AU2020101468A4 - Method for Preparing Reference Material with Turbot Muscle as Matrix for AOZ Residue Analysis - Google Patents
Method for Preparing Reference Material with Turbot Muscle as Matrix for AOZ Residue Analysis Download PDFInfo
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Abstract
of Description
The invention relates to a method for preparing reference material with turbot muscle as matrix
for AOZ residue analysis, which falls into the scope of detection of drug residues in aquatic
products. The preparation method comprises the following steps: a certain turbot is given a
medicated bath; part of furazolidone is left in the muscle tissue after absorption, accumulation and
metabolism in the turbot, to obtain positive matrix material with AOZ residue; matrix reference
material samples are prepared by homogenizing, freeze-drying, grinding, encapsulation and
sterilization of the positive matrix material, and are verified for its uniformity and stability. The
invention adopts the method of medicated bath to metabolize furazolidone in the turbot to generate
AOZ and obtain positive reference materials. The highly uniform and stable reference material for
AOZ residue analysis is obtained by homogenizing and freeze-drying steps. The matrix reference
material prepared by the invention is closer to the actual test sample. Therefore, it is more scientific
and accurate to evaluate the testing methods, spot the differences between laboratories and verify
the accuracy of operators' results.
Description
D e s crip tion
Method for Preparing Reference Material with Turbot Muscle as Matrix for AOZ Residue Analysis
Technical Field The invention relates to a method for preparing reference material with turbot muscle as matrix for AOZ residue analysis, which falls into the scope of detection of drug residues in aquatic products.
Technical Background Nitrofurans are synthetic broad-spectrum antibiotics of 5-nitro structure, mainly including furazolidone, furaltadone, nitrofurazone, nitrofurantoin, etc., which was widely used to treat and prevent gastrointestinal diseases of aquatic animals caused by Escherichia and Salmonella. Nitrofurans have a short half-life in animals, but their metabolites can be tightly bound to proteins and remain for a long time. Studies have shown that nitrofurans and their metabolites can induce gene mutations, especially their metabolites are highly carcinogenic. To be on the safe side, developed countries such as the European Union and the United States have issued a ban on the use of such veterinary drugs. Announcement No. 193 of the Ministry of Agriculture of the People's Republic of China, List of Veterinary Drugs and Their Compounds Banned for Use for Food Animals explicitly prohibits the use of nitrofurans broad-spectrum antibiotics.
However, due to their low price and good effect, the illegal use of nitrofurans in aquaculture still exists. Since the incident of "drug residues in turbot" in 2006, the problem of nitrofurans residues in aquatic products has been exposed to the public. In 2015, the media reported that nitrofuran was detected in turbot in Shandong and other places, which had a great impact on people's dining life. The routine monitoring results of aquatic products in China in recent years have shown that nitrofuran metabolites are still detected in aquatic products in China, most of which are AOZ and SEM. The illegal use of nitrofuran in aquaculture has long existed.
As the basis of quality assurance in the analysis process, reference materials are divided into pure reference materials and matrix reference materials. Traditional pure reference materials cannot be used to evaluate the effectiveness of analytical methods in a comprehensive and objective manner because they do not take into account the matrix effect during extraction and analysis and the efficiency of drug extraction from tissues. The matrix reference materials are an important tool to ensure the reliability of drug residue determination results due to the biological environment basically consistent with the tissue to be tested. However, the reference materials in aquatic
D e s crip tion
products for drug residue analysis are usually obtained by adding drugs directly to the matrix, with defects such as large matrix difference and different binding states of targets.
Invention Summary
The technical problem to be solved by the present invention is to provide a reference material with turbot muscle as matrix for AOZ residue analysis and its preparation method, wherein a certain turbot is given a medicated bath; part of furazolidone is left in the muscle tissue after absorption, accumulation and metabolism in the turbot, to obtain positive matrix material with AOZ residue.
The method for preparing reference material with turbot muscle as matrix for AOZ residue analysis includes the acquisition of matrix reference material, the preparation of matrix reference material samples, the uniformity verification of matrix reference material samples and the stability verification of matrix reference material samples, comprising the steps of:
(1) Acquisition of matrix reference material
Select a normal-sized healthy turbot, stop feeding 24 h before the medicated bath with the furazolidone initial concentration of 100-360 ng/mL for 2 h. Then take out the turbot and rinse the residual drug off the surface. Take the turbot muscle tissue samples, with the concentration of AOZ residues of 1.5~5 g/kg (3~10 times LOQ), and keep them at -20 °C to obtain the matrix reference material.
(2) Preparation of matrix reference material samples
The reference material with turbot muscle as matrix for AOZ residue analysis is obtained by homogenizing, freeze-drying, grinding, encapsulation and sterilization of the muscle tissue, with the content of AOZ residues of 6~20 g/kg.
Further, the homogenizing: the frozen turbot muscle tissue samples are homogenized by a meat mincer;
The freeze-drying: the homogenized reference material samples are pre-frozen in a refrigerator at -20°C, and then placed in a lyophilizer for freeze-drying;
The grinding and mixing: the lyophilized samples are ground with a grinder, pass through the -mesh sieve plate, and then further mixed.
D e s crip tion
The encapsulation and sterilization: brown glass bottles (with a sealed inner cap) are used for sample encapsulation, during which the samples are protected by filling nitrogen inside, sealed with caps and vacuum-sealed with aluminum bags. The lyophilized turbot muscle samples are sterilized by irradiation.
(3) Uniformity verification of matrix reference material samples
The packaging units are randomly selected in the early, middle and late stages of the whole encapsulation process. The random samples are numbered, and 3 duplicate samples are taken from each randomly selected unit to determine the AOZ residue in the lyophilized turbot muscle. The results are statistically analyzed by ANOVA, and the sample uniformity is determined by comparing the F test value with F critical value.
(4) Stability verification of matrix reference material samples
Stability verification includes long-term stability and short-term stability verification;
Long-term stability: long-term stability is tested at the 0, 1st, 2nd, f4th and 6th month, respectively. Three packaging units are randomly selected each time, and three parallel samples are measured for each unit. The average value of the measured results of the three packaging units is taken as the long-term stability test result, and the results are statistically analyzed.
Short-term stability: the random samples are stored in thermostats at -20 °C, 4 °C and 25 °C, respectively. Short-term stability is tested on the 0, 3rd, 6th, 9th, 12th and 15th day, respectively, and the results are statistically analyzed.
(5) The highly uniform and stable samples after passing the tests in step (3) and step (4) are the reference material for AOZ residue analysis.
The invention also provides a reference material for AOZ residue analysis prepared by the above method.
The invention also provides the application of the reference material for AOZ residue analysis prepared by the above method.
Compared with the prior art, the invention has the following beneficial effects: In the invention, a certain turbot is given a medicated bath. Positive matrix material with drug residue is obtained after absorption, enrichment and metabolism of the drug in the turbot, which is
D e s crip tion
highly reliable. The reference material is closer to the actual test sample. Therefore, it is more scientific and accurate to evaluate the testing methods, spot the differences between laboratories and verify the accuracy of operators' results.
Detailed Description of the Presently Embodiments Unless otherwise specified, the experiment methods hereinafter are all conventional in the art.
Unless otherwise specified, the materials and reagents hereinafter are all commercially available.
Embodiment 1. Preparation of reference material with turbot muscle as matrix for AOZ residue analysis
Follow the following steps to prepare reference material with turbot muscle as matrix for AOZ residue analysis:
(1) Acquisition of matrix reference material
Turbots, weighing 75050 g/piece, are kept in a mariculture system for two weeks under the following conditions: temperature: 16~18 °C, salinity: 31.7%o, dissolved oxygen: 4.7 mg/L, PH: 8.1, with circulating water, continuous air. Stop feeding 24 hours before the medicated bath.
Give furazolidone medicated bath with the initial concentration of 100 ng/mL for 2 h. then take out the turbot, and rinse any residual drugs off the surface with running water. Take the turbot muscle tissue samples with the concentration of AOZ residues of 1.75 g/kg (3.5 times LOQ), and keep them at -20 °C to obtain the matrix reference material.
In the above detection method, the LOQ is 0.5 g/kg as stipulated in the Determinationof the Metabolites of Nitrofuran Antibiotics Residues in Aquatic Products LC-MS/MS (ANNOUNCEMENT No. 783 -1-2006) of the Ministry of Agriculture of the People's Republic of China.
(2) Preparation of matrix reference material samples
The reference material with turbot muscle as matrix for AOZ residue analysis is obtained by homogenizing, freeze-drying, grinding, encapsulation and sterilization of the muscle tissue, with the content of AOZ residues of 7.0 [g/kg.
D e s crip tion
Homogenizing: the frozen turbot muscle tissue samples are thawed at room temperature. In order to facilitate the sample crushing, the turbot muscle tissue is cut into pieces of approximately 1 cm3 and homogenized by a meat mincer.
The freeze-drying: the homogenized reference material samples are spread evenly in a clean freeze-drying tray, pre-frozen in a refrigerator at -20°C, and then placed in a lyophilizer for freeze-drying of 48 h;
Grinding and mixing: the samples from the freeze-drying tray are crushed into pieces of 5 cm3
, ground with a grinder, and pass through 40 mesh sieve plate. Collect the freeze-dried muscle and mix the samples further.
Encapsulation and sterilization: brown glass bottles with sealed inner caps are used for sample encapsulation. For each packaging unit, 5 g freeze-dried powder is provided per bottle, a total of 500 bottles. Before encapsulation, each sample bottle is washed by acid, secondary water, and high-purity water, and then dried at 65 °C, and cooled to room temperature for encapsulation. In the encapsulation process, the samples are protected by filling nitrogen inside, sealed with caps and vacuum-sealed with aluminum bags. The freeze-dried turbot muscle samples are sterilized by r-ray irradiation of radioactive isotope Co with the radiation dose of 5.5 kGy for 4 h.
(3) Uniformity verification of matrix reference material samples
Twenty packaging units are randomly selected in the early, middle and late stages of the whole encapsulation process. The random samples are numbered from 1 to 20. Three parallel samples are taken for each randomly selected unit, and are number as 1-1, 1-2, 1-3, 2-1,2-2, ... , 20-1, 20-2, 20-3 to determine the AOZ residue in freeze-dried turbot. The results are statistically analyzed by ANOVA, and the sample uniformity is determined by comparing the F test value with F critical value. Table 1 shows the uniformity test results of the characteristic values of AOZ residues in freeze-dried turbot muscle.
Table 1 Uniformity Test Results of the Matrix Reference Material of Freeze-dried Turbot Muscle (pg/kg)
2 3 Average No. 1 value 1 6.89 6.97 7.16 7.01 2 6.92 7.07 7.24 7.08 3 7.26 7.14 6.97 7.12 4 7.17 7.04 6.88 7.03
D e s crip tion
5 6.72 6.86 7.02 6.87 6 7.25 7.12 6.92 7.10 7 6.92 7.24 7.14 7.10 8 7.07 6.76 6.89 6.91 9 6.84 6.93 7.14 6.97 10 7.09 6.95 7.29 7.11 11 6.93 7.12 7.24 7.10 12 7.13 6.96 6.87 6.99 13 6.94 7.05 6.82 6.94 14 6.72 6.93 7.06 6.90 15 7.24 7.03 6.95 7.07 16 6.76 7.04 6.86 6.89 17 7.26 7.08 6.91 7.08 18 6.72 6.89 7.04 6.88 19 7.08 6.97 7.01 7.02 20 7.01 7.12 6.95 7.03 Total average 7.01 value Total standard 0.15 deviation 2 0.02242
2s 0.02176 F 1.03 Fa.0 5(19, 40) 1.84 Conclusion F<Fa (v1, v2), raw material samples are uniform
The above data show that the characteristic values of AOZ residues in the freeze-dried turbot muscle have passed F test, indicating that the matrix reference materials are highly uniform.
(4) Stability verification of matrix reference material samples
Stability verification includes long-term stability and short-term stability verification;
Long-term stability: long-term stability is tested at the 0, 1st, 2nd, 4th and 6th month, respectively. Three packaging units are randomly selected each time, and three parallel samples are
D e s crip tion
measured for each unit. The average value of the measured results of the three packaging units is taken as the long-term stability test result, and the results are statistically analyzed, see Table 2.
Table 2 Long-term Stability Test Results (pg/kg) Time (month) 0 1 2 4 6 Bottle 1 6.94 7.09 6.95 7.17 6.94 2 7.15 6.81 7.21 7.02 6.76 3 6.72 6.92 6.83 6.87 7.11 Average value 6.94 6.94 7.00 7.02 6.94 Standard 0.22 0.14 0.19 0.15 0.18 deviation s bi 0.003276 bo 6.9576 S2 S2 0.001944 s(bi) 0.02894 to.9sn-2-sbi) 0.09203 Conclusion Ibil< t.95,n-2-s(bi), stable
The long-term stability results indicate that the reference material candidates for the analysis of AOZ residues in freeze-dried turbot have stable and reliable characteristic values when stored at -20 °C.
Short-term stability: the random samples are stored in thermostats at -20 °C, 4 °C and 25 °C, respectively. Short-term stability is tested on the 0, 3rd, 6th, 9th, 12th and 15th day, respectively, and the results are statistically analyzed, see Table 3.
Table 3 Short-term Stability Monitoring Results of Reference Materials (pg/kg) Time (d) -20°C 4°C 25°C 0 7.01 6.97 7.05 3 6.94 7.01 6.96 6 7.09 6.85 6.82 9 6.97 6.94 6.79 12 6.95 6.84 6.81 15 7.06 6.98 6.87 bi 0.001524 -0.00352 -0.01314
D e s crip tion
bo 6.9919 6.9581 6.9819 s2 0.004592 0.005782 0.006182 s(bi) 0.0054 0.006059 0.006265 to.95,n-2-s(bi) 0.01501 0.01684 0.01742
Conclusion b 1 < to.9 5 ,.-2-s(bi) b l< to.9 5 ,n-2-s(bi) I 1 bIl< to.9 5 ,n-2-s(bi) Stable Stable Stable
The short-term stability results show that the AOZ may remain unchanged for 15 days at ambient temperatures of -20 °C, 4 °C and 25 °C if transported in ice bags.
The reference materials in turbot muscle for AOZ residue analysis prepared according to this embodiment may be used for proficiency testing in the quality and safety of aquatic products, internal quality controlling in laboratory, inter-laboratory comparison and assessment, as well as comparison and verification of different detection methods, and assessment of different operators.
Claims (6)
1. A method for preparing reference material with turbot muscle as matrix for AOZ residue analysis, wherein a certain turbot is given a medicated bath; part of furazolidone is left in the muscle tissue after absorption, accumulation and metabolism in the turbot, to obtain positive matrix material with AOZ residue; matrix reference material samples are prepared by homogenizing, freeze-drying, grinding, encapsulation and sterilization of the positive matrix material, and are verified for their uniformity and stability.
2. A method for preparing reference material with turbot muscle as matrix for AOZ residue analysis as described in claim 1, wherein the method includes the acquisition of matrix reference material, the preparation of matrix reference material samples, the uniformity verification of matrix reference material samples and the stability verification of matrix reference material samples, comprising the steps of:
(1) Acquisition of matrix reference material
Select a normal-sized healthy turbot, stop feeding 24 h before the furazolidone medicated bath with the initial concentration of 100-360 ng/mL for 2 h. Then take out the turbot and rinse the residual drug off the surface. Take the turbot muscle tissue samples, with the concentration of AOZ residues of 1.5-5 [g/kg, and keep them at -20 °C to obtain the matrix reference material.
(2) Preparation of matrix reference material samples
The reference material with turbot muscle as matrix for AOZ residue analysis is obtained by homogenizing, freeze-drying, grinding, encapsulation and sterilization of the muscle tissue, with the content of AOZ residues of 620 g/kg.
(3) Uniformity verification of reference material samples
The packaging units are randomly selected in the early, middle and late stages of the whole encapsulation process. The random samples are numbered, and 3 duplicate samples are taken from each randomly selected unit to determine the AOZ residue in the freeze-drying turbot muscle. The results are statistically analyzed by ANOVA, and the sample uniformity is determined by comparing the F test value with F critical value.
(4) Stability verification of matrix reference material samples
Stability verification includes long-term stability and short-term stability verification;
C la i m s
Long-term stability: long-term stability is tested at the 0, 1st, 2nd, 4th and 6th month, respectively. Three packaging units are randomly selected each time, and three parallel samples are measured for each unit. The average value of the measured results of the three packaging units is taken as the long-term stability test result, and the results are statistically analyzed.
Short-term stability: the random samples are stored in thermostats at -20 °C, 4 °C and 25 °C, respectively. Short-term stability is tested on the 0, 3rd, 6th, 9th, 12th and 15th day, respectively, and the results are statistically analyzed.
(5) The highly uniform and stable samples after passing the tests in step (3) and step (4) are the reference material for AOZ residue analysis.
3. The method according to claim 2, wherein in step (1), the initial concentration of furazolidone medicated bath is 100-360 ng/mL, and the medicated bath time is 2 h.
4. A method for preparing reference material with turbot muscle as matrix for AOZ residue analysis according to claim 2, wherein the homogenizing is such that the frozen turbot muscle tissue samples are homogenized by a meat mincer;
The freeze-drying: the homogenized reference material samples are pre-frozen in a refrigerator at -20 °C, and then placed in alyophilizer for freeze-drying;
The grinding and mixing: the freeze-drying samples are ground with a grinder, pass through the 40-mesh sieve plate, and then further mixed.
The encapsulation and sterilization: brown glass bottles (with a sealed inner cap) are used for sample encapsulation, during which the samples are protected by filling nitrogen inside, sealed with caps and vacuum-sealed with aluminum bags. The freeze-drying turbot muscle samples are sterilized by irradiation.
5. The reference material for AOZ residue analysis prepared according to the method described in claim 2.
6. Application of the reference material for AOZ residue analysis prepared according to the method described in claim 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112816283A (en) * | 2020-12-30 | 2021-05-18 | 国家烟草质量监督检验中心 | Preparation method of tobacco specific N-nitrosamine standard substance |
| CN113514293A (en) * | 2021-04-16 | 2021-10-19 | 四川省食品药品检验检测院 | Preparation method and application of recessive malachite green standard sample in snakehead meat powder |
| CN114487168A (en) * | 2021-12-29 | 2022-05-13 | 中国计量科学研究院 | Preparation and value determination method of blood lactic acid standard substance |
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