CN111579320A - Egg standard substance containing chloramphenicol, preparation method and application - Google Patents
Egg standard substance containing chloramphenicol, preparation method and application Download PDFInfo
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Abstract
The invention discloses an egg standard substance containing chloramphenicol, a preparation method and application thereof, and relates to the technical field of preparation of chloramphenicol standard substances. The preparation of the standard substance comprises the following steps: selecting healthy hens in the laying period, simulating feeding conditions in a natural environment, and feeding chloramphenicol; collecting eggs; removing shell of egg, collecting egg liquid, and freeze drying egg liquid to obtain lyophilized powder; and (4) crushing the freeze-dried powder, subpackaging, and performing irradiation sterilization to obtain the standard substance. The standard substance provided by the invention is prepared in a natural pollution mode, and has good uniformity and strong stability. The standard substance can be used for quality control of related detection and analysis, improves the accuracy and reliability of detection results, and can also be used in the fields of development of detection methods, laboratory capability verification and the like.
Description
Technical Field
The invention relates to the technical field of preparation of chloramphenicol standard substances, and particularly relates to a chloramphenicol-containing egg standard substance, a preparation method and application thereof.
Background
Chloramphenicol, also called chloraminophen, is white or slightly yellowish green needle-like or sheet-like crystal, stable in property, easily soluble in organic solvent, slightly soluble in water, is one of chloramphenicol medicaments, has broad-spectrum antibacterial action, and the antibacterial categories comprise gram-positive bacteria, gram-negative bacteria, spirochete, chlamydia and rickettsia, are widely applied in the livestock industry in China, can treat various infectious diseases such as intestinal infection, mastitis and the like, and play an important role in controlling and treating diseases of livestock and poultry. However, chloramphenicol has serious toxic and side effects on human and livestock, and residue in animal food and edible tissues of animals caused by chloramphenicol abuse is transmitted to human through a food chain, so that harm to human health is possible, and drug resistance of pathogenic strains is generated, especially diseases such as aplastic anemia and leucopenia of human can be caused. The use of chloramphenicol and its salts and esters in all food animals is prohibited by the 235 bulletin of the Ministry of agriculture in China, which is banned from animal products of food origin in many countries, and cannot be detected in all edible animal tissues.
At present, a plurality of methods for detecting chloramphenicol residues are mainly adopted, such as a liquid chromatography-mass spectrometry combination method, a gas chromatography-mass spectrometry combination method, a high performance liquid chromatography and the like, and a plurality of detection standards are published in China, so that when chloramphenicol is detected, a standard substance plays a key role in quality control, and the quality control of a detection process by adopting a standard substance with a matrix is definitely proposed in detection methods for part of veterinary drug residues published in countries such as European Union, Japan and the like. The quality control of the detection process mainly comprises the steps of adding a pure chloramphenicol standard substance into a sample and then carrying out detection analysis, wherein the food composition is complex, and the interference of a matrix is avoided, so that the method cannot represent the real existing state of a target object in food, and the combination mode of the target object and the matrix is inconsistent with the real detection sample, so that the extraction rate is inconsistent during sample extraction, the detection result is deviated, the accuracy and the reliability of the result are influenced, and therefore, a standard substance with the matrix is required to assist in detection to ensure the accuracy of the detection result.
Chinese patent application 201710117226.3 discloses a preparation method of standard substance of fish powder containing chloramphenicol, which comprises the steps of obtaining fish raw material polluted by chloramphenicol through simulated feeding, pretreating the sample, mixing the pretreated sample with water in a proper proportion, homogenizing, obtaining freeze-dried fish by adopting a freeze-drying method, and preparing the freeze-dried fish powder with good uniformity through the steps of grinding, screening, uniformly mixing, irradiating and the like. Homogeneity verification and stability monitoring, as well as assignment of standard substances, were performed with high accuracy measurements by liquid chromatography isotope dilution mass spectrometry. The freeze-dried fish meal containing trace chloramphenicol prepared by the method has good uniformity and stability, but is limited to evaluation of a chloramphenicol trace residue analysis method in an aquatic product, quality control of an analysis process and laboratory capability assessment.
Chinese patent application 201710117076.6 discloses a preparation method of a standard substance of honey containing chloramphenicol, which comprises the steps of taking chloramphenicol blank honey as a substrate, utilizing the characteristics of viscosity reduction and fluidity increase of the honey at 60 ℃, heating and stirring in a water bath at 60 ℃, gradually diluting and uniformly mixing, and uniformly adding a chloramphenicol solution into the honey to obtain the standard substance of the honey substrate containing trace chloramphenicol residues of 2.3 mug/kg. The standard substance has good uniformity and stability, the use of the standard substance is beneficial to improving the accuracy and reliability of the trace residue detection of the chloramphenicol in the honey, and the standard substance can also be used for calibrating a measuring instrument and evaluating a measuring method.
However, the standard substances are only suitable for detecting the residue of chloramphenicol in specific substances, no published literature is available at present for introducing a preparation technology or a similar technology of the chloramphenicol standard substances in eggs, and the preparation of the related standard substances has important significance in the fields of quality control, laboratory capacity verification, comparison among laboratories and the like in the chloramphenicol detection process.
In view of the above, the invention provides an egg standard substance containing chloramphenicol and a preparation method thereof, and the standard substance has good uniformity and stability, wide application range, simple preparation method and strong operability.
Disclosure of Invention
The invention aims to provide an egg standard substance containing chloramphenicol and a preparation method thereof, wherein the standard substance is good in uniformity and stability, can be stored for a long time at the temperature of 2-8 ℃, and is simple in preparation method and strong in operability.
In view of the above object, the present invention provides the following technical solutions:
an egg standard substance containing chloramphenicol, which is prepared according to a method comprising the following steps:
(1) selecting laying hens, simulating feeding conditions in a natural environment, and feeding chloramphenicol;
(2) collecting eggs;
(3) removing shell of egg, and collecting egg liquid;
(4) preparing egg liquid into freeze-dried powder;
(5) pulverizing and sieving the freeze-dried powder, mixing for the second time, subpackaging and sterilizing to obtain the standard substance.
Wherein the content of the first and second substances,
the feeding condition in the step (1) is preferably single-cage feeding or captive feeding.
The conditions for feeding chloramphenicol in the step (1) are as follows: feeding the feed once or for many times according to the dose of 10-100mg/kg per day, wherein the feeding mode comprises feeding chloramphenicol capsules, feeding chloramphenicol drinking water and feeding chloramphenicol feed.
The feeding mode comprises feeding chloramphenicol capsules, feeding chloramphenicol-containing drinking water and feeding chloramphenicol-containing feed; the feeding is continuous fixed-point feeding.
The feeding time of the chloramphenicol is 3-15 days, preferably 4-10 days.
The content of the chloramphenicol in the eggs is mainly related to the feeding amount and the feeding time of the chloramphenicol, and if a standard substance with higher content of the chloramphenicol is to be prepared, the feeding amount of the chloramphenicol can be increased, and the feeding time of the chloramphenicol can be prolonged; if the standard substance with low content is prepared, the feeding amount of the chloramphenicol can be reduced, and the feeding time of the chloramphenicol can be reduced.
The time for collecting the eggs in the step (2) ranges from the 2 nd day of the administration to the 6 th day after the withdrawal of the medicine, and preferably ranges from the 2 nd day of the administration to the 4 th day after the withdrawal of the medicine.
The freeze-dried powder prepared in the step (4) is prepared by the following steps: mixing the egg liquid, homogenizing, and freeze drying to obtain lyophilized powder.
The mixing and homogenizing modes are mechanical stirring, the stirring speed is 30-40 r/min, and the stirring time is 1-2 h;
the freeze-drying conditions are as follows: the temperature is-40 to-50 ℃, the pressure is not higher than 0.1Mpa, the drying time is not less than 48 hours, preferably, the drying condition is that the temperature is-45 to-50 ℃, the pressure is not higher than 0.08Mpa, and the drying time is not less than 72 hours. The freeze drying condition is selected because the material drying speed is high under the condition, the original state of the standard substance can be well kept, the protein is not denatured, the rehydration is realized, the crushing is facilitated, and the later mixing efficiency can be improved.
The moisture content of the freeze-dried standard substance is not higher than 5 wt%. Controlling the moisture content of the dried standard substance to not more than 5% by weight can prevent the standard substance from caking during long-term storage and ensure the flowability of the pulverized powder.
Preferably, the method also comprises pre-freezing before freeze-drying, wherein the pre-freezing temperature is-40 ℃ to-50 ℃, the time is 1h to 2h, and the thickness of the egg liquid is not higher than 10 mm; more preferably, the pre-freezing temperature is-40 ℃ to-45 ℃, the time is 1h to 1.5h, and the thickness of the egg liquid is not higher than 8 mm.
The split charging mode in the step (5) is vacuum packaging, preferably aluminum foil bag vacuum packaging and PP plastic bottle nitrogen-filled packaging; the vacuum packaging is to isolate the standard substance from air, so that the standard substance can keep better stability.
The sterilization conditions in the step (5) are as follows: sterilizing by irradiation, wherein the radiation source is cobalt 60, the irradiation intensity is 8-10kGy, and the irradiation time is not less than 15 h; preferably, the irradiation intensity is 8kGy, and the irradiation time is not less than 15 h.
Preferably, the step (5) further comprises sieving and secondary mixing after crushing, and the mesh number of the sieve used for sieving is 40-50 meshes. The sieve with the size can well remove large particles and improve the later mixing efficiency, the materials with the mesh number larger than 50 meshes are difficult to sieve, the efficiency is low, the mesh number is smaller than 50 meshes, the large particles cannot be removed, and the uniformity of standard substances is influenced;
the secondary mixing adopts three-dimensional mixing, the rotating speed is not less than 40r/min, the mixing time is not less than 1h, and a high-rotating-speed short-time mode is adopted during mixing, so that the exposure time of standard substances in the air is reduced, and the pollution risk and the moisture regain risk are reduced.
In addition, the invention also provides the application of the egg standard substance in quality control of monitoring analysis, solving problems encountered in the analysis and detection process, development of a detection method, laboratory capability verification, comparison among laboratories, calibration of a measuring instrument and evaluation of the accuracy of a measurement method.
Compared with the prior art, the invention has the beneficial effects that:
(1) the standard substance of the eggs containing the chloramphenicol provided by the invention has good uniformity and strong stability, and can be stored at the temperature of 2-8 ℃ for more than 12 months;
(2) the standard substance for eggs containing chloramphenicol provided by the invention adopts a natural pollution mode, is consistent with the feeding process of laying hens, avoids the condition that the detection result is influenced by the combination of the standard substance and a matrix, and improves the accuracy and reliability of the detection result;
(3) the standard substance has wide application range, can be used for controlling the quality of the daily analysis and detection process of chloramphenicol, solving the problems encountered in the analysis and detection process, and can also be used in the fields of development of detection methods, laboratory capability verification, comparison among laboratories and the like;
(4) the preparation method is simple and has strong operability.
Drawings
FIG. 1 is a process flow chart of standard egg material containing chloramphenicol.
Detailed Description
The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
In the following examples, unless otherwise specified, all the procedures and equipment used were conventional procedures and equipment used was conventional equipment.
Example 1
1. Selecting laying hens: selecting 50 laying hens aged 150 days, simulating feeding conditions in a natural environment, feeding for one week in a single cage, and selecting 30 healthy laying hens with similar weight as feeding objects.
2. Feeding chloramphenicol: feeding each laying hen with chloramphenicol at a dose of 20mg/kg every day, and feeding for 5 days continuously by one-time feeding.
3. Collecting eggs: collecting eggs from day 2 after feeding chloramphenicol to day 4 after stopping drug feeding, removing damaged and rotten eggs, and collecting the rest matrix samples as standard substances.
4. And (3) egg treatment: removing shell of matrix sample, collecting egg liquid in jar of blender, and stirring at 40r/min for 1 hr to obtain uniform state.
5. Pre-freezing: transferring the homogenized egg liquid to a clapboard of a freeze dryer, laying the egg liquid with the thickness of 6mm, and pre-freezing the egg liquid in the freeze dryer for 1h at the temperature of minus 40 ℃.
6. And (3) freeze drying: after pre-freezing, freeze-drying in a freeze-drying machine for 60h at-45 deg.C under 0.08 MPa.
7. Crushing and secondary mixing: transferring a freeze-dried sample into a crusher in an environment with low humidity, crushing the sample, sieving the crushed sample by a 40-mesh sieve, transferring the sieved sample into a three-dimensional mixer for mixing, wherein the mixing conditions are as follows: three-dimensional mixing is carried out, the rotating speed is 40r/min, and the mixing time is 1 h.
9. Subpackaging: and (3) vacuum packaging by adopting aluminum foil bags, wherein each bag is 10g, the total number of the bags is 240, and the bags are refrigerated and stored at the temperature of 2-8 ℃.
10. And (3) sterilization: sterilizing a sample by adopting an irradiation sterilization method, wherein the sterilization conditions are as follows: and (4) irradiating the standard substance of the eggs with the irradiation source of cobalt 60 and the irradiation dose of 8KGy for 15h to obtain the standard substance of the eggs.
Example 2
Different from example 1, each layer of example 2 was fed with chloramphenicol at 100mg/kg per day.
Example 3
Different from example 1, example 3 each layer was fed with chloramphenicol at 10mg/kg per day.
Example 4
Unlike example 1, example 4 had a feeding period of 3 days.
Example 5
Unlike example 1, example 5 had a 15 day feeding period.
Example 6
Different from example 1, example 6 collects eggs from day 2 to day 6 after the termination of the feeding of chloramphenicol.
Comparative example 1
In contrast to example 1, comparative example 1 each laying hen was fed chloramphenicol at 120mg/kg per day.
Comparative example 2
Unlike example 1, comparative example 2 was fed for 17 days.
Comparative example 3
Unlike example 1, comparative example 3 collected eggs after the sixth day of administration.
Result detection
1. Uniformity test
Example 1
The number of the samples is 240, 15 representative samples are randomly extracted after the samples are numbered, 1g of the samples are weighed, 4mL of water is added for redissolving, the samples are detected according to a GB/T22338-2008 method for measuring residual quantity of chloramphenicol drugs in animal-derived food, each sample is detected for three times, and statistical analysis is carried out on data by adopting an F test method. The results of the tests and statistics for example 1 are shown in Table 1. As can be seen from table 1, the F value obtained from the uniformity experiment was less than the critical value F0.05(14,30) ═ 2.04, indicating that there was no significant difference between the samples and the resulting standard material was uniform.
TABLE 1 example 1 Standard substance homogeneity test and statistics
Examples 2 to 6
The same homogeneity test method was used to test the homogeneity of the standard substances prepared by the preparation methods provided in examples 2 to 6 and comparative example, and the same calculation method was used to obtain a statistical quantity F, the results of which are shown in Table 2.
TABLE 2 homogeneity test results statistics for the standard materials of examples 2-6
As can be seen from the results of the uniformity tests in tables 1 and 2, the F values obtained from the uniformity tests of the standard substances prepared by the standard substance preparation methods provided in examples 1 to 6 of the present invention are all less than the critical value F0.05(14,30) ═ 2.04, indicating that there is no significant difference between the samples, and the obtained samples are uniform, mainly different in the content of chloramphenicol in the prepared standard substances. Under the same conditions, the larger the feeding amount of the chloramphenicol is, the longer the time is, and the higher the content of the chloramphenicol in the prepared standard substance of the eggs is; the content of the chloramphenicol in the eggs is continuously reduced along with the time after the medicine is stopped, and the longer the egg collecting time is, the lower the content of the chloramphenicol in the standard substance is under the condition that the feeding amount is the same as the feeding time.
In comparative examples 1 and 2, the phenomena of laying rate reduction, morbidity and death of the fed subjects occur due to the large feeding amount and the long feeding time, so that the phenomena are not considered.
In comparative example 3, the content of chloramphenicol in the eggs collected after the stop of drug collection and after the 6 th day was measured, and the results are shown in table 3. As can be seen from the results, the content of chloramphenicol in the eggs after 6 days of drug withdrawal was low, and the chloramphenicol had no value as a standard substance matrix sample.
TABLE 3 summary of chloramphenicol content in eggs
2. Stability test
2.1 Long term stability test
Example 1
After the standard substance is subpackaged, refrigerating and storing at 2-8 ℃, randomly drawing 3 samples at each time point at time points of 0 month, 2 months, 4 months, 6 months, 8 months, 10 months and 12 months, parallelly measuring each sample for 2 times, taking the total average value of three bottles of samples as the time stability test result, using the total average value of uniformity test results as the test result at 0 month, and using the detection method to be consistent with the uniformity test method. The data statistical method adopts a linear fitting equation model in GB/T15000.3-2008 to carry out statistical analysis, and the stability of the sample is tested. The results of the tests and statistics for example 1 are shown in Table 4.
Table 4 long term stability test and statistics for the standard substances of example 1
As can be seen from Table 2, | b1I < t0.95, 5 × S (b1), so the slope is not significant, indicating good stability of the standard substance over 12 months.
Examples 2 to 6
The stability of the standard substances prepared by the preparation methods provided in examples 2 to 6 was tested by the same stability test method, and statistical analysis was performed by the same method, and the results showed that the standard substances provided in examples 1 to 6 had a stability statistic | b at 12 months1The I < t0.95 and 5 × S (b1) show better stability.
2.2 short term stability test
Taking example 1 as an example, a short-term stability test was performed. The short-term stability mainly investigates the stability under transportation conditions and the stability under extreme conditions, and the detection method comprises the following steps: 1. randomly extracting 6 samples, respectively placing 3 samples at 25 ℃ and 40 ℃ under each temperature condition, and detecting the content of chloramphenicol after placing for 14 days; 3. 6 samples are randomly extracted, sent to Ningxia and Guangzhou (3 samples per site) by western Ann through express delivery in a cold chain transportation mode, and then sent back to western Ann, and the total time is 7 days, and the content of chloramphenicol is detected. The test results were compared with the uniformity test results, and the data were statistically analyzed by the t-test, and the test and statistical results of example 1 are shown in tables 5 and 6. As can be seen from tables 5 and 6, the statistical values t are both smaller than the threshold value t, indicating that the stability of the sample is not affected by the storage of the sample at 25 ℃ and 40 ℃ for 14 days and the cold chain transportation.
TABLE 5 stability test and statistics for the standard substances of example 1 at 25 deg.C and 40 deg.C
TABLE 6 transport stability test and statistical results table for the standard substances of example 1
3. Standard substance constant value
Taking example 1 as an example, a standard substance quantitative test was performed. The fixed value of the standard substance is cooperatively completed by 8 laboratories, the fixed value method adopts a liquid chromatography tandem mass spectrometry method in GB/T22338-2008, wherein 2 samples are distributed in each laboratory, each sample is detected for 3 times, the average value of 6 detection data is taken as a laboratory fixed value result, and the detection data is shown in Table 7.
TABLE 7.8 results of the quantification of the standard substances of laboratory example 1
3.1 Cokelen test
Calculating the average value and the standard deviation of the detection result of the fixed value of each laboratory, carrying out the Kokern test on all data, and calculating the statistic C, wherein the calculation formula is as follows:
wherein: smax 2Is the maximum value of the standard deviation, Si 2Is the standard deviation for each laboratory.
The result determination rule is: if C is less than or equal to C (5 percent), the laboratory passes the Cocker test, all the data are accepted as correct values and can be included in the statistics of the next stage, and C is less than or equal to C (1 percent) when C (5 percent) or more than or equal to C (1 percent), the laboratory with the maximum standard deviation of the detection result does not pass the Cocker test. Where the number of laboratories P is 8, the number of measurements n is 4, the cutoff value for the charcot test C (5%) is 0.438, and the cutoff value for C (1%) is 0.521. As can be seen from table 5, the standard deviation is greatest in laboratory No. 5, the kokelen test statistic C is 0.180, C ≦ C (5%), and all of the test results from 8 laboratories passed the kokelen test.
3.2 Grabbs test
The Grabbs test was performed on all data to calculate the statistic G1P (G11) as:
or
Wherein: g1P(G11) The statistic of the maximum value (minimum value) in the average value of the detection results of each laboratory; XP (X1) is the maximum value (minimum value) of the mean value of the detection results of each laboratory;
the total average value of the detection results of each laboratory; s is the standard deviation of the mean. The result determination rule is: if G is1P(G11) G (5%) or less, which indicates that the laboratory of the maximum value (minimum value) of the average value of the detection results passes the single Grabs test, and all data are accepted as correct values; if G (5%) is less than or equal to G1P(G11) C (1%) or G1P(G11) G (1%) or more, indicating that the maximum (minimum) value of the mean values of the detection results failed the single Grabbs test in the laboratory. Where the number of laboratories P is 8, the number of measurements n is 4, the cutoff value for charbrobs test G (5%) is 2.126, and the cutoff value for G (1%) is 2.274. As can be seen from Table 5, the average value of the test results in laboratory No. 3 was the largest, the average value of the test results in laboratory No. 1 was the smallest, and the Grubbs test statistic G11＝1.77，G1P＝1.90，G11And G1PAll were less than G (5%), then the test results for 8 laboratories all passed the glabbus test.
4. Uncertainty assessment
The uncertainty of the standard substance mainly comprises: uncertainty introduced by short term stability, standard uncertainty of fixed value, uncertainty introduced by inter-vial variation, uncertainty introduced by long term stability. The uncertainty of the standard substance is represented by the synthetic uncertainty of the above uncertainties, and the calculation formula is:
wherein: mu.sCRMFor synthetic uncertainty, mucharIs the standard uncertainty of the measured value; mu.sbbUncertainty introduced by inter-vial differences; mu.sltsUncertainty introduced for long term stability, mustsUncertainty introduced for short term stability.
Since the standard substance is fixed in a cooperative manner, the uncertainty introduced by the short-term stability is included in the laboratory test values and is negligible, and the calculation results are shown in table 8.
TABLE 8 uncertainty evaluation results of example 1
| Name (R) | μchar | μbb | μlts | μsts | μCRM |
| Codeine | 0.00600 | 0.15373 | 0.003745 | 0 | 0.154 |
5. Result of constant value
Example 1 final values for the standard consisted of overall mean and uncertainty for fixed values, resulting in 6.18 ± 0.31 μ g/kg (where k is 2 at a 95% confidence level); the standard substance has a shelf life of 12 months, and if the stability of the standard substance is further proved, the effective period can be prolonged.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. An egg standard substance containing chloramphenicol is characterized in that the preparation method of the egg standard substance comprises the following steps:
(1) selecting laying hens, simulating feeding conditions in a natural environment, and feeding chloramphenicol;
(2) collecting eggs;
(3) removing shell of egg, and collecting egg liquid;
(4) preparing egg liquid into freeze-dried powder;
(5) and (4) crushing the freeze-dried powder, subpackaging and sterilizing to obtain the standard substance.
2. The egg standard substance according to claim 1, wherein the chloramphenicol feeding condition in the step (1) is: feeding the feed once or for many times according to the dose of 10-100mg/kg per day, wherein the feeding mode comprises feeding chloramphenicol capsules, feeding chloramphenicol drinking water and feeding chloramphenicol feed.
3. The egg standard substance according to claim 2, wherein the feeding time is 3-15 days.
4. The egg standard substance of claim 1, wherein the time period for collecting the eggs in step (2) ranges from day 2 of administration to day 6 after discontinuation of the administration.
5. The egg standard substance according to claim 1, wherein the freeze-dried powder prepared in step (4) is prepared by: mixing the egg liquid, homogenizing, and freeze drying to obtain lyophilized powder.
6. The egg standard substance according to claim 5, wherein the uniformly mixing and homogenizing mode is mechanical stirring, the stirring speed is 30-40 r/min, and the stirring time is 1-2 h; the freeze-drying conditions are as follows: the temperature is-40 to-50 ℃, the pressure is not higher than 0.1Mpa, and the drying time is not less than 48 hours.
7. The egg standard substance according to claim 5, further comprising a pre-freezing step before the freeze drying, wherein the pre-freezing temperature is-40 ℃ to-50 ℃, the pre-freezing time is 1h to 2h, and the thickness of the egg liquid is not higher than 10mm during the pre-freezing.
8. The egg standard substance of claim 1, wherein the dispensing in step (5) is vacuum packaging; the sterilization refers to irradiation sterilization, wherein a radiation source is cobalt 60, the irradiation intensity is 8-10kGy, and the irradiation time is not less than 15 h.
9. The egg standard substance of claim 1, wherein the step (5) further comprises the steps of: sieving and mixing for the second time; the sieving refers to sieving by a 40-50 mesh sieve; the secondary mixing adopts a three-dimensional mixing mode, the mixing rotating speed is not lower than 40r/min, and the mixing time is not lower than 1 h.
10. Use of an egg standard substance according to any one of claims 1 to 9 for monitoring quality control of analysis, solving problems encountered during analytical testing, development of testing methods, laboratory competence verification, laboratory-to-laboratory comparisons, calibration of metrology instruments and evaluation of accuracy of measurement methods.
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