Color developing agent for detecting sulphaguanidine medicine in aquatic products as well as preparation method and detection application of color developing agent

Abstract

The invention discloses a color developing agent for detecting sulphaguanidine medicine in aquatic products as well as a preparation method and detection application of the color developing agent, and the color developing agent is formed by mixing acetylacetone, a formaldehyde solution with the concentration of 36 wt%, a sodium acetate solution with the concentration of 0.2 mol / L and an acetic acid solution with the concentration of 0.2 mol / L according to the volume ratio of 7.8: 15: 16: 34: 27.2; the kit has the advantages of strong specificity and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of aquatic product detection, in particular to a color developer for detecting sulfaguanidine drugs in aquatic products, a preparation method thereof, and detection applications. Background Art

[0002] Sulfamidine has broad-spectrum antibacterial activity, showing excellent antibacterial effects against both Gram-positive and Gram-negative bacteria. It is an important drug for the prevention and treatment of bacterial infections. It is primarily used to treat bacillary dysentery and enteritis, and to prevent infections following intestinal surgery. It is listed in Volume 1 of the Ministry of Health's Drug Standards for Chemical Drugs and Preparations. Sulfamidine is also commonly used as a feed additive. As a sulfonamide, it accounts for a significant proportion of sulfonamide use. Caution is advised with sulfamidine use, particularly in animal-derived foods. Excessive sulfamidine residues may cause poisoning or allergic reactions in humans, and are potentially carcinogenic.

[0003] Currently, the main methods for detecting sulfaguanidine (SAC) raw material and residues include spectrophotometry, HPLC-UV detection, HPLC-tandem mass spectrometry, micellar liquid chromatography, voltammetry, and chemiluminescence. HPLC-UV detection uses liquid chromatography to separate sulfonamides and then detects them based on their absorption of ultraviolet light. However, this method has poor interference immunity and requires complex pretreatment steps, including solid-phase extraction of the sample solution to eliminate interference from oils and fats. HPLC-tandem mass spectrometry, based on liquid chromatography, separates SAC. The drug enters the mass spectrometer detector, generating fragmented ions. Quantitative analysis is performed by comparing the fragmented ions with a standard spectral library. This method offers the advantages of high resolution and selectivity, but the instrumentation is expensive and highly specialized. Therefore, the development of a simple, rapid, and sensitive method for the detection of SAC is of practical significance for the safety monitoring of animal-derived foods. Summary of the Invention

[0004] The purpose of the present invention is to provide a color developer with strong specificity and high sensitivity for detecting sulfaguanidine drugs in aquatic products, as well as a preparation method and detection application thereof.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a colorimetric reagent for detecting sulfaguanidine drugs in aquatic products, which is prepared by mixing acetylacetone, a 36wt% formaldehyde solution, a 0.2mol / L sodium acetate solution, and a 0.2mol / L acetic acid solution in a volume ratio of 7.8:15:16:34:27.2.

[0006] A method for preparing a color developer for detecting sulfaguanidine drugs in aquatic products comprises the following steps: mixing 7.8 mL of acetylacetone, 15 mL of a 36 wt% formaldehyde solution, 16 mL of a 0.2 mol / L sodium acetate solution, and 34 mL of a 0.2 mol / L acetic acid solution, keeping the mixture in a boiling water bath for 5 minutes, cooling the mixture to room temperature, adjusting the pH to 4.3, and diluting the mixture with water to 100 mL to obtain the color developer for detecting sulfaguanidine drugs in aquatic products.

[0007] A method for detecting sulfaguanidine drugs in aquatic products using a colorimetric reagent comprises the following steps:

[0008] Step 1: Take the muscle tissue portion of the aquatic sample to be tested, chop it with scissors, and then fully grind and mix the minced meat with a tissue homogenizer. Take 4-5g of the minced meat into a centrifuge tube, slowly add 20-40mL of formic acid-acetonitrile-water solution, place it on a vortex shaker, and extract it at a speed of 1800-2000rpm for 2-4 minutes to obtain a sample extract;

[0009] Step 2: Filter the sample extract obtained in step 1 with filter paper into a stoppered graduated cylinder containing 5 g of sodium chloride. After all the filtrate is filtered into the graduated cylinder, cover the bottle with the stopper, shake vigorously for 3 minutes, let it stand for 10 minutes, and then draw 5 mL of the supernatant into a glass test tube. Then, blow it dry with nitrogen, add 2-4 mL of the color developer, and store it in a water bath at 38-40°C for 15-25 minutes.

[0010] Step 3: If the color of the solution changes to red, it is determined that the sample to be tested contains sulfaguanidine; if the color of the solution does not change, it is determined that the sample to be tested does not contain sulfaguanidine.

[0011] Furthermore, the formic acid-acetonitrile-water solution is prepared as follows: formic acid, acetonitrile and ultrapure water are mixed in a volume ratio of 0.1:24.9:25, and then mixed evenly with a glass rod to obtain the formic acid-acetonitrile-water solution.

[0012] Compared with the prior art, the advantages of the present invention are: a color developer for detecting sulfaguanidine drugs in aquatic products, a preparation method thereof, and a detection application thereof; after the target substance sulfaguanidine is extracted from the aquatic product by means of a chemical reaction, it is filtered into a stoppered measuring cylinder to dissolve sodium chloride to form a saturated sodium chloride solution; the saturated sodium chloride solution has a greater polarity than water, which is conducive to the separation of the aqueous phase and the acetonitrile phase, forming salting out, further reducing the solubility of sulfaguanidine in the aqueous phase, and improving the extraction rate of acetonitrile relative to sulfaguanidine, which helps to improve the accuracy of the detection result; further utilizing the color development reaction of acetylacetone and formaldehyde aqueous solution with sulfaguanidine within a specific pH value range, the sulfaguanidine in the test liquid is identified, thereby realizing the detection of sulfaguanidine residues in the test sample. The method of the present invention has a simple and easy-to-understand principle, strong specificity, high sensitivity, intuitive detection results, high accuracy of detection results, does not require large-scale analytical instruments, consumes relatively little time, and has extremely low amortized analysis costs. It is specifically suitable for on-site rapid detection of sulfaguanidine residues in aquatic products and has strong practicality. DETAILED DESCRIPTION

[0013] The following will combine the contents of the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0014] Example 1: Preparation of a colorimetric reagent for detecting sulfaguanidine drugs in aquatic products.

[0015] 7.8 mL of acetylacetone, 15 mL of a 36 wt% formaldehyde solution (solvent: water), 16 mL of a 0.2 mol / L sodium acetate solution (solvent: water), and 34 mL of a 0.2 mol / L acetic acid solution (solvent: water) were mixed, kept in a boiling water bath for 5 minutes, cooled to room temperature, adjusted to pH 4.3, and diluted with water to 100 mL to obtain a colorimetric reagent for detecting sulfaguanidine drugs in aquatic products.

[0016] Example 2: A method for detecting sulfaguanidine using the color developing agent prepared in Example 1.

[0017] Step 1. Take the muscle tissue portion of the aquatic product sample to be tested, chop it with scissors, and then use a tissue homogenizer to fully grind and mix the minced meat. Weigh 4-5g of the minced meat into a 50mL plastic centrifuge tube, slowly add 20-40mL of formic acid-acetonitrile-water solution, place it on a vortex shaker, and extract it at a speed of 1800-2000rpm for 2-4min to obtain a sample extract. The formic acid-acetonitrile-water solution is prepared as follows: 0.4mL of formic acid and 99.6mL of acetonitrile are dissolved in 100mL of ultrapure water, and mixed evenly with a glass rod to obtain a formic acid-acetonitrile-water solution. The aquatic product samples are fish, shrimp, crab, shellfish, etc.

[0018] Step 2: Filter the sample extract obtained in step 1 through filter paper into a stoppered graduated cylinder containing 5 g of sodium chloride. After the filtrate is completely filtered into the graduated cylinder, cover the bottle with the stopper and shake vigorously for 3 minutes. After standing for 10 minutes, the solution separates into two layers, and 5 mL of the upper clear liquid is drawn into a glass test tube. The tube is then blown nearly dry with nitrogen. After adding 2-4 mL of the developer prepared in Example 1, the reaction mixture is placed in a water bath at 38-40° C. for 15-25 minutes to generate a compound containing a dihydropyridine ring. The reaction principle is as follows:

[0019]

[0020] Step 3: If the solution color changes to red, it is determined that the sample to be tested contains sulfaguanidine (bright red when the concentration is high and light red when the concentration is low). If the solution color does not change, it is determined that the sample to be tested does not contain sulfaguanidine.

[0021] Example 3: Sensitivity measurement was performed using the method of Example 2.

[0022] A homogenized fish sample without sulfaguanidine was used as a blank control sample. Different masses of sulfaguanidine were added to the homogenized fish sample without sulfaguanidine to obtain a series of spiked samples with sulfaguanidine addition amounts of 0.001 g / kg, 0.005 g / kg, 0.01 g / kg, and 0.05 g / kg, respectively. The determination method for sulfaguanidine in "Determination of sulfonamide residues in aquatic products - Liquid chromatography" in "Ministry of Agriculture Announcement No. 958-12-2007" was used as a control detection method and the method of Example 2. The above series of spiked samples were tested for sulfaguanidine content, and the test results are shown in Table 1 below:

[0023] Table 1 Spiked test results

[0024]

[0025] As can be directly seen from Table 1 above, the detection method of this embodiment gave a positive result for the spiked sample at a concentration of 0.005 g / kg, and a negative result for the spiked sample at a concentration of 0.001 g / kg, indicating that the detection limit of the detection method of the present invention for sulfaguanidine in aquatic products is 0.005 g / kg. The control detection method detected sulfaguanidine in the spiked sample at a concentration of 0.001 g / kg, indicating that the liquid chromatography detection method has a higher sensitivity than the chemical colorimetric detection method of the present invention. However, the liquid chromatography detection method is limited by the detection instrument, detection site, and the operating level of the detection personnel, and is not suitable for rapid on-site detection of aquatic products.

[0026] When the concentration of sulfaguanidine in the aquatic product sample to be tested is less than 0.005 g / kg, the sample solution is judged as negative because no color change is observed by naked eye. Therefore, the detection limit of the present invention's method for detecting sulfaguanidine in aquatic products is 0.005 g / kg. Comparison with the control method shows that when the sulfaguanidine content is greater than 0.005 g / kg, the detection results of the present method are the same as those of the control method, demonstrating that the method of Example 2 has high accuracy and sensitivity and is fully suitable for the rapid detection of chlordimeform in aquatic products.

[0027] Example 4: Specificity experiment was performed using the method of Example 2.

[0028] A fish homogenized sample without sulfadiazine or sulfapyridine was used as a blank control sample. Sulfonamides in an amount 10 times the detection limit of sulfaguanidine were added to the fish homogenized sample without the above two substances to obtain a series of spiked samples with sulfadiazine and sulfapyridine added in an amount of 0.05 g / kg, respectively. The samples were detected using the method of Example 2, and the color of the solution did not change, indicating that sulfadiazine and sulfapyridine, these sulfonamides, did not interfere with the detection of sulfaguanidine, and the method had strong anti-interference ability.

[0029] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A colorimetric reagent for detecting sulfaguanidine drugs in aquatic products, characterized in that: The mixture is prepared by mixing acetylacetone, a formaldehyde solution with a concentration of 36 wt%, a sodium acetate solution with a concentration of 0.2 mol / L, and an acetic acid solution with a concentration of 0.2 mol / L in a volume ratio of 7.8:15:16:34:27.

2.

2. A method for preparing a color developer for detecting sulfaguanidine drugs in aquatic products according to claim 1, characterized in that: 7.8 mL of acetylacetone, 15 mL of a 36 wt% formaldehyde solution, 16 mL of a 0.2 mol / L sodium acetate solution, and 34 mL of a 0.2 mol / L acetic acid solution were mixed, kept in a boiling water bath for 5 minutes, cooled to room temperature, adjusted to a pH of 4.3, and diluted with water to 100 mL to obtain a colorimetric reagent for detecting sulfaguanidine drugs in aquatic products.

3. A method for detecting sulfaguanidine drugs in aquatic products using the developer according to claim 1, characterized in that: The following steps are involved: Step 1: Take the muscle tissue portion of the aquatic sample to be tested, chop it with scissors, and then fully grind and mix the minced meat with a tissue homogenizer. Take 4-5g of the minced meat into a centrifuge tube, slowly add 20-40mL of formic acid-acetonitrile-water solution, place it on a vortex shaker, and extract it at a speed of 1800-2000rpm for 2-4 minutes to obtain a sample extract; Step 2: Filter the sample extract obtained in step 1 with filter paper into a stoppered graduated cylinder containing 5 g of sodium chloride. After all the filtrate is filtered into the graduated cylinder, cover the bottle with the stopper, shake vigorously for 3 minutes, let it stand for 10 minutes, and then draw 5 mL of the supernatant into a glass test tube. Then, blow it dry with nitrogen, add 2-4 mL of the color developer, and store it in a water bath at 38-40°C for 15-25 minutes. Step 3: If the color of the solution changes to red, it is determined that the sample to be tested contains sulfaguanidine; if the color of the solution does not change, it is determined that the sample to be tested does not contain sulfaguanidine.

4. The method for detecting sulfaguanidine drugs in aquatic products using a color developer according to claim 3, wherein: The formic acid-acetonitrile-water solution is prepared as follows: formic acid, acetonitrile and ultrapure water are mixed in a volume ratio of 0.1:24.9:25, and then mixed evenly with a glass rod to obtain the formic acid-acetonitrile-water solution.

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