CN112683613B - Preparation method of ivermectin matrix standard substance in goat milk - Google Patents
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Abstract
The invention discloses a preparation method of an ivermectin matrix standard substance in goat milk, which comprises the following steps: 1) performing subcutaneous injection of ivermectin to sheep in the lactation period, wherein the injection amount is 0.02mL per 1kg body weight and the ivermectin content is 10 mg/mL; 2) after 24h of injection, acquiring goat milk as experimental goat milk 1; 3) adjusting the content of ivermectin in the experimental goat milk to 15 microgrammes per milliliter (mu g/mL) by using the pure goat milk to obtain experimental goat milk 2; 4) subpackaging the experimental goat milk 2 into clean penicillin bottles, quickly placing the penicillin bottles in a refrigerator at the temperature of lower than-20 ℃ for pre-freezing for not less than 12 hours, and then carrying out vacuum freeze-drying; 5) after freeze-drying, the penicillin bottle is covered tightly, and the penicillin bottle is transferred to a temperature lower than-20 ℃ for storage. The method meets the requirements of quality control and method verification in the process of detecting the ivermectin in the fresh milk at home and abroad, and ensures the reliability, comparability and traceability of the detection result measured at home and abroad.
Description
Technical Field
The invention belongs to the field of food detection, and particularly relates to a preparation method of an ivermectin matrix standard substance in goat milk.
Background
The related primary standard substances of food in China comprise 145 types, the secondary standard substances comprise 452 types, and the development of the food matrix standard substances is relatively late and only 96 types. The Shanghai entry-exit inspection and quarantine bureau carries out homogenate, freeze drying and processing treatment after homogenization on muscle tissues containing drug residues through the metabolism research of the drug in bodies of river crucian carps, develops standard substances of chloramphenicol, clenbuterol and salbutamol in fish meal, which is the first veterinary drug residual matrix standard substance in China and fills the blank of the veterinary drug residual matrix standard substance in the field of domestic food safety detection; the Fujian entry-exit inspection and quarantine bureau carries out drug bath drug administration on the eels to ensure that furazolidone is metabolized in the bodies of the eels, so that the eels contain furazolidone metabolites with certain concentration in muscles, and a furazolidone metabolite 3-amino-2-oxazolidinone residual standard substance in eel muscle freeze-dried powder is developed, which is a first veterinary drug metabolite matrix standard substance issued in China; the research institute of the measurement and test technology in Shanghai city takes healthy and safe pig urine as a blank matrix, develops a set of clenbuterol hydrochloride standard substances in the pig urine freeze-dried powder with 4 concentration levels by using an adding method, meets the requirements of quality control and method verification of a clenbuterol residue laboratory in pig urine at home and abroad, fills the blank of clenbuterol matrix standard substances, and provides a quantity traceability basis for analysis of clenbuterol residue in pig urine.
Ivermectin belongs to antibiotic drugs, which are used cautiously, and can treat gastrointestinal nematodes, pulmonary nematodes, parasitic arthropods, canine intestinal nematodes and other diseases of cattle, sheep, horses and pigs, but the correct administration of ivermectin not only ensures the drug effect, but also ensures that the cultivated livestock is influenced as little as possible, does not remain in livestock products and does not influence human health.
Currently, the national standard substance resource sharing platform (www.ncrm.org.cn) does not find the standard substance of the ivermectin matrix in the goat milk, and does not find related reports of the standard substance of the ivermectin matrix in the goat milk at home.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing an ivermectin matrix standard substance in goat milk, provides a reliable matrix standard substance for the detection of the ivermectin in the goat milk, meets the requirements of quality control and method verification in the process of detecting the ivermectin in domestic and foreign fresh milk, ensures the reliability, comparability and traceability of the detection result in domestic and foreign measurement, fills the blank of the domestic ivermectin matrix standard substance, enhances the mutual recognition of the measurement results of different laboratories, and improves the level of detecting the ivermectin in domestic fresh milk.
The invention provides a preparation method of an ivermectin matrix standard substance in goat milk, which comprises the following steps:
1) performing subcutaneous injection of ivermectin to sheep in the lactation period, wherein the injection amount is 0.02mL per 1kg body weight and the ivermectin content is 10 mg/mL;
2) after 24h of injection, acquiring goat milk as experimental goat milk 1;
3) adjusting the content of ivermectin in the experimental goat milk to 15 mug/mL by using the pure goat milk to obtain experimental goat milk 2;
4) subpackaging the experimental goat milk 2 into clean penicillin bottles, quickly placing the penicillin bottles in a refrigerator at the temperature of lower than-20 ℃ for pre-freezing for not less than 12 hours, and then carrying out vacuum freeze-drying;
5) after freeze-drying, the penicillin bottle is covered tightly, and the penicillin bottle is transferred to a temperature lower than-20 ℃ for storage.
Wherein the amount of the ivermectin contained in the experimental goat milk 1 obtained in the step 2 is 16-20 mug/mL.
Wherein, in the step 4, 11.20g of experimental goat milk is packaged in each penicillin bottle.
Wherein, the vacuum freeze-drying conditions in the step 4 are that the temperature of a cold trap is minus 80 ℃, the vacuum degree is 1Pa, and the freeze-drying time is not less than 96 hours.
Wherein, the cover in the step 5 is a butyl rubber plug and an aluminum plastic cover.
The invention also provides a use method of the standard substance of the ivermectin matrix in the goat milk obtained by the preparation method, when in use, the penicillin bottle is recovered to room temperature, 10mL of pure water is added for dissolving, and the mixture is uniformly mixed.
Wherein, the content determination of the ivermectin in the goat milk in the step 3 is determined according to the method in GB/T29696-2013 high performance liquid chromatography for determining the avermectin medicine residue in the milk.
The obtained standard substance of the ivermectin matrix in the goat milk is subjected to property examination, and the following steps are carried out:
1. and (3) uniformity inspection: the uniformity is the most basic attribute of a standard substance and is the basis for smooth development of the standard substance, according to the relevant regulations of JJF 1006-1994-. The minimum sample size of the sample was 2.0g, and the uniformity of the sample was evaluated by performing one-way anova (F test) on the detection results using the above high performance liquid chromatography.
Table 1 unit of ivermectin uniformity test in sheep milk: mu g/kg
2. And (3) stability test: according to the related regulations of JJF 1006-1994 first-level standard substance technical specification and JJF 1343-2012 standard substance constant value general principle and statistical principle, short-term stability investigation in the sample transportation process and long-term stability investigation in the storage process are carried out according to the principle of density before density. The short-term stability of the samples (prepared according to the method of example 1) was examined by measuring the ivermectin content in the samples at 25 ℃ for 0, 1, 4, 7 and 14 days; the content of ivermectin in the sample is respectively detected at 4 ℃ and-20 ℃ in 0, 1, 2, 4 and 6 months, and the long-term stability of the sample is examined. At each detection, 3 bottles of standard substance samples are randomly taken, each bottle of sample is parallelly detected for 3 times, and the average value of the 3 times of data is taken as the final detection result. The detection method used by the quality safety supervision and inspection center of agricultural products in Qinhuang island is consistent with uniformity inspection.
Analyzing the measurement result by trend analysis method to examine the change value of the slope of the linear relation with the change of the stability of the sample, evaluating the stability of the standard substance by the change value, and fitting the straight line Y-b 0 +b 1 X, X represents stability test time, Y represents characteristic value of standard substance, b 0 、b 1 Represents a regression coefficient, if | -, b 1 ∣<t 0.95,n-2 ·s(b 1 ) The slope is not changed significantly, and the standard substance can be kept stable in the monitoring time. The slope calculation formula of the fitting straight line is as follows:
the intercept calculation formula is:
the linear standard deviation calculation formula is as follows:
the uncertainty of the slope is calculated by the formula:
table 2 long-term stability test results units: mu g/kg
Table 3 short term stability test results units: mu g/kg
3. Constant value of standard substance: the ivermectin matrix standard substance in the goat milk (prepared by the method of example 1) adopts 8 laboratories to perform synergistic setting, each laboratory measures 3 samples, each sample measures 2 times, the serial numbers and names of the 8 laboratories are 1 respectively, and the disease prevention and control center in Qinhuang island city; 2, a livestock product quality monitoring center in Shijiazhuang city; 3, a detection center for quality safety inspection of agricultural products in Tangshan city; 4, quality inspection monitoring center of agricultural products in Cangzhou city; 5, a quality supervision and management station for agricultural environment and agricultural and livestock products in the constant water city; 6, quality safety supervision and inspection center of agricultural products in Lulong county; 7, quality safety supervision and inspection center of agricultural products in Changli county; and 8, the safety supervision and inspection center for the quality of agricultural products in Qinhuang island city. The results of the laboratory tests are shown in Table 4.
TABLE 4 sheep milk ivermectin matrix standard substance fixed value data
The result of the xiapino-wilk test is W ═ 2.435, and the measured data bits are normally distributed because W (n, p) ═ 0.947 and W > W (n, p) are found by table lookup.
Dickson test result is r 1 ＝0.06，r n When f (0.05,48) is found to be 0.35, r 1 And r n Less than f (0.05,48), thus retaining all data.
The result of the cocklun test is C0.11, and the table lookup results in C (0.05,8,6) 0.3362, C < C (0.05,8,6), so the data are not suspicious.
Thus, the standard material rating results are the overall average of the 8 laboratory measurements: 15.3. mu.g/kg.
4. And (3) uncertainty evaluation: for the present standard, the uncertainty in the quantitative results is derived from the uncertainty introduced by the heterogeneity of the standard; instability-induced uncertainty of the standard substance; standard substance fixes the introduced uncertainty.
(1) Uncertainty of uniformity
Uncertainty u introduced by homogeneity test bb ＝S bb ＝0.185293μg/kg
Relative uncertainty u introduced by homogeneity test bb,rel ＝0.185293/15.35＝1.2071％
(2) Uncertainty of stability
Long term stability uncertainty u lts ＝s(b 1 )t＝0.0525896×6＝0.315538μg/kg
Short term stability uncertainty u sts ＝s(b 1 )t＝0.0203761×14＝0.285265μg/kg
Long term stability relative uncertainty u lts，rel ＝0.315538/15.35＝2.0556％
Short term stability relative uncertainty u sts,rel ＝0.285265/15.35＝1.8584％
Relative uncertainty of synthesis stability
(3) Uncertainty introduced by constant value
The data is free from abnormal values through the Kochron test and the Dickson test, the final definite value result is the cooperative definite value result of 8 laboratories, the average value of the 8 laboratories is taken as a new group of data, and the uncertainty u introduced by the definite value char 0.1263 mug/kg, the relative uncertainty u introduced is fixed char,rel ＝0.1263/15.35＝0.8228％。
In conclusion, the synthesis uncertainty of the quantitative result of the ivermectin matrix standard substance in the goat milk is as follows:
when k is 2, relative expansion uncertainty: u shape CRM,rel ＝ku CRM ＝6.27％。
Expanding uncertainty:
the result of the standard substance value determination of the ivermectin matrix in the goat milk (15.3 +/-1.0) is mu g/kg, and k is 2.
According to the inventionHas the advantages that: the product is an ivermectin matrix standard substance in the goat milk, can meet the requirements of quality control and method verification in the process of detecting the ivermectin in the domestic and foreign fresh goat milk, ensures the reliability, comparability and traceability of the detection result in domestic and foreign measurement, fills the blank of the domestic ivermectin matrix standard substance, and has important practical significance for enhancing the mutual recognition of the measurement results in different laboratories and improving the detection level of the ivermectin in the domestic fresh goat milk.
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FIG. 1 chromatogram of high performance liquid chromatography for detecting ivermectin in goat milk
FIG. 2 chromatogram of ultra-high performance liquid chromatography for detecting ivermectin in goat milk
Detailed Description
The invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention.
Example 1 preparation of Ivermectin matrix Standard in goat milk
Ivermectin (0.02 mL per 1kg body weight and containing 10mg/mL of the ivermectin) is injected subcutaneously into the sheep in the lactation period, and the sheep milk is taken after 24 injections. The content of the ivermectin in the goat milk is detected by referring to GB/T29696-2013 high performance liquid chromatography for detecting the multi-residue of the avermectins in the milk, and the content of the ivermectin in the goat milk is 16-20 mu g/mL. The content of the ivermectin in the experimental goat milk is adjusted by using pure goat milk (without the ivermectin) so that the content of the ivermectin in the goat milk is about 15 mu g/mL. And slowly and uniformly stirring the adjusted goat milk, and accurately subpackaging the goat milk into clean penicillin bottles by using one percent of electronic balance, wherein each bottle contains 11.20g of the goat milk. The split penicillin bottles are quickly placed in a refrigerator at the temperature of-20 ℃ for pre-freezing for 12 hours, and then vacuum freeze-drying is carried out in a freeze dryer until the penicillin bottles are in a dry state (the temperature of a cold trap is-80 ℃, the vacuum degree is 1Pa, and the freeze-drying time is 96 hours). After the goat milk is freeze-dried, a butyl rubber plug and an aluminum plastic cover are tightly covered on a penicillin bottle, and the goat milk is transferred to the temperature of minus 20 ℃ for storage. When in use, the penicillin bottle is restored to the room temperature, 10mL of pure water is added for dissolution, and the mixture is uniformly mixed. The goat milk should be used immediately after dissolution and should not be left.
EXAMPLE 2 preparation of Ivermectin matrix standards in goat milk
Ivermectin (0.02 mL per 1kg body weight and containing 10mg/mL of the ivermectin) is injected subcutaneously into the sheep in the lactation period, and the sheep milk is taken after 24 injections. The content of the ivermectin in the goat milk is detected by referring to GB/T29696-2013 high performance liquid chromatography for detecting the multi-residue of the avermectins in the milk, and the content of the ivermectin in the goat milk is 16-20 mu g/mL. The content of the ivermectin in the experimental goat milk is adjusted by using pure goat milk (without the ivermectin) so that the content of the ivermectin in the goat milk is about 15 mu g/mL. And slowly and uniformly stirring the adjusted goat milk, and subpackaging the goat milk into clean penicillin bottles by using one percent of electronic balance, wherein each bottle contains 11.20g of the goat milk. The subpackaged penicillin bottles are quickly placed in a refrigerator at the temperature of-8 ℃ for pre-freezing for 24 hours, and then are subjected to vacuum freeze-drying in a freeze dryer until the penicillin bottles are in a dry state (the temperature of a cold trap is-80 ℃, the vacuum degree is 1Pa, and the freeze-drying time is 100 hours). After the goat milk is freeze-dried, a butyl rubber plug and an aluminum plastic cover are tightly covered by a penicillin bottle, and the goat milk is transferred to a temperature lower than-20 ℃ for storage. When in use, the penicillin bottle is restored to the room temperature, 10mL of pure water is added for dissolution, and the mixture is uniformly mixed. The goat milk should be used immediately after dissolution and should not be left.
Example 3
Matrix standard materials prepared in the previous examples in multiple laboratories were subjected to cooperative quantification, homogeneity test results, short-term stability test and long-term stability test using simultaneous stability studies
The standard substance of ivermectin in the goat milk prepared according to the method of example 1 is tested according to the relevant regulations of JJF 1006-1994 primary standard substance technical Specification and JJF 1343-2012 general and statistical principles of standard substance fixed value, wherein the uniformity test result is shown in Table 1, the stability test result is shown in Table 2 and Table 3, the total average value of 8 laboratory combined fixed values is 15.3 mu g/kg, the uncertainty of expansion of the standard substance is 1.0 mu g/kg, and the fixed value result (15.3 +/-1.0) mu g/kg and k is 2 of the standard substance are finally obtained.
Example 4 quality control of assay results Using the Standard substance prepared according to the method of example 1
The standard substance is used for controlling the quality of the content of the ivermectin in the goat milk detected by a method GB/T29696-2013 high performance liquid chromatography for determining the avermectin medicine residue in the milk. The standard substance prepared by the method of example 1 was returned to room temperature, dissolved in 10mL of pure water, and mixed for further use. The dissolved standard substance is pretreated and chromatographed according to a standard method, and a chromatogram of the standard substance detected by high performance liquid chromatography is shown in figure 1.
The detection results show that the three detection results of the standard substance are 15.1 mug/kg, 14.8 mug/kg and 15.2 mug/kg respectively, the average detection result is 15.0 mug/kg, the average recovery rate is 98%, and the relative standard deviation in batches is 1.38%, so that the accuracy and precision of the detection result meet the requirements of the method.
Example 5 development of ultra high performance liquid chromatograph method for detecting ivermectin in goat milk using standard substance prepared according to the method of example 1
The standard substance prepared by the method of example 1 was returned to room temperature, dissolved in 10mL of pure water, and mixed for further use. The sample (goat milk to be detected) dissolved by the goat milk to be detected is preprocessed according to GB/T29696-2013 high performance liquid chromatography for detecting avermectins drug residue in milk, and then is analyzed by ultra high performance liquid chromatography. The method uses a Watt UPLC H-class, a chromatographic column BEH C18, the particle size is 1.7 mu m and 2.1 x 50mm, the mobile phase is acetonitrile plus water (90+10, volume ratio), the flow rate is 0.4mL/min, the column temperature is 30 ℃, the sample injection amount is 20 mu L, and the chromatogram is shown in figure 2.
As shown in figure 2, the time of peak emergence of the total ivermectin detected by the ultra-high performance liquid chromatography is 7.546min, and no interference peak exists near the target peak, and the method is shorter than the analysis time of a common high performance liquid chromatograph (13.7min), the flow rate of a mobile phase is low, and the use amount of organic reagents is small. Therefore, the method for detecting the ivermectin in the goat milk by using the ultra-high performance liquid chromatography has the advantages of good analysis effect, high efficiency and environmental protection.
Claims (2)
1. A method for preparing an ivermectin matrix standard substance in goat milk comprises the following steps:
1) performing subcutaneous injection of ivermectin to sheep in the lactation period, wherein the injection amount is 0.02mL per 1kg body weight and the ivermectin content is 10 mg/mL;
2) after 24h of injection, acquiring goat milk as experimental goat milk 1;
3) adjusting the content of ivermectin in the experimental goat milk to 15 mug/mL by using the pure goat milk to obtain experimental goat milk 2;
4) subpackaging the experimental goat milk 2 into clean penicillin bottles, quickly placing the penicillin bottles in a refrigerator at the temperature of lower than-20 ℃ for pre-freezing for not less than 12 hours, and then carrying out vacuum freeze-drying;
5) after freeze-drying, tightly covering a penicillin bottle, and transferring to the temperature of minus 20 ℃ for storage;
wherein the amount of ivermectin contained in the experimental goat milk 1 obtained in the step 2 is 16-20 mug/mL; the content determination of the ivermectin in the goat milk in the step 3 is determined according to a method in GB/T29696-2013 high performance liquid chromatography for determining the avermectin drug residue in the milk; in the step 4, 11.20g of experimental goat milk is filled in each penicillin bottle; in the step 4, the vacuum freeze-drying condition is that the temperature of a cold trap is-80 ℃, the vacuum degree is 1Pa, and the freeze-drying time is not less than 96 hours; in the step 5, the cover is a butyl rubber plug and then an aluminum-plastic cover.
2. The use method of the ivermectin matrix standard substance in the goat milk obtained by the preparation method according to claim 1, is characterized in that a penicillin bottle is restored to room temperature during use, 10mL of pure water is added for dissolving, and the mixture is uniformly mixed.
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