Method for determining florfenicol residue content in eggs through chemiluminescence analysis

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for determining the content of florfenicol residues in eggs through chemiluminescence analysis, which comprises the following steps: preparing an egg sample solution, a KMnO4 solution, a florfenicol standard stock solution and a luminol stock solution; the KMnO4 solution and the luminol stock solution are mixed, and a luminol-KMnO4 chemiluminescence system is prepared; the luminol-KMnO4 chemiluminescence system liquid is respectively added into the florfenicol standard stock and the egg sample liquid for reaction, detection and comparison are carried out through a detector, the florfenicol residue content in the egg is detected, and the method comprises the following steps: detecting through the detector, recording a luminescence signal through a computer, and determining the chemiluminescence intensity three times in parallel. The florfenicol has an obvious inhibition effect on chemiluminescence of a luminol-KMnO4 system, and the concentration of the florfenicol is in direct proportion to the inhibition intensity, so that quantitative detection is carried out, the method is simple and rapid, the sensitivity is relatively high, and the problem of low sensitivity of an existing florfenicol determination method is solved.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis. Background Art

[0002] Florfenicol, also known as fluprofen, has a molecular formula of C 12 H 14 Cl 2 FNO 4 S, Florfenicol is a derivative formed by replacing one hydrogen of thiamphenicol with fluorine. It is an amide antibiotic. Florfenicol is a broad-spectrum antibacterial drug of chloramphenicol for veterinary use. It exerts its antibacterial effect by reducing the activity of enzymes to hinder the formation of peptide chains. Due to its high efficiency, rapid absorption and wide distribution, it is widely used in the treatment of animal diseases. It can treat bacterial respiratory diseases and foot rot in cattle and sheep; it can be used to treat respiratory infectious diseases and nasal diseases in pigs that may suffocate and die due to Actinobacillus pleuropneumoniae. It can effectively inhibit intestinal infections in pigs caused by certain bacteria, and can treat chronic respiratory diseases in chickens and enhance immune responses.

[0003] However, florfenicol is toxic, so the safe dosage of florfenicol is extremely important. And when it is used for animals and poultry, the metabolism of florfenicol in the animal body and the residue in animal and poultry products must also be considered. Eating animal and poultry products with excessive antibiotic veterinary drug residues will have adverse effects on the human body. Therefore, it is necessary to monitor the antibiotic drug florfenicol residues in animal-derived foods.

[0004] According to reports, the main methods for determining florfenicol include enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry and spectrophotometry. All of the above detection methods can perform qualitative and accurate quantitative analysis of antibiotics, but they also have shortcomings. For example, enzyme-linked immunosorbent assay requires specific antibodies and the reagents are expensive. Instrument detection is cumbersome and time-consuming, and requires professional counterpart detection staff. The sensitivity of spectrophotometry is not high.

[0005] Therefore, there is an urgent need to establish a new method for monitoring and detecting florfenicol residues that solves the above problems. Summary of the invention

[0006] The object of the present invention is to provide a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis, aiming to solve the problem that the existing florfenicol determination method has low sensitivity.

[0007] To achieve the above object, the present invention provides a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis, comprising the following steps:

[0008] Preparation of egg sample solution, KMnO 4 solution, florfenicol standard stock solution, and luminol stock solution;

[0009] The KMnO 4 The solution was mixed with the luminol stock solution to prepare luminol-KMnO 4 Chemiluminescent system;

[0010] The luminol-KMnO 4 The chemiluminescent system liquid is added to the florfenicol standard reserve and the egg sample liquid respectively for reaction, and the detection and comparison are performed through a detector to realize the detection of the florfenicol residual content in the eggs.

[0011] The method for preparing the egg-like liquid includes purchasing 3 batches of different local eggs from a supermarket, randomly selecting 3 eggs from each batch, and evenly mixing the egg white liquid and egg yolk liquid, respectively adding acetonitrile organic solvent to completely denature the protein, collecting the supernatant after filtering, and diluting the supernatant 10 times before placing it in a 4°C refrigerator for standby use.

[0012] Wherein, the preparation of KMnO 4 The solution method includes weighing 0.0142 g KMnO 4 Dissolve the granules in a 100 mL volumetric flask, make up to volume with deionized water, shake well, and obtain 9.0 × 10 -4 mol / L KMnO 4 Solution.

[0013] The method for preparing the florfenicol standard stock solution comprises the steps of transferring 1.0 mL of the florfenicol standard to a 100 mL volumetric flask to obtain a quasi-stock solution, storing the quasi-stock solution for later use, and transferring 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask using a pipette, and respectively transferring the 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask, respectively using deionized water to make up the volume, i.e., forming 2.0×10 -5 4.0×10 -5 , 6.0×10 -5 , 8.0×10 -5 , 10.0×10 -5 g / mL florfenicol solution.

[0014] Among them, the method for preparing the luminol stock solution includes weighing 1.7720g of luminol, first taking an appropriate amount of 1.0mmol / LNaOH solution in a beaker to dissolve the luminol, then transferring the dissolved solution to a 1000mL volumetric flask to make up the volume, and placing it away from light to obtain a 0.01mol / L florfenicol standard stock solution. When necessary, dilute with deionized water to obtain the target mass concentration.

[0015] The present invention provides a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis, preparing egg sample liquid, KMnO 4 solution, florfenicol standard stock solution and luminol stock solution; the KMnO 4 The solution was mixed with the luminol stock solution to prepare luminol-KMnO 4 Chemiluminescent system: the luminol-KMnO 4 The chemiluminescent system liquid is added to the florfenicol standard reserve and the egg sample liquid for reaction, and the residual content of florfenicol in the egg is detected by comparison through the detector. The method uses a peristaltic pump A to mix luminol with KMnO 4 The two solutions in the passage are first mixed in the three-way pipe, and the carrier (H 2 O) diluting the florfenicol solution by a peristaltic pump B, and mixing the diluted florfenicol solution with the egg sample solution and the luminol-KMnO in the reaction coil. 4 The chemiluminescence system reacts, the photomultiplier tube captures the light signal and converts it into an electrical signal, which is then detected by the detector, and the computer records the luminescence signal. The chemiluminescence intensity is measured three times in parallel. 4 The chemiluminescence of the system has an obvious inhibitory effect, and the concentration of florfenicol is proportional to the inhibitory intensity, so that quantitative detection is simple, rapid and highly sensitive, solving the problem of low sensitivity of existing florfenicol determination methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The present invention provides a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis, and a schematic diagram of the flow path for determining florfenicol.

[0018] Figure 2 The present invention provides a method flow chart for determining the residual content of florfenicol in eggs by chemiluminescence analysis. DETAILED DESCRIPTION

[0019] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0020] See also Figure 1 to Figure 2 The present invention provides a method for determining the residual content of florfenicol in eggs by chemiluminescence analysis, comprising the following steps:

[0021] S1 Preparation of egg sample solution, KMnO 4 solution, florfenicol standard stock solution, and luminol stock solution;

[0022] Specifically, the method for preparing the egg-like liquid includes purchasing 3 batches of different free-range eggs from a supermarket, randomly selecting 3 eggs from each batch, and evenly mixing the egg white liquid and egg yolk liquid, respectively adding acetonitrile organic solvent to completely denature the protein, collecting the supernatant after filtering, and diluting the supernatant 10 times before placing it in a 4°C refrigerator for standby use.

[0023] The preparation of KMnO 4 The solution method includes weighing 0.0142 g KMnO 4 Dissolve the granules in a 100 mL volumetric flask, make up to volume with deionized water, shake well, and obtain 9.0 × 10 -4 mol / L KMnO 4 Solution.

[0024] The method for preparing a florfenicol standard stock solution comprises the steps of transferring 1.0 mL of the florfenicol standard to a 100 mL volumetric flask to obtain a quasi-stock solution, storing the quasi-stock solution for later use, and transferring 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask using a pipette, and respectively transferring the 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask to obtain a quasi-stock solution .... -5 4.0×10 -5 , 6.0×10 -5 , 8.0×10 -5 , 10.0×10 -5 g / mL florfenicol solution.

[0025] The method for preparing the luminol stock solution comprises weighing 1.7720 g of luminol, first taking an appropriate amount of 1.0 mmol / L NaOH solution in a beaker to dissolve the luminol, then transferring the dissolved solution to a 1000 mL volumetric flask to make up the volume, and placing it away from light to obtain a 0.01 mol / L florfenicol standard stock solution, and when necessary, making up the volume with deionized water to obtain the target mass concentration.

[0026] S2 is the KMnO 4 The solution was mixed with the luminol stock solution to prepare luminol-KMnO 4 Chemiluminescent system;

[0027] Specifically, the luminol stock solution and the KMnO 4 The two solutions in the solution path are first mixed in a three-way pipe to obtain the luminol-KMnO 4 Chemiluminescent system.

[0028] S3 converts the luminol-KMnO 4 The chemiluminescent system liquid is added to the florfenicol standard reserve and the egg sample liquid respectively for reaction, and the detection and comparison are performed through a detector to realize the detection of the florfenicol residual content in the eggs.

[0029] Specifically, (H 2 O) diluting the florfenicol solution by a peristaltic pump B, and mixing the diluted florfenicol solution with the egg sample solution and the luminol-KMnO in the reaction coil. 4 The chemiluminescence system reacts, the photomultiplier tube captures the light signal and converts it into an electrical signal, which is then detected by the detector, and the computer records the luminescence signal. The chemiluminescence intensity is measured three times in parallel. 4 The chemiluminescence of the system was significantly inhibited, and the concentration of florfenicol was proportional to the inhibition intensity, so quantitative detection was performed. The detection results showed that under the optimized adjustment, the optimized conditions were: the concentration of the alkaline medium NaOH was 4.0×10 -5 mol / L, luminol-KMnO 4 The concentration of the system is 1.0×10 -5 mol / L, oxidant KMnO 4 The concentration was 9.0×10-4mol / L, the pump speed parameter was set to 60, and at 1.0×10 -6 ~1.0×10 -4 In the mass concentration range of g / mL, there was a good linear relationship between the mass concentration of florfenicol and the chemiluminescence intensity, and the detection limit (3σ) was 3.0×10 -7 g / mL. For mass concentration 8.0×10 -5 The florfenicol concentration of g / mL was determined 11 times in parallel, with a relative standard deviation of 4.88%. The detection is simple and rapid, with high sensitivity and reliable method.

[0030] The above disclosure is only a preferred embodiment of the method for determining the residual content of florfenicol in eggs by chemiluminescence analysis of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for determining the residual content of florfenicol in eggs by chemiluminescence analysis. It is characterized in that The following steps are involved: Preparation of egg sample solution, KMnO 4 solution, florfenicol standard stock solution, and luminol stock solution; The KMnO 4 The solution was mixed with the luminol stock solution to prepare luminol-KMnO 4 Chemiluminescent system; The luminol-KMnO 4 The chemiluminescent system liquid is added to the florfenicol standard reserve and the egg sample liquid respectively for reaction, and the detection and comparison are performed through a detector to realize the detection of the florfenicol residual content in the eggs.

2. The method for determining the residual content of florfenicol in eggs by chemiluminescence analysis according to claim 1, It is characterized in that The preparation of KMnO 4 The solution method includes weighing 0.0142 g KMnO 4 Dissolve the granules in a 100 mL volumetric flask, make up to volume with deionized water, shake well, and obtain 9.0 × 10 -4 mol / L KMnO 4 Solution.

3. The method for determining the residual content of florfenicol in eggs by chemiluminescence analysis as claimed in claim 1, It is characterized in that The method for preparing the egg sample liquid comprises purchasing 3 batches of different free-range eggs from a supermarket, randomly selecting 3 eggs from each batch, and evenly stirring the egg white liquid and egg yolk liquid, respectively adding acetonitrile organic solvent to completely denature the egg white, collecting the supernatant after filtering, and diluting the supernatant 10 times before placing it in a 4°C refrigerator for standby use.

4. The method for determining the residual content of florfenicol in eggs by chemiluminescence analysis according to claim 1, It is characterized in that The method for preparing the luminol stock solution comprises weighing 1.7720 g of luminol, first taking an appropriate amount of 1.0 mmol / L NaOH solution in a beaker to dissolve the luminol, then transferring the dissolved solution to a 1000 mL volumetric flask to make up the volume, and placing it away from light to obtain a 0.01 mol / L florfenicol standard stock solution, and when necessary, making up the volume with deionized water to obtain the target mass concentration.

5. The method for determining the residual content of florfenicol in eggs by chemiluminescence analysis according to claim 1, It is characterized in that The method for preparing a florfenicol standard stock solution comprises the steps of transferring 1.0 mL of the florfenicol standard to a 100 mL volumetric flask to obtain a quasi-stock solution, storing the quasi-stock solution for later use, and transferring 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask using a pipette, and respectively transferring the 1.0, 2.0, 3.0, 4.0 and 5.0 mL of the florfenicol standard stock solution to a 50 mL volumetric flask to obtain a quasi-stock solution .... -5 4.0×10 -5 , 6.0×10 -5 8.0×10 -5 , 10.0×10 -5 g / mL florfenicol solution.

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