CN114414321A - Fipronil sulfone residue analysis standard substance candidate in egg powder and preparation method thereof - Google Patents
Fipronil sulfone residue analysis standard substance candidate in egg powder and preparation method thereof Download PDFInfo
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- CN114414321A CN114414321A CN202111458804.2A CN202111458804A CN114414321A CN 114414321 A CN114414321 A CN 114414321A CN 202111458804 A CN202111458804 A CN 202111458804A CN 114414321 A CN114414321 A CN 114414321A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
The invention discloses a preparation method of a fipronil sulfone residual analysis standard substance candidate in egg powder, which comprises the following steps: animal experiment, homogenizing raw materials, freeze-drying, grinding and sieving, uniformly mixing dry powder, primarily inspecting the dry powder, packaging products and sterilizing by irradiation. The invention also provides a fipronil sulfone residual analysis standard substance candidate in the egg powder prepared by the method. The preparation method can be used for mass production of fipronil sulfone residual analysis standard substance candidates in the egg powder, the raw materials are from natural egg samples, the target substance is tightly combined with the egg content matrix, the egg powder uniformity initial check is qualified, and the formed matrix standard substance product can better reflect the accuracy and comparability of related detection technologies when used for training assessment, capability verification and laboratory comparison. For some metabolites without standard reference substances, quality control samples close to actual samples can be obtained according to the technical route of the invention, and the problem of the bottleneck in the field of research of matrix standard substances is mainly solved.
Description
Technical Field
The invention relates to the field of food detection and processing, in particular to a fipronil sulfone residue analysis standard substance candidate in egg powder and a preparation method thereof.
Background
Fipronil is a phenylpyrazole pesticide and has been widely used in agriculture, veterinary medicine and other fields. However, fipronil is easy to accumulate in water, soil and crops due to repeated use, causes toxic action on some non-target organisms (bees, poultry, aquatic organisms and the like), and affects human bodies through food chains. Researches show that after the laying hens take the feed polluted by fipronil, most fipronil is converted into fipronil sulfone and is excreted into eggs. Fipronil sulfone is a main metabolite of fipronil in laying hens and is also a residual marker of fipronil in eggs.
GB 2763 and 2019, maximum limit of pesticide residue in national food Standard for food safety stipulates that fipronil residues are as follows: fipronil, fluoronitrile, fipronil sulfone, fipronil sulfoxide, expressed as fipronil in total; the maximum residual limit of fipronil in eggs is 20 mug/kg. The pharmacokinetic experiment shows that the fluoronitrile and fipronil sulfoxide are rarely detected in the eggs; even if fipronil is detected, the content of fipronil is mostly lower than that of fipronil sulfone; furthermore, when fipronil is not detectable in the eggs, there is still a possibility of higher residual fipronil sulfone.
Fipronil sulfone is much more toxic than fipronil and ingestion of large amounts can damage the liver, thyroid, kidney, and reproductive and nervous systems of the human body. Its chemical property is similar to fipronil, it is easily dissolved in organic solvent of acetonitrile and acetone, etc., and is easily negatively charged, and its molecular formula is C12H4Cl2F6N4O2S, the chemical structural formula is as follows:
the effective safety evaluation system is beneficial to ensuring the safety of agricultural products and food. The standard substance is a measurement standard in a physical form with the main characteristics of uniformity, stability, accuracy and the like of characteristic quantity values, and is an important support for guaranteeing the accuracy and reliability of an analysis and detection system. The matrix standard substance is a standard substance with actual sample characteristics, is mainly used for analytical method evaluation, operator level assessment, detection process quality monitoring and the like, and is a reference material for capability verification, assessment and evaluation of a detection mechanism. The standard substance candidate is a product form which is prepared by a special production and processing technology and is not endowed with a standard value and uncertainty in order to meet the characteristic requirements of uniformity and stability of the standard substance and the requirements of actual detection work, and is a substance circulated in the standard substance market. The standardized preparation method of the standard substance candidate is beneficial to ensuring the continuous and stable supply of the standard substance product, thereby effectively meeting the requirements of detection and quality control.
Through retrieval, no method system and technical specifications for preparing fipronil sulfone residue analysis standard substance candidates in egg powder through animal experiments and special processing technology are available at home and abroad.
Disclosure of Invention
The invention provides a fipronil sulfone residue analysis standard substance candidate in egg powder.
The invention particularly provides a preparation method of a fipronil sulfone residual analysis standard substance candidate in egg powder, which comprises the following steps:
(1) animal experiments: the method comprises the following steps of (1) screening an egg liquid raw material containing 15-25 mug/kg of fipronil sulfone through a pharmacokinetic test of fipronil in a laying hen by taking the limit of fipronil and metabolite residues thereof in eggs as a target, wherein the limit of fipronil residues is 20 mug/kg;
(2) homogenizing raw materials: stirring the egg liquid raw material obtained by screening uniformly;
(3) and (3) freeze drying: pre-freezing and vacuum freeze-drying the uniformly stirred egg liquid raw material in batches to constant weight;
(4) grinding and sieving: crushing and sieving the freeze-dried sample until the grain size of the egg powder is not more than 40 meshes;
(5) and (3) uniformly mixing dry powder: mixing all the collected sieved egg powder, stirring and uniformly mixing;
(6) primary detection of dry powder: performing primary uniformity detection on the uniformly mixed egg powder;
(7) and (3) product packaging: subpackaging the egg powder qualified by the primary uniformity inspection into sample bottles, and then carrying out vacuum-pumping packaging;
(8) irradiation sterilization: and sterilizing the packaged product to obtain a fipronil sulfone residue analysis standard substance candidate in the egg powder, and storing at low temperature.
The technical scheme is as follows:
a preparation method of a fipronil sulfone residual analysis standard substance candidate in egg powder comprises the following steps:
(1) and (5) animal experiments. By taking the residue limit (20 mug/kg) of fipronil and metabolites thereof in eggs as targets, the egg liquid raw material containing 15-25 mug/kg fipronil sulfone is screened through a pharmacokinetic test of fipronil in laying hens. The raw materials are screened near the target content, so that the homogenization treatment of the later processing is facilitated, the minimum sampling amount is reduced, and the final product is more consistent with a natural sample.
The pharmacokinetics test is that fipronil is filled into a medicinal capsule according to the required dosage, the laying hen is orally taken into the capsule and is administrated at one time, the administration dosage is 0.005-2 mg/egg, and the test period is 28 days. Collecting eggs every day, measuring the content of fipronil sulfone in each group of egg liquid every day, and drawing a residue elimination curve. According to the residual situation of fipronil sulfone at each time point after different dosages are administrated, egg liquid samples with the content closer to the target content of 20 mug/kg are screened.
Preferably, the fipronil is administrated to healthy 24-week-old 'Dawangjinfeng' laying hens with the average body weight of 1.46 kg: 0.005, 0.01 and 0.1 mg/piece. When the dosage is 1mg or 2mg, the fipronil sulfone content in the egg liquid is always far higher than 25 mug/kg from the 2 nd to the 28 th day after the administration.
Preferably, when the dosage of the egg liquid raw material containing 15-25 mug/kg fipronil sulfone is 0.005 mg/egg, the egg collection time is 6 days and 7 days after administration; when the dosage is 0.01 mg/egg, the egg collection time is 4-10 days after administration; when the dosage is 0.1 mg/egg, the egg collection time is 2 days and 17-22 days after administration. The use amount of the laying hens can be reduced through a mode of collecting a plurality of doses for a plurality of days, and the cost of raw materials is reduced.
(2) And (4) homogenizing the raw materials. And (3) placing the egg liquid raw material obtained by screening in the step (1) into a clean stainless steel drum, and uniformly stirring at a fixed rotating speed by using a commercial electric eggbeater.
Preferably, the parameters of the commercial electric eggbeater are as follows: the double-shaft has the capacity of 15L, the power of 200W and the rotating speed of 467 r/min.
And (3) uniformly stirring, namely extracting 3 parts of egg liquid samples from the upper part, the middle part and the lower part of a stainless steel drum respectively, detecting, and judging that the raw materials are basically and uniformly mixed when the fipronil sulfone content is 17.0-20.5 mu g/kg.
Preferably, the material slurry is stirred, cooled, defoamed and stirred at an ambient temperature of 20. + -. 2 ℃ and a humidity of 40. + -. 10% RH. Stirring for 30min, cooling and defoaming in a refrigerator at-80 deg.C for 30min, and stirring for 2 hr. Because the egg liquid has rich nutrition and overlong stirring time, the protein is easy to denature. In addition, the stirring process is easy to foam, the volume is increased, and the freeze drying is not facilitated. Freezing at-80 deg.C for 30min to remove foam and reduce microbial contamination of egg liquid.
(3) And (5) freeze drying. And (3) packaging the egg liquid raw material uniformly stirred in the step (2) into a clean stainless steel disc, packaging the egg liquid raw material by using aluminum foil paper with small holes, and pre-freezing the egg liquid raw material in a refrigerator at the temperature of-80 ℃. Then put into a freeze dryer in batches, and freeze-dried under vacuum at the temperature of minus 80 ℃ to constant weight. Each batch of egg liquid is dried to constant weight for 6 days. The raw material of the egg liquid is frozen and dried, the average dehydration rate is 75 percent, namely 1.25g of egg powder is added with 3.75g of water, and the egg liquid can be recovered into 5g of egg liquid which is directly used for quality control of national standard method (GB23200.115-2018) detection.
Preferably, the parameters of the freeze dryer are as follows: the water catching capacity is 6kg/24h, the power is 950W, and the freeze-drying area is 0.18-0.27 m2Degree of vacuum of<10Pa。
The constant weight refers to that when the sample is frozen and dried for 6 days, the sample is weighed in a tray, continuously frozen and dried overnight (16 hours), and weighed again, wherein the weight difference between the two times is less than 0.1 percent, and the sample is regarded as the constant weight.
Preferably, the vacuum freeze drying technology is adopted to remove the water in the egg liquid, the dehydration rate is required to reach 75%, the water content of the final egg powder product is not higher than 1.5%, and the product is always in a loose state in a sealed state and cannot be agglomerated. The vacuum freeze drying technology can keep the basic shape of the product, ensure that nutrient substances, vitamins and the like are not damaged, and after the product is restored by adding water, the color, the taste, the material structure and the like of the product are basically the same as those of the product which is fresh before. Meanwhile, the technology can lower the water content of the product, inhibit the growth and proliferation of microorganisms and improve the stability of the product.
Preferably, the egg liquid is recovered, namely, the egg powder is accurately weighed, purified water is accurately added according to the ratio of 1:3(w/w), vortex oscillation is carried out for 1min, ultrasonic treatment is carried out for 10min, and water bath is carried out at 30 ℃ in a dark place for 1h, so that the egg liquid after homogenization is recovered. The content of fipronil sulfone in the product is finally counted by egg powder. The adding amount of the purified water is 3 times of the egg powder, so that the recovered egg liquid is almost the same as the actual sample. The sampling amount of each egg powder is recommended to be 1.25g, and the excessive sampling amount can increase the burden of sample extraction and purification and influence the repeatability of results. The water bath is beneficial to the egg liquid to be fully recovered, but the water bath time is not more than 2 hours because fipronil and the metabolite thereof are in the risk of slow degradation under the illumination of light in the water solution.
(4) Grinding and sieving. And (4) placing the honeycomb-shaped egg sample subjected to freeze drying in the step (3) into a large-scale crusher, and crushing at a high speed for 30 s. And (4) screening the crushed sample by a 40-mesh sieve and collecting. And pouring the sample which is not sieved into a small-sized pulverizer to pulverize for 30 seconds again, sieving the sample with a 40-mesh sieve again, and collecting the sample again. The grinding and sieving loss is controlled within 2 percent. The particle size of the final product is not larger than 40 meshes.
Preferably, the parameters of the large-scale crusher are 4500g of capacity, 550W of rated power and 50HZ of frequency. The parameters of the small-sized pulverizer are as follows: the capacity is 500g, the rated power is 500W, and the frequency is 50 HZ.
Preferably, the environmental temperature is 20 plus or minus 2 ℃ and the humidity is less than 30 percent RH when the powder is ground and sieved, so as to prevent the product from absorbing moisture and deteriorating.
(5) Mixing the dry powder. And (4) pouring all the sieved egg powder collected in the step (4) into a mixing machine, mixing and stirring for 16 hours, and fully and uniformly mixing.
Preferably, the parameters of the mixer are as follows: the capacity is 8kg, the rotating speed of the mixing barrel is 0-26 r/min, the rotating speed of the stirring shaft is 0-100 r/min, the power of the mixing barrel is 40W, the power of the stirring shaft is 25W, the working mode is V-shaped convection mixing, and automatic circulation positive and negative rotation is realized.
Preferably, the rotation speed of the mixing barrel is set to be 18r/min, the rotation speed of the stirring shaft is set to be 36r/min, and a mode of forward rotation for 1h and reverse rotation for 1h is adopted.
Preferably, the environment temperature is 20 plus or minus 2 ℃ and the humidity is less than 30 percent RH in the process of uniformly mixing so as to prevent the product from absorbing moisture and deteriorating.
(6) And (5) performing primary detection on the dry powder. And (4) sampling the egg powder uniformly mixed in the step (5) on an upper sampling layer and a lower sampling layer according to a trisection method, wherein 6 parts of samples are sampled in total, and performing primary uniformity detection. The initial inspection is qualified, and the product can be subpackaged.
And (3) the initial detection is qualified, namely the fipronil sulfone content detection is carried out on 6 extracted samples, and each sample is parallelly detected for 3 times. And (4) carrying out statistical analysis on the detection result by adopting a one-factor analysis of variance method (F-test method). Is formed by F <αAnd P-value is more than 0.05, and the initial detection of the uniformity of the standard substance candidate is judged to be qualified.
Preferably, the initial uniformity test is performed with a sample size of 1.25 g. In a minimum sampling amount test, the sampling amount of 0.50-1.50 g can meet the repeatability requirement of the detection method, and the product is proved to be sufficiently uniform. However, in order to recover 5g of egg liquid and meet the requirement of quality control in the national standard method, the minimum sampling amount of the standard substance candidate is set to 1.25 g. And finally, analyzing fipronil sulfone residues in the egg powder formed by the candidate to obtain a standard substance product, wherein the standard value and the uncertainty are results obtained under the condition of minimum sample quantity.
(7) And (6) packaging the product. And (4) pouring the egg powder qualified by the uniformity primary inspection in the step (6) into a clean computer intelligent packaging machine, and automatically packaging into sample bottles through vibration and infrared induction. Then vacuum packaging is carried out by a vacuum packaging machine.
The parameters of the intelligent racking machine are as follows: the rated power is 180W, the subpackaging speed is 4-8 s/bottle, the weighing range is 1-30 g, and the error range is 0.1-0.2 g.
The parameters of the vacuum packaging machine are as follows: the packaging speed is 1-3 bags/min, the power is 120W, and the size of the vacuum chamber is 330 multiplied by 275 multiplied by 50 mm.
Preferably, the system is set to have a specification of 10.5g for intelligent split charging, so that the net weight of each bottle of egg powder is more than 10 g. About 8 samples of each bottle of product can be weighed for detection and quality control based on 1.25g of each sample to be detected.
The used sample bottle needs to be tested to have good sealing performance, and the material of the sample bottle has no influence on the product.
Preferably, the used sample bottle is an acrylic bottle, the volume of the sample bottle is 60mL, the inner container is silver, the sample bottle is completely shielded from light, each bottle is provided with a pressure-sensitive gasket, and the sealing performance is good. The packaging bottle has no adsorption or degradation effect on fipronil sulfone through inspection; can be stored in a refrigerator at-20 deg.C for a long time without frost cracking.
Preferably, during vacuum packaging, the mouth of the sample bottle is filled into an aluminum foil bag, air in the bottle is removed through vacuumizing, and the gasket can be further compressed through reverse vacuumizing, so that air infiltration is avoided, and the stability of the product is improved.
(8) And (5) performing irradiation sterilization. And (4) sterilizing the packaged product in the step (7) to obtain a fipronil sulfone residue analysis standard substance candidate in the egg powder, and storing the candidate in a refrigerator at the temperature of-20 ℃ for a long time. The eggs are rich in nutrition and are easy to pollute microorganisms in the production and processing processes. The sterilization treatment is to thoroughly eliminate the egg powder deterioration caused by microbial contamination, thereby prolonging the shelf life of the product.
Preferably, it is used60Co irradiation for sterilizationAnd (6) processing. In contrast to other sterilization processes, the sterilization process,60co irradiation sterilization is more thorough, unsealing is not needed, the penetrating power is stronger, the economy is higher, the treatment mode is reliable and efficient, the processing confirmation is simple, no harmful substance is left under a certain dosage, sterilization post-treatment is not needed, and the operation is convenient and rapid.
Said60Co irradiation is carried out, the irradiation dose is 500-2000 Gy, the irradiation dose is too low, and the sterilization is not thorough; the irradiation dose is too high, and the product is easy to deteriorate. The environmental temperature is 22 +/-2 ℃ and the humidity is less than 37 percent RH during irradiation.
As a preference, the first and second liquid crystal compositions are,60the Co irradiation dose is 1000Gy, and the actual measurement value is 959-1009 Gy.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fipronil sulfone residue analysis standard substance candidate in the egg powder provided by the invention is the product form of the currently unique fipronil sulfone residue analysis standard substance in the egg powder at home, and the standard substance product formed by the method finally determines the standard value and the uncertainty through uniformity test, stability test and 6 laboratory combined value setting, and meets the national standard substance evaluation requirement.
(2) The fipronil sulfone residue analysis standard substance candidate in the egg powder provided by the invention relates to a metabolite of fipronil which is metabolized by the egg body and remained in the egg, and is tightly combined with the egg content matrix, and the obtained egg liquid raw material containing the fipronil sulfone belongs to a natural sample and is completely different from an artificial labeling sample; through the process of adding water for restoration, the matrix standard substance candidate can be reduced into egg liquid containing fipronil sulfone, is basically consistent with a natural sample, and can better reflect the accuracy and comparability of related detection technologies when being used for training assessment, capability verification and comparison among laboratories.
(3) The preparation method of the fipronil sulfone residual analysis standard substance candidate in the egg powder comprises a whole set of technical process and related technical parameters, and can be used for mass production of fipronil sulfone residual analysis standard substance products in the egg powder; aiming at carrying out animal experiments and obtaining positive samples of a residual limited target, product processing is carried out around the requirements of uniformity and stability of a standard substance, and the problems that the obtaining of the positive samples is uncontrollable and the uniformity and the stability are difficult to guarantee in the detection and quality control process are solved.
(4) The invention has technical particularity on key control points of the processing process of the matrix standard substance candidate, has innovativeness on standardizing process flow and product result, and has guiding significance and application prospect in the research and production of other animal-derived matrix standard substance products. Especially for metabolites without standard reference substances, the quality control sample close to the actual sample can be obtained according to the technical route of the invention, and the problem of the bottleneck in the field of research of matrix standard substances is mainly solved.
Drawings
FIG. 1 is a flow chart of the preparation of fipronil sulfone residual analysis standard substance candidates in egg powder.
FIG. 2 is a curve showing the residual elimination of fipronil sulfone in egg liquid after administration of fipronil at different dosages, wherein the dosage in graph A is 0.005, 0.01, 0.1 mg/egg, and the dosage in graph B is 1, 2 mg/egg.
FIG. 3 shows the chemical structure of fipronil sulfone stable isotope internal standard.
Detailed Description
The invention is further elucidated with reference to the figures and the examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of matrix Standard substance candidates
1.1 obtaining raw egg liquid
53 egg samples were randomly drawn from Hangzhou farmer markets and supermarkets, and the related information is shown in Table 1. All samples do not contain fipronil and its metabolites (fipronil sulfone, fipronil sulfoxide and fluoroform) according to GB23200.115-2018 & determination of residual quantity of fipronil and its metabolites in eggs of national food safety Standard liquid chromatography-mass spectrometry combined usage. Therefore, the positive egg samples are obtained in a sampling mode, the controllability is poor, and the quantity and the target content of the positive samples often cannot meet the requirements of batch product preparation scale and detection quality control.
TABLE 1 market spot check egg source information
The invention adopts animal pharmacokinetic experiments to obtain a positive sample containing a target analyte, establishes a production technical specification and is beneficial to the continuous and stable supply of animal-derived matrix standard substance products.
As shown in fig. 1, the preparation method of the standard substance candidate for analyzing fipronil sulfone residue in egg powder provided by the invention comprises the following technical routes: animal experiment, homogenizing raw materials, freeze-drying, grinding and sieving, uniformly mixing dry powder, primarily inspecting the dry powder, packaging products and sterilizing by irradiation.
In the embodiment, the egg liquid raw material with the fipronil sulfone content close to 20 mug/kg is obtained through a pharmacokinetic experiment by taking the residual limit as a screening target.
The specific method comprises the following steps: selecting 60 healthy 24-week-old and similar-physique 'grand gold phoenix' laying hens with the average weight of 1.46kg, adopting 3-layer full-step cage rearing, randomly dividing the laying hens into 3 groups, and each group comprises 20 laying hens; filling fipronil into medicinal capsule according to required dosage, and making the laying hen orally take the medicinal capsule. The dose for each group was: 0.005, 0.01, 0.1, 1 and 2 mg/piece, and the test period is 28 d; collecting eggs every day, cleaning, wiping, beating eggs in groups, bottling with clean food-grade plastic samples, and storing in a refrigerator at-20 deg.C; returning to room temperature before use, fully homogenizing, synchronously measuring the content of fipronil sulfone in each group of egg liquid every day, and drawing to obtain a residue elimination curve shown in figure 2. When the dosage is 1mg or 2mg, the fipronil sulfone content in the egg liquid is always far higher than 25 mug/kg from the 2 nd to the 28 th day after the administration.
Under the effective administration dosage (0.005, 0.01 and 0.1 mg/egg), the egg liquid raw material containing 15-25 mug/kg fipronil sulfone can be obtained in a specific collection time. As can be seen from Table 2, when the dosage is 0.005 mg/egg, the egg collection time is 6 and 7 days after administration; when the dosage is 0.01 mg/egg, the egg collection time is 4-10 days after administration; when the dosage is 0.1 mg/egg, the egg collection time is 2 days and 17-22 days after administration. The law of eliminating the residual metabolites can be known through animal experiments, and the mode of collecting multiple doses for multiple days can obtain enough amount of various target residual egg liquid raw materials under the condition of using less laying hens.
TABLE 2 administration dose, acquisition time and corresponding content determined by animal experiments
1.2 determination of the recovery mode of egg powder
In order to facilitate storage and prolong the validity period, the free water in the egg liquid is removed by adopting a vacuum freeze drying homogenization technology to prepare the egg powder. The specific operation is as follows: the homogenized egg liquid raw material is packaged in a clean stainless steel disc, the upper surface of the disc is packaged by aluminum foil paper with small holes, and the disc is pre-frozen in a refrigerator at the temperature of minus 80 ℃. Then put into a Creatrust freeze dryer in batches and vacuum freeze-dried at-80 ℃. In this example, each batch of egg liquid was dried to a constant weight for 6 days. Before and after drying, the weight was taken and the dehydration rate is shown in Table 3. The average dehydration rate was calculated to be 75%. When the product is used, 1.25g of sample is accurately weighed, 3.75g of purified water is accurately added, vortex oscillation is carried out for 1min, ultrasound is carried out for 10min, and water bath is carried out in a dark place at 30 ℃ for 1h, so that 5g of egg liquid can be recovered, and the egg liquid is basically consistent with a homogenized natural sample.
TABLE 3 detection results of the vacuum freeze-drying dehydration rate of egg liquid
In this embodiment, after the water is added to the egg powder, the recovery of the egg liquid is promoted by means of the auxiliary oscillation, ultrasonic and water bath. The difference between the immediate extraction of 6 samples after ultrasonic treatment in a dark water bath at 30 ℃ for 1h and the random extraction within 0-2 h after standing at room temperature is compared. Detecting the restored egg liquid according to GB23200.115-2018, performing t test on all measured values between groups, wherein the p value is more than 0.05, and indicating that the residue detection of fipronil sulfone in the egg powder does not have significant difference by water bath operation and non-water bath treatment during restoration. But the addition of the water bath operation can better promote the redissolution of the egg powder, and the repeatability of the detection result is relatively good. Therefore, the invention adopts a mode of adding water, oscillating, carrying out ultrasonic treatment, and carrying out water bath in a dark place at 30 ℃ for 1h to fully recover the egg powder into egg liquid, and then the egg liquid is used for detection and quality control.
1.3 analysis of Key control points
And (4) screening, homogenizing, freeze-drying, grinding and screening the egg liquid to obtain egg powder, mixing uniformly again, and performing primary uniformity inspection. And only the egg powder raw materials which pass the primary uniformity inspection can be packaged, and if the egg powder raw materials do not pass the primary uniformity inspection, the mixing is continued. The specific operation is as follows: sampling 6 parts of samples in the upper and lower sampling layers of the mixer container according to a trisection method, wherein the sampling quantity of each sample is 1.25g, and parallel detecting for 3 times according to GB 23200.115-2018. The results of the tests of this example are shown in Table 4. And (4) carrying out statistical analysis on the detection result by adopting a one-factor analysis of variance method (F-test method). As can be seen from Table 5, F < FαAnd the P-value is more than 0.05, and the initial inspection of the uniformity of the egg powder raw material is judged to be qualified.
Table 4 preliminary examination result of uniformity of egg powder raw material
TABLE 5 egg powder raw material homogeneity evaluation-one-way ANOVA
Remarking: SS is sum of squared deviations, MS is mean square, df is degree of freedom, F is statistic, F isαIs a critical statistic at the alpha level and P-value is a value that characterizes significance.
When the sampling amount is too small, the characteristic quantity of the substance is easy to generate a non-uniform phenomenon; when the sampling amount is too large, the waste of the sample is caused, and the burden is caused to the extraction and the subsequent purification.
The minimum sampling amount test is carried out in the embodiment, and the specific operations are as follows: 6 portions of egg powder samples are taken according to different sampling quantities respectively and detected according to GB23200.115-2018, and the results are shown in Table 6. T test is carried out on all the measured values among groups, the p value is greater than 0.05, and the result shows that the influence of 0.50 g-1.50 g of sample quantity on the detection result of fipronil sulfone is not obvious. From the repeatability of the measured values, the RSD values were very small and less than 2% when measured in 1g and 1.25g samples, respectively, indicating that relatively small uncertainty can be obtained when experiments are performed with these two samples. And the sampling amount of the egg liquid in the national standard method (GB23200.115-2018) is 5 g. In order to meet the requirement of national standard method for quality control, the minimum sampling amount of the standard substance candidate is set to 1.25 g. And finally, analyzing fipronil sulfone residues in the egg powder formed by the candidate to obtain a standard substance product, wherein the standard value and the uncertainty are results obtained under the condition of minimum sample quantity.
TABLE 6 detection results of fipronil sulfone as raw material of egg powder at different sampling amounts
The eggs are rich in nutrition and are easy to pollute microorganisms in the production and processing processes. The invention adopts60Co irradiation sterilization is carried out to avoid the deterioration of the product during the storage and transportation processes.
This example compares the effect of irradiation dose on egg powder. The specific operation is as follows: 12 bottles of the product were randomly sampled, of which 9 bottles were equally divided into 3 groups as irradiation groups, and were subjected to irradiation sterilization at doses of 500Gy, 1000Gy, and 2000Gy, respectively, and the other 3 bottles were used as control groups. Total colonies were determined according to GB 4789.2-2016. 1g of the sample was weighed under sterile conditions, diluted with 10mL of physiological saline, 0.1mL of the coated PCA plate was incubated at 37 ℃ for 2 days, observed and counted. As a result, it was found that the average bacteria content of the product was 2.25X 10 without irradiation4CFU/g; after the irradiation treatment of 500Gy, the average bacteria content of the product is 200CFU/g, which is obviously reduced compared with a control group, but the sterilization is not thorough; the products were sterile after irradiation with 1000Gy and 2000Gy, respectively. Each group is composed of the upper layer and the lower layer of the sample bottleAnd 6 samples are extracted, and the content of fipronil sulfone is detected according to GB 23200.115-2018. T test between the groups is carried out on the measured values of the irradiation group and the control group, and the p values are all larger than 0.05, which shows that the irradiation dose has no significant influence on the quantity value of fipronil sulfone in the egg powder. However, the 2000Gy treatment may have an impact on the nutritional quality of the egg powder, as the yellow color of the product becomes lighter after irradiation of this dose and a smelly egg taste is slightly emitted. Therefore, the irradiation dose is preferably 1000Gy, the measured value is 959-1009 Gy, the irradiation environment temperature is 22 ℃ and the humidity is 37% RH. The product after irradiation is stored in a refrigerator at the temperature of 20 ℃ below zero for a long time.
Considering that a small amount of moisture may still exist in the egg powder when the egg powder is freeze-dried in vacuum to constant weight and the subsequent processing process may absorb moisture, the embodiment further detects the moisture of the final product: and 6 parts of vacuum-packaged egg powder is randomly extracted and detected by adopting a direct drying method in GB 5009.3-2016. The average water content of the egg powder is 1.5% and the relative standard deviation RSD value is 1.62%. Namely, under the corresponding state, the egg powder is in loose powder shape, and is frozen and stored in a refrigerator at the temperature of minus 20 ℃ after being packed in vacuum, so that the egg powder cannot be caked and the detection of the product cannot be influenced.
Since egg powder is easily hygroscopic and contaminated with microorganisms when exposed to air at room temperature, it is recommended that the product be disposable after opening. Each bottle of product should be returned to room temperature before use. 6 portions of egg powder test samples (each portion is 1.25g) are weighed at one time and placed in a clean 50mL centrifuge tube, after sealing, the centrifuge tube is immediately placed in a refrigerator at 20 ℃ below zero and kept in the dark, 2 portions of samples are respectively taken for detection in 0 day, 3 days and 7 days, the difference of the average value of 3 times of detection is small, the RSD value of 6 portions of samples is less than 2%, and the egg powder after unsealing is stored by the method, detected in one week and stable in value.
1.4 selection of detection methods
The invention adopts a national standard method for sample detection, wherein the national standard method is a stable isotope dilution liquid chromatography-mass spectrometry (ID-LC-MS/MS) combined method which is properly modified on the basis of GB 23200.115-2018. The ID-LC-MS/MS is rarely interfered by various physical and chemical factors, and can overcome the influence of matrix effect. It is stable in the same molecular structureAnd determining the isotope-labeled organic compound as an internal standard, and performing accurate mass spectrometry measurement of isotope abundance on the internal standard and the target object to convert so as to obtain the accurate content of the target object in the sample to be detected. In the experimental process, the ID-LC-MS/MS method measures not the concentration but the ratio of the molar quantity of the substance to be measured and the internal standard in the sample, so that the measurement can be directly traced back to a basic unit. The fipronil sulfone reference substance is national standard substance (GBW (E)084271), and is developed and produced by agricultural academy of sciences in Zhejiang province. Isotopic internal standard fipronil sulfone-13C6Provided by Shanghai' an spectrum, and the chemical structural formula is shown in figure 3.
In this embodiment, the instrument conditions and pretreatment method of the national standard method are optimized appropriately, and the results are as follows:
(1) sample pretreatment
Egg liquid recovery: the product was taken out of the refrigerator and returned to room temperature. Accurately weighing 1.25g egg powder (accurate to 0.0001g) and placing in a 50mL plastic centrifuge tube, accurately adding 3.75g water, vortex oscillating for 1min, performing ultrasound for 10min, and performing light-proof water bath at 30 deg.C for 1 h.
Extraction: accurately adding 50 mu L of fipronil sulfone-13C6And (3) placing the standard substance and 20mL of acetonitrile in a 50mL plastic centrifuge tube filled with the egg liquid to be detected, performing vortex oscillation for 1min, and performing ultrasonic treatment for 30 min. Continuously adding 2g of sodium chloride and 6g of anhydrous sodium sulfate, carrying out vortex oscillation for 1min, centrifuging at 5000r/min for 5min, and allowing the supernatant to be purified.
Purifying: accurately pipette 2mL of supernatant into a pre-filled 100mg PSA powder, 100mg C18And (3) in a 10mL plastic centrifuge tube containing the powder and 300mg of anhydrous magnesium sulfate, carrying out vortex oscillation for 1min, centrifuging for 5min at 5000r/min, filtering supernate through a 0.22 mu m microporous filter membrane, and waiting for determination on a machine.
(2) LC-MS/MS detection
Liquid chromatography conditions:
the instrument model is as follows: a Thermo Fisher UltiMate 3000 high performance liquid chromatograph;
a chromatographic column: ACE Excel 2C18Column (2.1 mm. times.100 mm, 2 μm);
column temperature: 35 ℃;
mobile phase: the phase A is water, the phase B is methanol, gradient elution is carried out, and the elution procedure is shown in Table 7;
flow rate: 0.25 mL/min;
sample introduction amount: 5 μ L.
TABLE 7 procedure for mobile phase gradient elution
Mass spectrum conditions:
the instrument model is as follows: thermo Fisher TSQ Quantum Discovery triple quadrupole mass spectrometer;
the scanning mode is as follows: anion scanning (ESI)－)；
Capillary voltage: 3800V;
ion source temperature: 320 ℃;
sheath gas: 25 arb;
auxiliary gas: 4 arb;
collision gas: 2 mTorr;
and (3) monitoring mode: reaction Monitoring (SRM) was selected and the monitoring conditions are shown in Table 8.
TABLE 8 Mass Spectrometry information for analytes and their internal standards
The accuracy, precision and sensitivity of the finally established detection method can meet the research requirements of national standard methods and related standard substances.
EXAMPLE 2 use of matrix reference candidate
Samples were extracted according to the random number table of JJF 1343-2012 "general principle and statistical principle of standard substance quantitation" for homogeneity test. And (3) performing statistical analysis on the detection result by adopting a one-factor analysis of variance method (F-test method), and judging that the uniformity of the candidate is good according to P-value > 0.05.
The stability of long-term storage and the stability of short-term transport were examined as JJF 1343-. Extracting samples at different time points according to the principle of dense before sparse for detection, performing statistical analysis on detection results by adopting a trend analysis method (t-test method), and judging that the stability of the candidate is good according to the P-value > 0.05.
The candidates were jointly evaluated by 6 laboratories according to JJF 1343-2012, using the stable isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS) described above. The detection data provided by each laboratory is respectively checked by using a Dixon (Dixon) criterion to check whether an outlier exists. The accuracy between each set of data was checked by the Cochran method. The average value of the measured data of 6 laboratories is regarded as a single measurement value to form a new set of measurement data, and the dixon criterion is used for checking again to judge whether an abnormal value exists. All the data tested in 6 laboratories were tested for normality using the Agostino method (D' Agostino). Through statistical analysis, all detection data provided by 6 laboratories are effective, and the finally obtained content standard value of the fipronil sulfone residual analysis standard substance in the egg powder is as follows: 77 μ g/kg.
And according to JJF 1343-. The numerical value reduction rule is carried out according to GB8170-2008 'representation and judgment of numerical value reduction rule and limit numerical value' and JJF 1059.1-2012 'evaluation and representation of measurement uncertainty'. The expanded uncertainty of the fipronil sulfone residual analysis standard substance in the finally obtained egg powder is as follows: 6 μ g/kg (k 2).
The fipronil sulfone residue analysis standard substance candidate in the egg powder provided by the invention has uniformity and stability meeting the standard substance requirements, and a standard value and uncertainty are obtained through subsequent analysis, so that a standard substance product with a magnitude traceability is formed, and the standard substance candidate can be used for quality monitoring, analysis method evaluation, operator level assessment and the like in the egg fipronil sulfone detection process.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a fipronil sulfone residual analysis standard substance candidate in egg powder comprises the following steps:
(1) animal experiments: the method comprises the following steps of (1) screening an egg liquid raw material containing 15-25 mug/kg of fipronil sulfone through a pharmacokinetic test of fipronil in a laying hen by taking the limit of fipronil and metabolite residues thereof in eggs as a target, wherein the limit of fipronil residues is 20 mug/kg;
(2) homogenizing raw materials: stirring the egg liquid raw material obtained by screening uniformly;
(3) and (3) freeze drying: pre-freezing and vacuum freeze-drying the uniformly stirred egg liquid raw material in batches to constant weight;
(4) grinding and sieving: crushing and sieving the freeze-dried sample until the grain size of the egg powder is not more than 40 meshes;
(5) and (3) uniformly mixing dry powder: mixing all the collected sieved egg powder, stirring and uniformly mixing;
(6) primary detection of dry powder: performing primary uniformity detection on the uniformly mixed egg powder;
(7) and (3) product packaging: subpackaging the egg powder qualified by the primary uniformity inspection into sample bottles, and then carrying out vacuum-pumping packaging;
(8) irradiation sterilization: and sterilizing the packaged product to obtain a fipronil sulfone residue analysis standard substance candidate in the egg powder, and storing at low temperature.
2. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in the step (1), the pharmacokinetics test is to put fipronil into the medicinal capsule according to the required dosage, so that the laying hens take the medicinal capsule orally, the medicinal capsule is administrated at one time, the administration dosage is 0.005-2 mg/egg, and egg liquid samples containing 15-25 microgram/kg fipronil sulfone are screened.
3. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 2, wherein the method comprises the following steps: aiming at an egg liquid sample containing 15-25 mug/kg fipronil sulfone, when the dosage is 0.005 mg/egg, the egg collection time is 6 days and 7 days after administration; when the dosage is 0.01 mg/egg, the egg collection time is 4-10 days after administration; when the dosage is 0.1 mg/egg, the egg collection time is 2 days and 17-22 days after administration.
4. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in the step (2), the raw material homogenate is carried out in a stirring-cooling defoaming-stirring manner under the conditions of the ambient temperature of 20 +/-2 ℃ and the humidity of 40 +/-10% RH.
5. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in the step (3), the water in the egg liquid is removed by adopting a vacuum freeze drying technology, the dehydration rate is required to reach 75%, and the water content of the final egg powder product is not higher than 1.5%.
6. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in the step (4) and the step (5), the environment temperature is required to be 20 +/-2 ℃ and the humidity is required to be less than 30% RH when the powder is ground and sieved and the dry powder is uniformly mixed.
7. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in the step (6), the sampling amount of the uniformity initial test is 0.50-1.50 g.
8. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 1, wherein the method comprises the following steps: in step (8), radiation sterilization is adopted60Co irradiation is carried out for sterilization treatment.
9. The method for preparing a fipronil sulfone residual analysis standard substance candidate in egg powder according to claim 8, wherein the method comprises the following steps: in step (8)Described in60The irradiation dose of Co irradiation is 500-2000 Gy.
10. The method for preparing an analysis standard substance candidate for fipronil sulfone residue in egg powder according to any one of claims 1 to 9.
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| CN107917974A (en) * | 2017-11-10 | 2018-04-17 | 韩超 | The detection method of ethiprole and its metabolite residue amount in egg |
| CN111579320A (en) * | 2020-04-26 | 2020-08-25 | 西安文理学院 | Egg standard substance containing chloramphenicol, preparation method and application |
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| CN115628958A (en) * | 2022-11-21 | 2023-01-20 | 中检科(北京)测试认证有限公司 | Egg powder matrix standard sample containing rimantadine and preparation method thereof |
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