).

[0041] Figure 5 Comparison of the peak areas of the derivatives under room temperature oscillation and ultrasound-assisted conditions at different time points (n=3).

[0042] Figure 6 This is a graph showing the variation trend of the peak area of ​​the derivatives under ultrasound-assisted conditions as a function of ultrasound time (n=3).

[0043] Figure 7 Selected ion chromatogram of blank grass carp matrix spiked samples (5.0 μg kg -1 ). DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0045] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] The push rod filter type purification column used in the following examples is the application number: 201920261816.8, and the invention name is "Push rod filter type purification column". A polyethylene (PE) porous sieve plate is placed at the bottom of the purification column; 100 mg C 18 As a purifying filler (C 18 is a commercially available filler), and a polyethylene (PE) porous sieve plate is placed on the upper layer.

[0048] The standard substances used in the following examples, semicarbazide hydrochloride (SEM·HCl), 3-amino-2-oxazolidinyl ketone (AOZ), 1-amino-2-hydantoin hydrochloride (AHD·HCl), and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone (AMOZ), were purchased from Dr. Ehrenstorfer Company, Germany.

[0049] The grass carp samples described in the following examples are grass carp meat.

[0050] Example 1

[0051] (1) Extraction process: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and add 0.05 mL of mixed internal standard working solution (containing SEM- 13 C- 15 N2, AOZ-D4, AHD-13 C3, AMOZ-D5, all at 100 μg / L) and 0.1 mL of mixed standard working solution (containing SEM, AOZ, AHD, AMOZ, all at 100 μg / L), vortexed for 30 seconds. 5.0 mL of 0.2 mol / L hydrochloric acid solution and 0.15 mL of 1.0 mol / L derivatization reagent (2-pyrrole carboxaldehyde solution) were added in sequence, vortexed for 1 minute, placed in an ultrasonicator (power 200 W), and derivatized for 5 minutes at room temperature in the dark to carry out the derivatization reaction (the derivatization process of the four nitrofuran metabolites is shown in Figure 1 ). After the derivatization, add 2g of neutral alumina and 6mL of 1.0mol / L potassium dihydrogen phosphate, vortex mix at 2500r / min for 1min to adjust the pH (Note: the role of adding potassium dihydrogen phosphate is to neutralize the hydrochloric acid solution added during the extraction process) to 7.0-7.5; centrifuge at 10000r / min for 10min at 4℃, take out the upper aqueous phase into a 50mL centrifuge tube; add 8mL of ethyl acetate, vortex mix at 2500r / min for 1min; centrifuge again at 4000r / min at 4℃ for 10min. Take the upper clear liquid and blow dry with nitrogen at 40℃. Accurately add 1.0mL of 5% methanol-water solution (V / V=5:95) to dissolve, vortex and oscillate fully, and wait for purification.

[0052] (2) Purification process: Pull the piston of the push rod filter purification column upward to the sample loading port, use a pipette to add 1 mL of the sample to be purified into the column tube through the loading port; push the piston to transfer the purified liquid to a 2 mL centrifuge tube (the piston can be pushed multiple times to completely transfer the purified liquid to the injection bottle), collect all the outflow liquid, pass it through a 0.22 μm nylon microporous filter membrane, and then test it on the machine.

[0053] Liquid chromatography tandem mass spectrometry was used to detect it, and the liquid chromatography conditions for the above detection were as follows:

[0054] Chromatographic column: BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm); liquid automatic sampler: injection volume 5 μL; mobile phase flow rate: 0.30 mL / min; binary mobile phase: phase A is aqueous solution (containing 0.1% formic acid, V / V), phase B is methanol (containing 0.1% formic acid, V / V); gradient elution conditions are shown in Table 1.

[0055] Table 1 HPLC elution program

[0056] time Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0 0.30 95.0 5.0 1.00 0.30 95.0 5.0 8.00 0.30 25.0 75.0 10.00 0.30 25.0 75.0 10.10 0.30 95.0 5.0 12.00 0.30 95.0 5.0

[0057] The mass spectrometry conditions for the above detection are as follows:

[0058] Ion source: electrospray ion source (ESI); scanning mode: positive ion scanning; detection mode: multiple reaction monitoring (MRM); ion source temperature: 650°C; nebulizer gas (Gas1, zero-order air): 60.0psi; nebulizer auxiliary heating gas (Gas2, zero-order air): 55.0psi; curtain gas (CUR, nitrogen): 30.0psi; spray voltage 5500V; scanning time: 50.0msec; selected ion detection: one quantitative ion and one qualitative ion are selected for each compound.

[0059] Table 2 Molecular formula, retention time, parent ion, product ion and collision energy of target compounds

[0060]

[0061]

[0062] Note: *Quantitative ion

[0063] (3) Effect of temperature under constant temperature oscillation conditions

[0064] Sample processing and detection: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and then use the method of (1) extraction process and (2) purification process, except that different derivatization temperatures are used in a constant temperature oscillator (speed: 150 rpm) in the dark for 60 min, and then it is detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion measured on the mass spectrum. The peak area trend of the derivatives of the four nitrofuran metabolites with temperature (n = 3) is shown in Figure 2 .

[0065] Depend on Figure 2 It can be seen that the peak area of ​​SEM reaches a peak value when the derivatization temperature is 20°C; the peak area of ​​AOZ reaches a peak value when the derivatization temperature is 30°C; the peak area of ​​AHD has no obvious change at different derivatization temperatures; the peak area of ​​AMOZ reaches a peak value when the derivatization temperature is 70°C; but the overall difference is not large. Based on the overall situation of the peak areas of the derivatives of the four nitrofuran metabolites, room temperature of about 25°C was finally selected as the derivatization temperature for subsequent optimization experiments.

[0066] (4) Effect of hydrochloric acid concentration under shaking conditions at room temperature (25°C)

[0067] Sample processing and detection: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and then use the method of (1) extraction process and (2) purification process, except that different concentrations of hydrochloric acid are added during the derivatization reaction, and the derivatization is carried out in a constant temperature oscillator (speed: 150 rpm) at room temperature in the dark for 60 minutes, and then it is detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion measured on the mass spectrum. The peak area trend of the derivatives of the four nitrofuran metabolites as a function of hydrochloric acid concentration (n = 3) is shown in Figure 3 .

[0068] Depend on Figure 3 It can be seen that the peak areas of SEM and AMOZ reached their peak values ​​when the hydrochloric acid concentration was 0.15 mol / L; the peak areas of AOZ and AHD reached their peak values ​​when the hydrochloric acid concentration was 0.3 mol / L; based on the overall situation of the peak areas of the derivatives of the four nitrofuran metabolites, 0.2 mol / L was finally selected as the hydrochloric acid concentration for subsequent optimization experiments.

[0069] (5) Effect of derivatization reagent concentration under shaking conditions at room temperature (25°C)

[0070] Sample processing and detection: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and then use the method of (1) extraction process and (2) purification process. The difference is that different concentrations of derivatization reagents are added during the derivatization reaction. Derivatization is carried out in a constant temperature oscillator (speed: 150 rpm) at room temperature in the dark for 60 minutes, and then it is detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion measured on the mass spectrum. The peak area trend of the derivatives of the four nitrofuran metabolites as a function of the concentration of the derivatization reagent (n = 3) is shown in Figure 4 .

[0071] Depend on Figure 4 It can be seen that when the concentration of the derivatizing agent is greater than 1 mol / L, the peak areas of the derivatives of the four nitrofuran metabolites remain basically unchanged. Therefore, 1 mol / L was selected as the concentration of the derivatizing agent for subsequent optimization experiments.

[0072] (6) Effect of room temperature (25°C) shaking and ultrasound-assisted conditions

[0073] Sample processing and detection: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and then use the method of (1) extraction process and (2) purification process, except that the derivatization reaction is carried out in a constant temperature oscillator (speed: 150 rpm) at room temperature in the dark for 60 min and in an ultrasonicator (power: 200 W) at room temperature in the dark for 10 min, and then detect it by liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion measured on the mass spectrum. The peak area of ​​the derivatives of the four nitrofuran metabolites changes with the derivatization time (n = 3) is shown in Figure 5 .

[0074] Depend on Figure 5 It can be seen that the peak areas of the derivatives of the four nitrofuran metabolites under room temperature ultrasound for 10 minutes are all higher than those under room temperature oscillation for 60 minutes. Obviously, ultrasound-assisted derivatization can achieve better derivatization effects in a shorter derivatization time.

[0075] (7) Effect of derivatization time under ultrasound-assisted conditions

[0076] Sample processing and detection: Weigh 2.0 g (accurate to 0.01 g) of blank grass carp sample into a 50 mL centrifuge tube, and then use the method of (1) extraction process and (2) purification process, except that the sample is derivatized in an ultrasonic instrument (power: 200 W) at room temperature in the dark for different time periods, and then detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion measured on the mass spectrum. The peak area of ​​the derivatives of the four nitrofuran metabolites changes with the derivatization time (n = 3) is shown in Figure 6 .

[0077] Depend on Figure 6 It can be seen that the peak area of ​​SEM reaches a peak value when the derivatization time is 10 minutes, but the overall difference is not large compared with 5 minutes; the peak area of ​​AOZ reaches a peak value when the derivatization time is 5 minutes; the peak area of ​​AHD has no obvious change at different derivatization times; the peak area of ​​AMOZ reaches a peak value when the derivatization time is 5 minutes, but the overall difference is not large. Based on the overall situation of the peak areas of the derivatives of the four nitrofuran metabolites, 5 minutes can be selected as the ultrasonic derivatization time for subsequent optimization experiments.

[0078] Example 2

[0079] (1) Preparation of standard curve

[0080] Extraction process: In a 50mL centrifuge tube, accurately add 100.0μL of mixed standard working solution (including SEM, AOZ, AHD, AMOZ, all with a concentration of 10.0μg / L, taking the concentration point of the standard curve with a concentration of 1.0μg / L as an example). Accurately add 0.05mL of mixed internal standard working solution (including SEM-13 C- 15 N2, AOZ-D4, AHD- 13 C3, AMOZ-D5, concentrations of 100.0 μg / L), vortexed for 30 seconds. 5.0 mL of 0.2 mol / L hydrochloric acid solution and 0.15 mL of 1.0 mol / L derivatization reagent (2-pyrrole carboxaldehyde solution) were added in sequence, vortexed for 1 minute, and placed in an ultrasonicator (power: 200 W) for 5 minutes at room temperature in the dark to carry out the derivatization reaction. After the derivatization, add 2g neutral alumina and 6mL 1.0mol / L potassium dihydrogen phosphate, vortex mix at 2500r / min for 1min to adjust the pH (Note: the role of adding potassium dihydrogen phosphate is to neutralize the hydrochloric acid solution added during the extraction process) to 7.0-7.5; centrifuge at 10000r / min for 10min at 4℃, take out the upper aqueous phase into a 50mL centrifuge tube; add 8mL ethyl acetate, vortex oscillate at 2500r / min for 1min, centrifuge at 4000r / min for 10min at 4℃; take the upper clear liquid and blow dry with nitrogen at 40℃. Accurately add 1.0mL 5% methanol-water solution (V / V=5:95) to dissolve, vortex oscillate fully, and pass through a 0.22μm nylon microporous filter membrane for detection.

[0081] The same method as above was used to prepare mixed standard working solutions with concentrations of 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, and 20.0 μg / L, which were detected by liquid chromatography tandem mass spectrometry. The concentration value was used as the horizontal axis, 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 axis. A linear equation was prepared and the correlation coefficient was obtained, as shown in Table 3.

[0082] Table 3 Standard curve equations and correlation coefficients of target compounds

[0083]

[0084] (2) Sample testing

[0085] The grass carp used for development and verification of the present invention was purchased from the farmers' market in Beijing. The collected samples were transported back to the laboratory in cold storage. After skinning and bone removal, the edible parts were taken, ground and crushed with a homogenizer, and frozen at -20°C for later use. A blank grass carp sample was spiked (5.0 μg / kg), and after the same extraction and purification treatment as in Example 1 and detection on the machine, the chromatograms of the derivatives of the four nitrofuran metabolites in the sample were as follows: Figure 7 As shown; the measured concentrations were 4.7, 5.6, 5.2, and 4.4 μg / kg, respectively (see Table 4 below).

[0086] (3) The accuracy test of the method of the present invention uses the sample spike recovery rate and relative standard deviation as indicators. The experimental method is the same as that of Example 1. The spike recovery rate of the blank grass carp sample is calculated based on the percentage of the concentration measured by the instrument to the target concentration; the relative standard deviation is calculated by the percentage of the relative deviation of the concentration measured in 3 parallel samples of the same compound to the average value, see Table 4. The recovery rates of the four target compounds are 89.0-111.4%, which meets the requirements of GB / T27404-2008 "Laboratory Quality Control Standards for Physical and Chemical Inspection of Foods" that when the content of the measured component is less than 0.1 mg / kg, the recovery rate range is 60%-120% and when the content of the measured component is 1-10 μg / kg, the relative standard deviation range is 30%-21%, indicating that the method has high correctness and precision and good accuracy.

[0087] Spiked recovery rate = C / C0×100%

[0088] Where C is the concentration measured by the instrument and C0 is the spiked concentration.

[0089]

[0090] In the formula, S is the standard deviation of the measured concentration, is the average value of the measured concentrations.

[0091] Table 4 Recovery and precision test results of four nitrofuran metabolite derivatives (n=3)

[0092]

[0093] The liquid chromatography and mass spectrometry conditions for detection are the same as those in Example 1.

Claims

1. A method for detecting four nitrofuran metabolites in aquatic products, comprising the following steps: (1) 2-pyrrole carboxaldehyde solution was used as a derivatization reagent to carry out a derivatization reaction on four nitrofuran metabolites in aquatic product samples, and then an organic solvent was used to extract them to obtain the sample to be purified; (2) purifying the sample to be purified obtained in step (1), and detecting it by liquid chromatography tandem mass spectrometry after purification; The four nitrofuran metabolites are semicarbazide, 3-amino-2-oxazolidinyl ketone, 1-amino-2-hydantoin and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone.

2. The detection method according to claim 1, characterized in that: In step (1), the concentration of the 2-pyrrolecarboxaldehyde solution is 1.0 mol / L; The mass volume ratio of the aquatic product sample to the 2-pyrrole carboxaldehyde solution is 40:3 g / mL; The organic solvent is ethyl acetate.

3. The detection method according to claim 1, characterized in that: The method in step (1) comprises the following steps: mixing the aquatic product sample with a mixed internal standard working solution, a hydrochloric acid solution, and a 2-pyrrole carboxaldehyde solution and performing a derivatization reaction; after the derivatization reaction is completed, adding neutral alumina and a potassium dihydrogen phosphate solution to adjust the pH to 7.0-7.5; Centrifuge to obtain a supernatant; add ethyl acetate to extract, centrifuge, blow dry the supernatant, and then add methanol-water solution to re-dissolve to obtain a sample to be purified.

4. The detection method according to claim 3, characterized in that: The mass volume ratio of the aquatic product sample to 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; 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 the hydrochloric acid solution is 2:5 g / mL; The concentration of the hydrochloric acid is 0.15-0.6 mol / L; The mass ratio of the aquatic product sample to the neutral alumina is 1:1; The concentration of the dipotassium hydrogen phosphate solution is 1.0 mol / L; The mass volume ratio of the aquatic product sample to the ethyl acetate is 1:4 g / mL; The mass volume ratio of the aquatic product sample to the methanol-water solution is 2:1 g / mL; In the methanol-water solution, the volume ratio of methanol to water is 5:

95.

5. The detection method according to claim 4, characterized in that: The concentration of the hydrochloric acid is 0.2 mol / L.

6. The detection method according to claim 1, characterized in that: The temperature of the derivatization reaction is 20-50°C and the time is 5-60 min; The derivatization reaction is carried out under ultrasonic conditions in the dark or under shaking conditions in the dark.

7. The detection method according to claim 6, characterized in that: The derivatization reaction time is 5 min.

8. The detection method according to claim 1, characterized in that: In step (2), purification is performed using a push rod filter type purification column; The filler in the push rod filter type purification column is 100 mg C 18 .

9. The detection method according to claim 1, characterized in that: In step (2), in the detection of the liquid chromatography tandem mass spectrometer, the liquid chromatography detection conditions are as follows: chromatographic column: BEH C18 chromatographic column, 2.1 mm×100 mm, 1.7 µm; liquid automatic sampler: injection volume 5 μL; mobile phase flow rate: 0.30 mL / min; binary mobile phase: phase A is an aqueous solution containing 0.1% by volume of formic acid, and phase B is a methanol solution containing 0.1% by volume of formic acid; gradient elution conditions are: 0-1min, phase B volume concentration: 5%-5%; 1-8min, phase B volume concentration: 5%-75%; 8-10min, phase B volume concentration: 75%-75%; 10-10.1min, phase B volume concentration: 75%-5%; 10.1-12min, phase B volume concentration: 5%-5%; The mass spectrometry detection conditions were as follows: ion source: electrospray ion source; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring mode; ion source temperature: 650°C; nebulizer gas: zero-grade air: 60.0 psi; nebulizer auxiliary heating gas: zero-grade air: 55.0 psi; curtain gas: nitrogen: 30.0 psi; spray voltage: 5500 V; scanning time: 50.0 msec; selected ion detection: select one quantitative ion and one qualitative ion for each compound; The parent ions of the four target compounds SEM, AOZ, AHD, and AMOZ were 153.0, 180.1, 193.0, and 279.1, respectively; the quantitative ions were 136.1, 93.0, 92.9, and 235.1, respectively, and the collision energies V were 15.0, 24.0, 31.0, and 14.0, respectively; the qualitative ions were 110.1, 119.1, 122.3, and 125.0, respectively, and the collision energies V were 14.0, 23.0, 30.0, and 16.0, respectively; the isotopic internal standards SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 The parent ions of C3 and AMOZ-D5 were 156.1, 184.0, 196.1, and 284.2, respectively; the product ions were 139.1, 93.0, 93.0, and 240.1, respectively, and the collision energies V were 15.0, 24.0, 31.0, and 14.0, respectively.

10. The detection method according to any one of claims 1 to 9, characterized in that: The method further comprises the steps of: (1) Steps for drawing a standard curve In the step of drawing the standard curve, standard substances, semicarbazide hydrochloride, 3-amino-2-oxazolidinyl ketone, 1-amino-2-hydantoin hydrochloride and 5-methylmorpholine-3-amino-2-oxazolidinyl ketone, are weighed, and then treated by the derivatization and extraction method in the detection method according to any one of claims 1 to 9, and detected by liquid chromatography tandem mass spectrometry, 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, to prepare a linear equation and obtain a correlation coefficient; (2) Substituting the area ratio of the quantitative ion chromatographic peak of the target compound in the aquatic product sample and the corresponding isotope internal standard peak into the standard curve, the concentration of the target compound in the aquatic product sample is obtained.

11. The detection method according to any one of claims 1 to 9, characterized in that: The aquatic product sample is edible tissue of fish, shrimp or crab.

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