Method for detecting zeranol in meat sample

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting zeranol in a meat sample. The detection method comprises the following steps: pretreating a meat sample by adopting a magnetic dispersive solid-phase extraction method, and then detecting zeranol by adopting an immunochromatography technology, the magnetic dispersion solid-phase extraction method adopts an HLB magnetic nano material. The detection method provided by the invention has good accuracy, sensitivity and specificity, and can meet the requirement of rapid detection of zeranol in meat samples. The method is simple and convenient to operate, does not need professional detection equipment, can be operated by common law enforcement officers or food supervisors through simple training, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and particularly relates to a detection method of zearalanol in meat samples. BACKGROUND

[0002] As a banned growth promoter of beta-receptor agonist, zearalanol is prone to be illegally used and then to be left in pork, and long-term intake of which can interfere with the endocrine system of human body and harm health.

[0003] Although traditional detection methods such as liquid chromatography-mass spectrometry (LC-MS) are highly accurate, they are expensive and complicated to operate, and are difficult to meet the demand of on-site rapid screening. Magnetic dispersive solid phase extraction (MDSPE) can significantly simplify the sample pretreatment process and reduce matrix interference due to the high adsorption efficiency and rapid separation characteristics of magnetic materials. The colloidal gold immunochromatography technology (GICA) has the advantages of simple operation, visual interpretation and rapid detection. The combination of the two can not only realize efficient enrichment and purification of zearalanol in complex matrix, but also meet the demand of on-site detection, and provide a technical solution with sensitivity and convenience for pork quality and safety monitoring.

[0004] Therefore, it is a technical problem to be solved to develop a detection method of zearalanol in meat samples based on magnetic solid phase extraction-colloidal gold immunochromatography. SUMMARY

[0005] The present application innovatively combines magnetic dispersive solid phase extraction technology with colloidal gold immunochromatography test strips suitable for dairy products, and successfully establishes a detection method of zearalanol in meat samples, which comprises: pretreating the meat sample by magnetic dispersive solid phase extraction method, and then detecting zearalanol by immunochromatography technology; the magnetic dispersive solid phase extraction method uses HLB magnetic nanomaterial as a functional adsorbent.

[0006] By screening HLB magnetic nanomaterial as a functional adsorbent, the present application can combine the high separation capacity of magnetic dispersive solid phase extraction and the rapid and sensitive characteristics of colloidal gold immunochromatography technology, and realize rapid detection of zearalanol in meat samples.

[0007] Preferably, the HLB magnetic nanomaterial is a polystyrene matrix magnetic nanomaterial.

[0008] Preferably, the pretreatment step comprises: mixing the meat sample with acetonitrile and centrifuging, taking the supernatant and mixing with activated HLB magnetic nanomaterial, using a magnet to magnetically attract the HLB magnetic nanomaterial, collecting the remaining liquid, and using nitrogen blowing to redissolve for immunochromatography technology to detect zearalanol.

[0009] The present application finds that the detection limit of the zearalanol colloidal gold test strip for milk and dairy products in raw milk, milk and milk powder is 1 μg / kg, which can meet the experimental requirements, but is not suitable for pork matrix, therefore, through a large number of combination and optimization of pretreatment conditions, it is found that the colloidal gold immunochromatographic rapid detection method is suitable for pork matrix after pretreatment by the method of reverse magnetic dispersion solid phase extraction using HLB magnetic nanomaterial, and the accurate detection of zearalanol is realized.

[0010] The present application adopts reverse magnetic dispersion solid phase extraction: after extracting protein from the sample, only the sample loading step is needed, without the steps of sample loading, elution and elution, so that the sample pretreatment time can be shortened. The traditional solid phase extraction (SPE) operation process includes several steps of activation, sample loading, elution and elution, which is similar to the traditional SPE. The magnetic dispersion solid phase extraction (MDSPE) improves the solid phase particle extraction agent on the basis of the SPE, and realizes the dispersion extraction by migrating inert magnetic core in each SPE particle. Both the SPE and the MDSPE realize the dispersion and extraction of the target compound by using the different interaction mechanisms between the filler and the target compound and impurities. According to the specific adsorption and elution process, the positive and reverse solid phase extraction are divided according to the different use modes. In the process of the MDSPE, the magnetic adsorbent is added to the solution or suspension of the sample, the target compound is adsorbed to the surface of the dispersed magnetic adsorbent by using a strong magnet in a few seconds or tens of seconds, the target analyte is migrated together with the adsorbent by the external magnetic field, and finally the target analyte is eluted by using a suitable solvent. The reverse solid phase extraction only needs to be activated and loaded, most of the impurities and adsorbents are loaded at the same time, the target analyte is eluted with the solution, and finally the sample is detected by using the test strip after nitrogen blowing and enrichment.

[0011] Preferably, the mass-volume ratio of the meat sample to acetonitrile is 1 g: (2.5-3.5) mL. Preferably, the centrifugal speed is above 12000 r / min, and preferably the centrifugal time is above 10 min.

[0012] Preferably, the activation is to activate the HLB magnetic nanomaterial by using methanol and water in sequence. Preferably, 1 mL of methanol or water is added to activate every 10 mg of HLB magnetic nanomaterial.

[0013] Preferably, the nitrogen blowing is carried out in a water bath at 45-55 DEG C.

[0014] Under the above pretreatment conditions, the interfering impurities can be removed to the greatest extent, the extraction efficiency is high, the target compound can be separated and enriched from the complex matrix, and the subsequent high sensitivity and high specificity detection by using the colloidal gold immunochromatographic technology is facilitated.

[0015] Preferably, the immunochromatography technique is a colloidal gold immunochromatography technique.

[0016] Preferably, the colloidal gold immunochromatography test paper is used to detect zearalanol.

[0017] Further, the present application provides the use of the detection method in detecting zearalanol in meat samples.

[0018] Preferably, the meat sample includes but is not limited to pork and the like.

[0019] The detection method of the present application has the advantages of simple operation, rapidness, accuracy, sensitivity and the like.

[0020] Compared with the prior art, the present application has the following beneficial effects: The detection method of the present application has good accuracy, sensitivity (detection limit of 5 μg / kg) and specificity (no cross-reaction with common antibiotics), and can meet the demand for rapid detection of zearalanol in meat samples. Moreover, the method of the present application is simple to operate, and does not require professional detection equipment. Ordinary law enforcement personnel or food supervision personnel can operate it after simple training, and has a broad application prospect.

[0021] In actual application scenarios, when cracking food and drug crime, the public security organ often needs to quickly determine whether the food contains prohibited drugs on the spot. The detection method of the present application can help the police to quickly detect meat samples in food production and processing dens, farmers' markets and other places, timely lock criminal evidence, and improve the efficiency of cracking crime. For the market supervision and management department, in daily law enforcement inspection, meat products can be quickly sampled, and products with zearalanol added in violation can be timely found, so as to protect the rights and interests of consumers. In terms of food safety protection, whether it is the self-checking link of food enterprises or the daily monitoring of grass-roots food safety detection stations, the detection method of the present application can provide convenient and efficient detection means, provide strong support for building a strict food safety defense line, and effectively improve the timeliness and accuracy of food safety supervision, which has very high practical value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a schematic diagram of the detection method of the present application; wherein A is a schematic diagram of a pretreatment step; B is a schematic diagram of result determination of a colloidal gold immunochromatography test strip.

[0023] Figure 2 is a specificity evaluation result of the detection method of the present application.

[0024] Figure 3 is a detection limit test result of the detection method of the present application. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present application. In the embodiments provided in the present specification, the specific techniques or conditions not noted are implemented according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not noted by the manufacturers are conventional products that can be purchased through regular channels. The brand and model number of the test strip used in the following examples are Corn Zeranol Rapid Test Strip (Milk Sample) 1 ppb (Beijing Vydun Biotechnology Co., Ltd.); the brand and model number of the HLB magnetic nanomaterial are Polystyrene Matrix Magnetic Extractant (Bonnacats-MA) 20-40 μm; 1 g / pk (Zhonglang Hongtai (Beijing) Intelligent Technology Co., Ltd.).

[0026] Example 1 The present embodiment provides a method for detecting zearalanol in a meat sample (pork), and a schematic diagram of the detection method is shown in Figure 1 The steps are as follows: 1. Sample pretreatment 1.1 Sample extraction 0.5 g of pork sample (accurate to 0.01) was weighed into a 15 mL plastic centrifuge tube, vortexed for 30 s, and allowed to stand for 10 min. 1.5 mL of acetonitrile was added, and oscillated for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was taken into another 15 mL centrifuge tube.

[0027] 1.2 Sample purification 10 mg of HLB magnetic nanomaterial was taken into a 15 mL centrifuge tube, activated with 1 mL of methanol and 1 mL of water in sequence, and the sample extraction liquid in 1.1 was loaded, vortexed and oscillated for 1 min, and the remaining liquid was collected in a 15 mL centrifuge tube by using a magnet to magnetically attract the HLB magnetic nanomaterial, and then dried by nitrogen blowing in a 50°C water bath, and redissolved with 0.5 mL of water, vortexed and mixed for 1 min.

[0028] 2. Sample detection 200 μL of the test sample was taken into a gold label microwell (a special microwell matched with the test strip), and the purple red particles at the bottom of the well were completely dissolved by careful blowing, and the gold label microwell was placed in a 50°C incubator for incubation for 3 min, the test strip was inserted into the gold label microwell, the sample pad was fully immersed in the test sample, and the test strip was taken out of the gold label microwell, the sample pad at the lower end of the test strip was discarded, and the result was determined.

[0029] Example 2 The embodiment evaluates the specificity of the detection method of embodiment 1, and the specific steps are as follows: The T line position of the test strip of embodiment 1 uses a specific antibody designed for zearalanol, and other substances cannot be combined with this special antibody because of the structural and chemical properties different from zearalanol. Fendiline meglumine, chloramphenicol, florfenicol and zearalanol were added to pork to make the concentration reach 200 μg / kg, and then the detection method of embodiment 1 was used for detection, and the results are shown in Figure 2 The results show that except for zearalanol which is positive on the test strip, the rest are negative. The experimental results show that the test strip has good specificity and no cross reaction with other compounds.

[0030] Embodiment 3 The embodiment evaluates the detection limit of the detection method of embodiment 1, and the specific steps are as follows: Different contents of zearalanol were added to pork, and then the detection method of embodiment 1 was used for detection, and the results are shown in Figure 3 When the content of zearalanol in pork is ≥5 μg / kg, the T line does not appear purple red band and is positive; when the content of zearalanol in pork is <5 μg / kg, the T line appears purple red band and is negative. Therefore, the detection limit of the detection method of the present application is 5 μg / kg.

[0031] Finally, it should be pointed out that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for the detection of zearalanol in a meat sample, characterized in that, include: Meat samples were pretreated using magnetic dispersion solid-phase extraction, followed by immunochromatography to detect zearalenone. The magnetic dispersion solid-phase extraction method uses HLB magnetic nanomaterials as functional adsorbents.

2. The detection method according to claim 1, characterized in that, The HLB magnetic nanomaterial is a polystyrene-based magnetic nanomaterial.

3. The detection method according to claim 1, characterized in that, The pretreatment steps include: mixing meat samples with acetonitrile and centrifuging, taking the supernatant and mixing it with activated HLB magnetic nanomaterials, using a magnet to magnetically attract the HLB magnetic nanomaterials, collecting the remaining liquid, resolving it by nitrogen blowing, and then using it for immunochromatographic detection of zearalenone.

4. The detection method according to claim 3, characterized in that, The mass-to-volume ratio of the meat sample to acetonitrile was 1 g: (2.5~3.5) mL.

5. The detection method according to claim 3, characterized in that, The activation process involves sequentially activating the HLB magnetic nanomaterials with methanol and water.

6. The detection method according to claim 3, characterized in that, The nitrogen blowing is carried out in a water bath at 45℃~55℃.

7. The detection method according to claim 3, characterized in that, The resolution is performed using water.

8. The detection method according to claim 1, characterized in that, The immunochromatographic technique is colloidal gold immunochromatographic technique.

9. The detection method according to claim 8, characterized in that, Zearalenone was detected using colloidal gold immunochromatographic strips.

10. The application of the detection method according to any one of claims 1 to 9 in the detection of zearalenone in meat samples.

Images