Method for detecting heavy metal content in livestock and poultry serum and application

Abstract

The invention relates to the technical field of livestock and poultry product quality detection, and particularly discloses a method for detecting the content of heavy metal in livestock and poultry serum and application. According to the method, a serum sample is subjected to digestion treatment by utilizing a persulfate advanced oxidation technology, so that organic matters in the serum are flocculated and metal ions combined with the organic matters are released at the same time, and quantitative analysis and detection on heavy metals in the serum are realized. SO4 <->. Has strong oxidizability and can trigger macromolecular organic matters to generate an oxidation cross-linking reaction when the concentration of SO4 <->. Is relatively high, so that the organic matters are flocculated and removed; meanwhile, H < + > can be continuously generated in the oxidation flocculation process, and heavy metal ions complexed with organic matters are replaced into the solution. The problem of matrix interference of the serum sample in a conventional method is solved, rapid, simple and efficient pretreatment of the serum sample can be realized under a mild condition by utilizing the method, the reagent dosage is reduced, the experimental operation is simplified, and the method has practical significance in quality control of livestock and poultry agricultural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of livestock and poultry product quality detection, and particularly relates to a method for detecting heavy metal content in livestock and poultry serum and its application. Background Art

[0002] The samples usually selected for heavy metal detection in livestock and poultry are muscles, internal organs, hair, blood and serum. Compared with other test samples, serum has the advantages of being less harmful, not causing the death of the tested animals, stable metal ion content, relatively low organic matter, not being affected by diet and excretion times of the day, and easy to collect, so it is easily selected as the sample to be tested. However, serum still contains a certain amount of organic substances such as proteins, lipids, peptides and fats. These components will cause matrix effects during the test and affect the test results. Therefore, it is necessary to select an appropriate pretreatment method to effectively remove organic matter in the serum. At present, in the pretreatment process of serum samples, digestion and removal of organic matter in serum is a key step, which can break the complex and dissociate metal ions bound to proteins or other metabolites.

[0003] Currently, the national standard method for detecting heavy metal content in biological fluid samples generally uses wet digestion and direct dilution methods to process blood samples. The wet digestion method often uses nitric acid, hydrochloric acid, sulfuric acid, and hydrogen peroxide as digestion reagents, using a hot plate or microwave digester to destroy the organic matter in the sample under heating or high pressure conditions, thereby digesting it. Hot plate digestion can process a large number of samples, but it is time-consuming, has poor sealing, easily loses volatile elements, and produces a large amount of acidic gas that causes pollution. Microwave digestion can avoid the loss of volatile elements in a closed environment, but microwave digesters are relatively expensive, cumbersome to operate, and the digestion tank needs to be cleaned and soaked afterwards, otherwise it is easy to cause sample contamination. The direct dilution method uses a diluent to dilute samples with low organic content, and then directly detects them after dilution. Commonly used diluents include dilute nitric acid, Triton X-100, tetramethylammonium hydroxide, etc. The direct dilution method is relatively simple to operate, but it cannot eliminate sample matrix interference. In order to reduce matrix interference, a large amount of diluent is often added during the dilution process. If the dilution multiple is too large, it is difficult to ensure the accuracy of the test, and the applicable sample categories are limited.

[0004] It can be seen that the commonly used pretreatment methods now usually have problems such as long time consumption, high energy consumption, large amount of reagents, environmentally unfriendly waste liquid, narrow scope of application, and limited accuracy. Therefore, there is an urgent need to study a pretreatment method with a wide range of applications, high efficiency, greenness, and convenience. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a method and application for detecting the heavy metal content in livestock and poultry serum. The method uses an oxidant to flocculate organic matter in the serum and simultaneously release metal ions bound to the organic matter, thereby achieving quantitative analysis and detection of heavy metals in the serum.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention is to provide a method for detecting heavy metal content in livestock and poultry serum, comprising the following steps:

[0008] S1. Collect blood from livestock and poultry, centrifuge it, collect the upper liquid, and obtain a serum sample A to be tested; dissolve a certain amount of oxidant in deionized water to obtain solution B; the oxidant is peroxymonosulfate or peroxydisulfate;

[0009] S2. Add serum A, solution B, and a certain amount of deionized water to a centrifuge tube in sequence, shake well, and adjust the acidity of the solution to obtain solution C;

[0010] S3, placing the mixed solution C in a constant temperature water bath oscillator for oscillation reaction to form a suspension D;

[0011] S4. After centrifuging solution D, the supernatant was filtered to obtain solution E to be tested, and solution E was tested using an atomic absorption spectrophotometer.

[0012] Furthermore, the livestock and poultry include any one of pigs, cattle, sheep, rabbits, chickens, and ducks.

[0013] Furthermore, the heavy metal includes any one of copper, zinc, manganese, lead and cadmium.

[0014] Furthermore, in step S1, the centrifugal separation process is performed at a speed of 5000 to 15000 rmin. -1 , centrifuge for 3 to 10 minutes.

[0015] Furthermore, in step S1, the concentration of the oxidant is 0.01 to 0.3 mol L -1 .

[0016] Furthermore, in step S2, the amount of serum sample A is 0.2-5 mL, the amount of solution B is 0.2-5 mL, the amount of deionized water added is 0.2-2 mL, and the concentration is 0.01-2 mol L -1 The pH value of the solution is adjusted to 5.0-6.5 by adding HNO3.

[0017] Furthermore, in step S3, the water bath heating temperature is 30-90°C, and the oscillation frequency is 50-250 r min -1, the oscillation time is 10 to 60 minutes.

[0018] Furthermore, in step S4, the centrifugal speed is 3000-8000 rpm. -1 The centrifugation time is 1 to 10 minutes.

[0019] Furthermore, in step S4, the filtration is performed using a water membrane filtration with a pore size of 0.22 to 0.45 μm.

[0020] The second aspect of the present invention is to provide the application of the above method in the quality control of livestock and poultry agricultural products. Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0021] (1) The present invention provides a method for detecting heavy metal content in livestock and poultry serum. The method utilizes persulfate advanced oxidation technology to digest serum samples, causing them to flocculate organic matter in the serum while releasing metal ions bound to the organic matter, and based on this method, quantitative analysis and detection of heavy metals in the serum is performed. The method is characterized in that by using persulfate to oxidatively flocculate serum samples, a rapid, simple, and efficient pre-treatment of serum samples is achieved under mild conditions, significantly improving detection efficiency and accuracy, and reducing detection costs;

[0022] (2) Sulfate radical advanced oxidation technology can degrade small molecular organic matter. At the same time, when the persulfate concentration is high, it can induce the oxidation cross-linking reaction of large molecular organic matter and flocculate and remove organic matter from the solution. In addition, this reaction can continuously generate H + , replacing the heavy metal ions complexed with organic matter into the solution. According to the above reaction characteristics, when the sulfate radical advanced oxidation technology is introduced into the serum system: a) After adding an appropriate amount of persulfate, the SO4 generated by activation -· It has strong oxidizing properties and can analyze a large number of metabolites in the serum, such as proteins, lipids, and peptides, breaking the long chains of macromolecular metabolites (saturated organic matter) and converting them into free radicals. These free radicals in the system will spontaneously undergo cross-linking polymerization to complete the oxidative cross-linking reaction. At the same time, some small molecule metabolites may be degraded under a strong oxidizing environment. The two reactions above work together to significantly remove organic matter in the serum and reduce the chemical oxygen demand (COD) of the sample; b) During the reaction, H + , weakening the binding ability of metal ions and organic matter in serum. When the pH reaches a certain level, the organic matter is protonated, prompting the metal ions and organic matter to dechelate and transform from a coordinated state to a free state;

[0023] (3) The present invention uses advanced oxidation technology, which does not require a complicated digestion process compared to other detection methods, has high detection efficiency, is simple and convenient, and takes less time;

[0024] (4) The present invention uses an oxidant to digest the sample, which can reduce interference caused by a series of reasons such as matrix effect, and the quantitative results are more accurate;

[0025] (5) The present invention can simultaneously process and detect five elements, namely copper, zinc, manganese, lead and cadmium, in a variety of livestock and poultry samples, which is more efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the operating flow of a method for detecting heavy metal content in livestock and poultry serum provided by the present invention;

[0027] Figure 2 Comparison of COD degradation before and after treatment with pig and chicken serum;

[0028] Figure 3 These are SDS-PAGE bands of serum and flocculation products after treatment. DETAILED DESCRIPTION

[0029] To make the objects, technical solutions, and advantages of the present invention more apparent, the following describes the specific embodiments of the present invention in further detail with reference to the specific examples and accompanying drawings. Where specific test methods, instruments, or conditions are not specified in the examples, the methods or conditions described in the literature in the art or the product specifications were used. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0030] like Figure 1 The figure shows the operation flow diagram of the method for detecting heavy metal content in livestock and poultry serum using advanced oxidation technology provided by the present invention. By using persulfate to oxidize and flocculate the serum sample, a rapid, simple, and efficient pre-treatment of the serum sample is achieved under mild conditions, which significantly improves the detection efficiency and accuracy and reduces the detection cost. The specific process is as follows:

[0031] 1. The collected blood was centrifuged at 5000~15000rpm -1 Centrifuge and collect the upper serum sample;

[0032] 2. The concentration is 0.01~0.3mol L -1 Oxidant solution; the oxidant is persulfate, including peroxymonosulfate (PMS, whose molecular formula is HSO 5- ) and persulfate (PS, whose molecular formula is S2O8 2- );

[0033] 3. In a centrifuge tube, mix 0.2-5 mL of serum sample, 0.2-5 mL of oxidant solution, and 0.2-2 mL of deionized water. -1 HNO3 solution adjusts the acidity of the mixed solution to pH 5.0-6.5;

[0034] 5. Place the mixed solution in a constant temperature water bath oscillator and maintain at 30-80°C, 50-250 r min -1 Oscillate for 10 to 60 minutes;

[0035] 6. Take out the solution for 3000~8000min -1 Centrifuge for 1 to 10 minutes, take the supernatant and filter it through a 0.22 to 0.45 μm water filter. The resulting liquid is tested using an atomic absorption spectrophotometer.

[0036] The atomic absorption instrument model used in the embodiments provided by the present invention is: PinAAcle 900H Platinum Elmer Enterprise Management (Shanghai) Co., Ltd.

[0037] The instrument parameters for copper detection were: wavelength 324.75 nm, slit width 0.7 nm, lamp current 10 mA, acetylene flow rate 2 L / min, and air flow rate 10 L / min.

[0038] The instrument parameters for zinc detection were: wavelength 213.86 nm, slit width 0.7 nm, lamp current 7 mA, acetylene flow rate 2.5 L / min, and air flow rate 10 L / min.

[0039] The instrument parameters for detecting manganese were as follows: wavelength 279.48 nm, slit width 0.7 nm, lamp current 8 mA, acetylene flow rate 2.5 L / min, and air flow rate 10 L / min.

[0040] The instrument parameters for lead detection are: wavelength 283.31 nm, slit width 0.7 nm, lamp current 8 mA, and the heating program is shown in Table 1:

[0041] Table 1. Instrument conditions for lead determination by graphite furnace atomic absorption spectrometry

[0042]

[0043] The instrument parameters for cadmium detection are: wavelength 228.80nm, slit width 0.2nm, lamp current 6mA, and the heating program is shown in Table 2:

[0044] Table 2. Instrument conditions for cadmium determination by graphite furnace atomic absorption spectrometry

[0045]

[0046] like Figure 2 The figure shows the comparison of COD degradation before and after treatment with pig and chicken serum. After adding an appropriate amount of persulfate, the SO4 produced by activation -· It has strong oxidizing properties, which can analyze a large number of metabolites in serum, such as proteins, lipids, and peptides, breaking the long chains of macromolecular metabolites (saturated organic matter) and converting them into free radicals. These free radicals in the system will spontaneously undergo cross-linking polymerization, completing the oxidative cross-linking reaction. At the same time, some small molecule metabolites may degrade in a strong oxidizing environment. The combined effect of these two reactions can significantly remove organic matter from serum and reduce the chemical oxygen demand of the sample.

[0047] like Figure 3 Shown are SDS-PAGE bands of serum and flocculation products after treatment, indicating that when sulfate radical advanced oxidation technology is introduced into the serum system, organic matter in the serum can be flocculated.

[0048] Example 1

[0049] This example uses cow blood as the test object, and the specific steps are as follows:

[0050] 1. Use the collected bovine blood at 15000r min -1 Centrifuge for 3 min and collect serum;

[0051] 2. Prepare 0.05 mol L -1 PMS solution, specifically potassium peroxymonosulfate solution;

[0052] 3. Take 0.5mL serum, 3mL PMS solution and 0.5mL deionized water and place them in a centrifuge tube, mix them evenly, and add 0.02mol L -1 Adjust the solution to pH 5.0 with HNO3;

[0053] 4. Place the centrifuge tube in a water bath at 30°C, 100 rpm -1 , oscillate for 50 min;

[0054] 5. Take out 3500r min -1 After centrifugation for 8 minutes, the supernatant was filtered through a 0.45 μm water filter membrane to obtain the test solution, which was tested on an instrument.

[0055] The metal element concentrations in the samples were calculated based on the standard curve and were Cu 593 μg L -1 、Zn 655μg L -1 、Mn 284μg L -1 、Cd 91μg L -1 、Pb 84μg L -1 .

[0056] Example 2

[0057] This example uses chicken blood as the test object, and the specific steps are as follows:

[0058] 1. Use the collected chicken blood for 5000r min -1 After centrifugation for 10 min, serum was collected;

[0059] 2. Prepare 0.2 mol L -1 PS solution, specifically potassium peroxodisulfate solution;

[0060] 3. Take 3 mL of serum, 1 mL of PS solution, and 2 mL of deionized water and place them in a centrifuge tube, mix them evenly, and add 0.5 mol L - 1 Adjust the solution to pH 6.0 with HNO3;

[0061] 4. Place the centrifuge tube in a water bath at 50°C, 200 rpm -1 , oscillate for 30 min;

[0062] 5. Take out 6000r min -1 After centrifugation for 5 minutes, the supernatant was filtered through a 0.45 μm water filter membrane to obtain the test solution, which was tested on an instrument.

[0063] The metal element concentrations in the samples were calculated based on the standard curve and were Cu 341 μg L -1 、Zn 763μg L -1 、Mn 359μg L -1 、Cd 110μg L -1 、Pb 96μg L -1 .

[0064] Example 3

[0065] This example uses rabbit blood as the test object, and the specific steps are as follows:

[0066] 1. Use the collected rabbit blood at 15000 r min -1 Centrifuge for 5 min and collect serum;

[0067] 2. Prepare 0.1 mol L -1 PMS solution, specifically potassium peroxymonosulfate solution;

[0068] 3. Take 1mL serum, 0.5mL PMS solution and 1mL deionized water and place them in a centrifuge tube, mix them evenly, and add 0.1molL -1 Adjust the solution to pH 6.5 with HNO3;

[0069] 4. Place the centrifuge tube in a water bath at 80°C, 250 rpm -1 , oscillate for 20 min;

[0070] 5. Take out 8000r min -1 After centrifugation for 3 minutes, the supernatant was filtered through a 0.22 μm water filter membrane to obtain the test solution, which was tested on an instrument.

[0071] According to the standard curve, the metal element concentrations in the samples were calculated to be Cu 413 μg L -1 、Zn 383μg L -1 、Mn 550μg L -1 、Cd 104μg L -1 、Pb 115μg L -1 .

[0072] Example 4

[0073] In this example, 5 metal ions were tested at 10-1000 μg L -1 concentration, draw the standard working curve, and obtain the linear regression equation and R 2 , blank samples were tested 11 times continuously to obtain the detection limit and quantification limit, see Table 3.

[0074] Table 3. Regression equation, linear range, detection limit, and quantification limit

[0075]

[0076] Example 5

[0077] This example is a spike recovery test. 20.00 μg L -1 , 50.00 μg L -1 and 100.00 μg L -1 The test results of three metal ion concentration levels are as follows, see Table 4:

[0078] Table 4. Spike recovery

[0079]

[0080]

[0081] The results showed that the recoveries at all spike levels were 88.6% to 114.0%, and the RSDs of three replicate samples were between 0.36% and 11.91%, indicating that the method had high accuracy and precision.

[0082] Comparative Example 1

[0083] Blood was pre-treated using the existing national standard method: wet digestion, followed by quantitative detection using an inductively coupled plasma mass spectrometer (ICP-MS). Referring to the "Forensic Science Test for Chromium and Five Other Elements in Blood and Urine - Inductively Coupled Plasma Mass Spectrometry" (GA / T 1630-2019), the specific implementation steps are as follows:

[0084] Two 0.25 mL serum samples were placed in digestion tubes. 800 μL of 65% concentrated nitric acid and 200 μL of 30% hydrogen peroxide were added to each tube. The tubes were sealed and allowed to stand at room temperature for 10 minutes. The samples were digested in a heating block at 90°C for 3 hours, then cooled to room temperature. The digestion solution was removed and the tubes were rinsed three times with deionized water. The rinses were combined with the digestion solution. Finally, the test solution was brought to a final volume of 10 mL with deionized water and analyzed using the internal standard method using ICP-MS.

[0085] Blood was pretreated using the existing national standard method—Triton-X dilution—before quantitative analysis using ICP-MS. Reference was made to "Determination of Lead in Blood Part 2: Inductively Coupled Plasma Mass Spectrometry" (GBT / Z 316.2-2018) and "Determination of Cadmium in Blood Part 2: Inductively Coupled Plasma Mass Spectrometry" (GBT / Z 317.2-2018). The sample was thoroughly shaken, 0.5 mL of serum sample was transferred to a centrifuge tube, 4.5 mL of diluent was added, and the mixture was shaken thoroughly before analysis using the ICP-MS internal standard method. The diluent was prepared as follows: 10 mL of 1% Triton X-100 solution was placed in 1 L of 0.5% nitric acid solution.

[0086] The test results of the implementation case and the comparative example are shown in Table 5 below:

[0087] Table 5. Comparison between Example 1 and two methods in national standards

[0088]

[0089] The concentrations of the five metal elements Cu, Zn, Mn, Cd, and Pb obtained in this example were analyzed for significance compared with the concentrations obtained after treatment using the national standard digestion method and dilution method. The results in Table 5 show that the significance p values for the five metal elements are 0.53, 0.22, 0.45, 0.50, and 0.10, respectively, all greater than 0.05, indicating that there are no significant differences in the concentrations of the five metals measured after testing the samples treated using this pretreatment method and the other two pretreatment methods specified in the national standard.

[0090] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0091] 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 in the scope of protection of the present invention.

Claims

1. A method for detecting heavy metal content in livestock and poultry serum, characterized in that: The following steps are involved: S1. Collect blood from livestock and poultry, centrifuge it, collect the upper liquid, and obtain a serum sample A to be tested; dissolve a certain amount of oxidant in deionized water to obtain solution B; the oxidant is peroxymonosulfate or peroxydisulfate; S2. Add serum A, solution B, and a certain amount of deionized water to a centrifuge tube in sequence, shake well, and adjust the acidity of the solution to obtain solution C; S3, placing the mixed solution C in a constant temperature water bath oscillator for oscillation reaction to form a suspension D; S4. After centrifuging solution D, the supernatant was filtered to obtain solution E to be tested, and solution E was tested using an atomic absorption spectrophotometer.

2. The method according to claim 1, wherein The livestock and poultry include any one of pigs, cattle, sheep, rabbits, chickens and ducks.

3. The method according to claim 2, wherein The heavy metals include any one of copper, zinc, manganese, lead and cadmium.

4. The method according to claim 3, wherein In step S1, the centrifugal separation process is carried out at a speed of 5000 to 15000 rpm. -1 , centrifuge for 3 to 10 minutes.

5. The method according to claim 3, wherein In step S1, the concentration of the oxidant is 0.01 to 0.3 mol / L -1 .

6. The method according to claim 4, wherein In step S2, the amount of serum sample A is 0.2-5 mL, the amount of solution B is 0.2-5 mL, the amount of deionized water added is 0.2-2 mL, and the concentration is 0.01-2 mol L -1 The pH value of the solution is adjusted to 5.0-6.5 by adding HNO3.

7. The method according to claim 6, wherein In step S3, the water bath heating temperature is 30-90°C and the oscillation frequency is 50-250 r / min. -1 , the oscillation time is 10 to 60 minutes.

8. The method according to claim 7, wherein In step S4, the centrifugal speed is 3000-8000 rpm -1 The centrifugation time is 1 to 10 minutes.

9. The method according to claim 8, wherein In step S4, the filtration is performed using a water membrane filtration with a pore size of 0.22 to 0.45 μm.

10. Use of the method according to any one of claims 1 to 9 in quality control of livestock and poultry agricultural products.

Images