Method for detecting sulfonamides in animal-derived food through capillary electrophoresis-laser-induced fluorescence

Abstract

The invention provides a method for detecting sulfonamides in animal-derived food through capillary electrophoresis-laser-induced fluorescence, and belongs to the technical field of detection. Comprising the following steps: (1) mincing a sample, adding an extracting solution for extraction, and carrying out solid-phase extraction column extraction and methanol elution to obtain an eluted sample solution; (2) preparing a sulfanilamide drug standard substance and fluorescamine into gradient solutions by using methanol and acetonitrile respectively, and stirring at room temperature for reaction to obtain a solution A; (3) mixing the elution sample solution with a fluorescent amine-acetonitrile solution, and stirring and reacting at room temperature to obtain a solution B; and (4) detecting the fluorescence intensity of the sulfonamides in the solution A by adopting capillary electrophoresis-laser induced fluorescence to obtain a linear equation for detecting the concentration of the sulfonamides in the solution B. The invention establishes a novel method for detecting sulfonamide residues in animal-derived food by using high-efficiency capillary electrophoresis-laser-induced fluorescence, and realizes high-sensitivity detection of three sulfonamides in animal-derived food.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a method for detecting sulfonamides in animal-derived food by capillary electrophoresis-laser-induced fluorescence. BACKGROUND

[0002] Sulfonamides are a class of broad-spectrum antibiotics with p-aminobenzene sulfonamide structure. Because of its excellent antibacterial performance and low price, it is widely used to resist bacterial and protozoan infections in animals to improve the survival rate and yield of livestock and poultry. In the process of feeding edible animals, improper use of drugs or insufficient drug holiday will lead to the presence of sulfonamide residues in edible animals or their by-products. However, sulfonamides have low solubility in human body fluids and are not easily metabolized. Long-term intake of food containing sulfonamide residues can cause damage to human organs, make the body resistant to drugs, and even have the risk of causing cancer. In order to ensure food safety and prevent drug residue problems from endangering public health, our country stipulates that the total amount of sulfonamides in animal tissues should not exceed 100 μg / kg.

[0003] At present, the detection methods of sulfonamides include high performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC / MS), thin layer chromatography (TLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC / MS) and enzyme-linked immunosorbent assay (ELISA), etc. Each of them has its own advantages and disadvantages. High performance liquid chromatography is a commonly used detection method with high sensitivity and accuracy, but the cost is high and the operation is complex; liquid chromatography-mass spectrometry combines liquid chromatography and mass spectrometry technology, which can provide higher sensitivity and specificity, but the equipment is expensive and the operation is complex; thin layer chromatography is simple and low in cost, but its sensitivity is low and it is only suitable for preliminary screening; gas chromatography and gas chromatography-mass spectrometry are more suitable for volatile compounds, and the detection effect of sulfonamides is not as good as that of liquid chromatography; enzyme-linked immunosorbent assay has high sensitivity and short detection time, but the operation is complicated and there are many factors that interfere with the results. Therefore, it is of great practical significance to develop a rapid and efficient method for detecting sulfonamide residues in food.

[0004] Capillary electrophoresis-laser induced fluorescence detection (CE-LIF) as a kind of efficient, fast, high sensitivity analysis technology, gradually applied in the field of pharmaceutical analysis. Capillary electrophoresis is a kind of analysis method which realizes separation by ion migration behavior in capillary under the action of current. It is based on the electroosmotic flow theory in capillary, and the drug sample is brought into the capillary with specific filler for separation by applying electric field. Capillary electrophoresis has high separation efficiency, short separation time, and is very advantageous for the case of extremely low concentration detection in pharmaceutical analysis, and the sample usage of capillary electrophoresis is very small, which is very suitable for the analysis of expensive drug samples. SUMMARY

[0005] The purpose of the present application is to provide a method for detecting sulfonamides in animal-derived food by capillary electrophoresis-laser induced fluorescence. The present application designs a method for separating and analyzing sulfonamides by capillary electrophoresis-laser induced fluorescence (CE-LIF, Capillary Electrophoresis-Laser Induced Fluorescence) using fluorescent amine as a derivative reagent and a 405 nm laser as a light source, which realizes high sensitive detection of three kinds of sulfonamides in animal-derived food.

[0006] The technical solution of the present application is as follows: The present application provides a method for detecting sulfonamides in animal-derived food by capillary electrophoresis-laser induced fluorescence, comprising the following steps: (1) grinding the sample, adding extraction solution for extraction, extracting through a solid phase extraction column, eluting with methanol to obtain an eluted sample solution; (2) preparing a gradient solution by using methanol and acetonitrile to prepare sulfonamide standard and fluorescent amine respectively, and stirring and reacting at room temperature to obtain solution A; (3) mixing the eluted sample solution with the fluorescent amine-acetonitrile solution, stirring and reacting at room temperature to obtain solution B; (4) detecting the fluorescence intensity of sulfonamides in solution A by capillary electrophoresis-laser induced fluorescence to obtain a linear equation, which is used to detect the concentration of sulfonamides in solution B.

[0007] As a further improvement of the present application, the extraction solution in step (1) is a 0.05-0.15 mol / L potassium dihydrogen phosphate solution.

[0008] As a further improvement of the present application, the solid phase extraction column in step (1) is a C18 solid phase extraction column.

[0009] As a further improvement of the present application, the sulfonamides in step (2) are sulfadiazine, sulfamethazine and sulfamethazine.

[0010] As a further improvement of the application, the molar ratio of the sulfonamide standard to the fluorescent amine in step (2) is 1:2.

[0011] As a further improvement of the application, the room temperature stirring reaction time in step (2) is 30-50 min.

[0012] As a further improvement of the application, the room temperature stirring reaction time in step (3) is 30-50 min.

[0013] As a further improvement of the application, the detection conditions in step (4) are that the running buffer solution is a Tris buffer solution with a pH of 7-8 and a concentration of 10-20 mmol / L, the separation voltage is 10-20 kV, the injection time is 5-15 s, and the capillary specifications are 20-40 cm in length and 70-80 µm in inner diameter.

[0014] As a further improvement of the application, the linear equation for detecting sulfadiazine in step (4) is y=0.0008x+0.0689, the linear range is 0.1-1000 ng / mL, the linear coefficient is 0.9990, and the detection limit is 0.056 ng / mL; the linear equation for detecting sulfamethazine is y=0.0013x+0.0797, the linear range is 0.1-1000 ng / mL, the linear coefficient is 0.9996, and the detection limit is 0.035 ng / mL; and the linear equation for detecting sulfadimethazine is y=0.0018x+0.0689, the linear range is 0.1-1000 ng / mL, the linear coefficient is 0.9994, and the detection limit is 0.025 ng / mL.

[0015] The application has the following beneficial effects: The application derives sulfonamides by using the derivative reagent fluorescent amine, separates and quantifies the derivative products by using a capillary electrophoresis-laser-induced fluorescence detection system with a 405 nm laser as a light source, establishes a new method for detecting sulfonamide residues in animal-derived food by using high-efficiency capillary electrophoresis-laser-induced fluorescence, and realizes high-sensitivity detection of three kinds of sulfonamides in animal-derived food. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative labor.

[0017] Figure 1This is an electrophoresis image of sulfonamide drugs in bullfrogs and crayfish analyzed using a capillary electrophoresis-laser-induced fluorescence detection method. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Bullfrog Sample This invention preprocesses and enriches bullfrog muscle tissue samples, then uses a CE-LIF detection system to detect any sulfonamide drugs they may contain. The specific experimental steps are as follows: Weigh 5 g of bullfrog muscle tissue, mince it, and place it in a stoppered centrifuge tube. Add 2 mL of extraction buffer (0.1 M KH2PO4), vortex for 5 min, centrifuge at 10000 r / min for 10 min, and discard the supernatant. Repeat the extraction three times with the bottom precipitate. Combine the supernatants and mix well. Take 5 mL of the combined supernatant and inject it into a C18 solid-phase extraction column activated with 3 mL methanol and 3 mL water. Then rinse with 3 mL water, vacuum dry, and elute with 3 mL methanol. Set aside for use.

[0020] Take a 5.0 mL centrifuge tube and add 0.1 mL of KH₂PO₄ solution (pH=3.0), 0.2 mL of fluorescein solution (1 mg / mL), and 1.7 mL of sample processing solution in sequence. After reacting at room temperature for 40 min, perform separation and detection using CE-LIF. (CE-LIF detection conditions: derivatizing reagent is fluorescein, buffer solution is 15 mmol / L Tris solution at pH=7.50, separation voltage is 15 kV, and injection time is 10 s.) Three sulfonamide drugs were not detected in bullfrog muscle tissue samples. A spiking experiment was used to test the feasibility and accuracy of this method. The results are as follows: Figure 1 (a, b, d) and as shown in Table 1, the relative standard deviations were 3.3%-4.7%, and the recovery rates were 91.2%-101.1%, indicating that the detection method proposed in this invention can be used for the quantitative analysis of sulfonamide drugs in actual bullfrog samples.

[0021] Table 1. Analytical results of sulfonamides in bullfrog samples

[0022] Example 2: Crayfish Sample This invention preprocesses and enriches crayfish muscle tissue samples, then uses a CE-LIF detection system to detect any sulfonamide drugs they may contain. The specific experimental steps are as follows: Take 5 g of crayfish muscle tissue, mince it, and put it into a stoppered centrifuge tube. Add 2 mL of extraction buffer (0.1 M KH2PO4), vortex for 5 min, centrifuge at 10000 r / min for 10 min, and discard the supernatant. Repeat the extraction three times with the bottom precipitate. Combine the supernatants and mix well. Take 5 mL of the combined supernatant and inject it into a C18 solid-phase extraction column activated with 3 mL methanol and 3 mL water. Rinse with 3 mL water, vacuum dry, and elute with 3 mL methanol. Set aside for use.

[0023] Take a 5.0 mL centrifuge tube and add 0.1 mL of KH₂PO₄ solution (pH=3.0), 0.2 mL of fluorescein solution (1 mg / mL), and 1.7 mL of sample processing solution in sequence. After reacting at room temperature for 40 min, perform separation and detection using CE-LIF. (CE-LIF detection conditions: derivatizing reagent is fluorescein, buffer solution is 15 mmol / L Tris solution at pH=7.50, separation voltage is 15 kV, and injection time is 10 s.) Three sulfonamide drugs were not detected in crayfish muscle tissue samples. A spiked assay was used to test the feasibility and accuracy of this method. The results are attached. Figure 1 As shown in (a, c, e) and Appendix Table 2, the relative standard deviation of the determination results is 2.7%-4.6%, and the recovery rate is 92.1%-108.2%, indicating that the detection technology proposed in this invention can be used for the quantitative analysis of sulfonamide drugs in actual crayfish samples.

[0024] Table 2. Analytical results of sulfonamides in crayfish samples.

[0025] Test Example 1 The established CE-LIF detection method was compared with the traditional fluorescence detection method. It was found that the CE-LIF detection method significantly improved sensitivity and reduced the detection limit by three orders of magnitude, providing a new method and approach for the detection of trace sulfonamide antibiotics.

[0026] 1. Fluorescence detection method: Steps: (1) Prepare gradient solutions of sulfonamide drug standard and fluorescent amine respectively using methanol and acetonitrile, stir at room temperature for 40 min to obtain solution A; (2) The fluorescence intensity of sulfonamide drugs in solution A was detected by a fluorescence spectrometer, and a linear equation was obtained; (3) Mix the methanol solution without sulfonamide drug standard with fluorescent amine-acetonitrile, stir at room temperature for 40 min to obtain solution C; (4) The fluorescence intensity in solution C was detected by a fluorescence spectrometer. Five consecutive measurements were taken to obtain the standard deviation of the blank. (5) Combine the above standard deviation with the linear equation in step (2) to obtain the detection limit.

[0027] The results show that the linear equation for detecting sulfadiazine is y = 176.0x + 29.64, with a linear range of 0.25–5 μg / mL, a linear coefficient of 0.9966, and a limit of detection of 0.081 μg / mL; the linear equation for detecting sulfamethazine is y = 213.8x + 62.10, with a linear range of 0.25–5 μg / mL, a linear coefficient of 0.9942, and a limit of detection of 0.077 μg / mL; and the linear equation for detecting sulfadiazine is y = 201.0x + 118.6, with a linear range of 0.25–5 μg / mL, a linear coefficient of 0.9969, and a limit of detection of 0.062 μg / mL.

[0028] 2. The CE-LIF detection method of the present invention: Steps: (1) Prepare gradient solutions of sulfonamide drug standard and fluorescent amine respectively using methanol and acetonitrile, stir at room temperature for 40 min to obtain solution A; (2) The fluorescence intensity of sulfonamide drugs in solution A was detected by capillary electrophoresis-laser induced fluorescence, and a linear equation was obtained; (3) Mix the methanol solution without sulfonamide drug standard with fluorescent amine-acetonitrile, stir at room temperature for 40 min to obtain solution C; (4) The fluorescence intensity in solution C was detected by capillary electrophoresis-laser induced fluorescence detection. The measurement was repeated 5 times to obtain the standard deviation of the blank. (5) Combine the above standard deviation with the linear equation in step (2) to obtain the detection limit.

[0029] The results show that the linear equation for detecting sulfadiazine is y = 0.0008x + 0.0689, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9990, and a limit of detection of 0.056 ng / mL; the linear equation for detecting sulfamethazine is y = 0.0013x + 0.0797, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9996, and a limit of detection of 0.035 ng / mL; and the linear equation for detecting sulfadiazine is y = 0.0018x + 0.0689, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9994, and a limit of detection of 0.025 ng / mL.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting sulfonamide drugs in animal-derived foods using capillary electrophoresis-laser-induced fluorescence, characterized in that... Includes the following steps: (1) Grind the sample, add the extraction solution for extraction, extract through a solid phase extraction column, and elute with methanol to obtain the eluent. (2) Prepare gradient solutions of sulfonamide drug standards and fluorescent amine respectively using methanol and acetonitrile, and stir the reaction at room temperature to obtain solution A; (3) Mix the eluent with the fluorescent amine-acetonitrile solution and stir at room temperature to obtain solution B; (4) The fluorescence intensity of sulfonamide drugs in solution A was detected by capillary electrophoresis-laser induced fluorescence detection, and a linear equation was obtained, which was used to detect the concentration of sulfonamide drugs in solution B; The linear equation for the detection of sulfadiazine was y = 0.0008x + 0.0689, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9990, and a limit of detection of 0.056 ng / mL; the linear equation for the detection of sulfamethazine was y = 0.0013x + 0.0797, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9996, and a limit of detection of 0.035 ng / mL; the linear equation for the detection of sulfadiazine was y = 0.0018x + 0.0689, with a linear range of 0.1–1000 ng / mL, a linear coefficient of 0.9994, and a limit of detection of 0.025 ng / mL.

2. The method for detecting sulfonamide drugs in animal-derived food by capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The extraction solution mentioned in step (1) is a 0.05-0.15 mol / L potassium dihydrogen phosphate solution.

3. The method for detecting sulfonamide drugs in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The solid phase extraction column mentioned in step (1) is a C18 solid phase extraction column.

4. The method for detecting sulfonamides in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The sulfonamide drugs mentioned in step (2) are sulfadiazine, sulfamethazine and sulfadiazine.

5. The method for detecting sulfonamides in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The molar ratio of the sulfonamide drug standard to the fluorescent amine in step (2) is 1:

2.

6. The method for detecting sulfonamides in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The stirring time at room temperature in step (2) is 30-50 min.

7. The method for detecting sulfonamides in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, The stirring time at room temperature in step (3) is 30-50 min.

8. The method for detecting sulfonamides in animal-derived foods using capillary electrophoresis-laser-induced fluorescence according to claim 1, characterized in that, In step (4), the detection conditions are as follows: the running buffer solution is a Tris buffer solution with pH=7-8 and a concentration of 10-20 mmol / L; the separation voltage is 10-20 kV; the injection time is 5-15 s; and the capillary used has a length of 20-40 cm and an inner diameter of 70-80 µm.

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