CN112903875B - Ractopamine standard substance with conjugated state and free state coexisting in pig urine matrix as well as preparation method and application thereof - Google Patents
Ractopamine standard substance with conjugated state and free state coexisting in pig urine matrix as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a ractopamine standard substance with conjugated state and free state coexisting in a pig urine matrix, and a preparation method and application thereof. Feeding animal ractopamine to obtain raw materials of pig urine, and obtaining ractopamine standard substances which coexist in free state and conjugated state in the freeze-dried powder of pig urine after setting values; adding water to redissolve and reduce into liquid, pretreating by a mixed biological enzyme enzymolysis method, and determining the content of ractopamine in the matrix standard substance by a liquid chromatography-tandem mass spectrometry/isotope internal standard method combined method. The standard substance prepared by the method provided by the invention can truly reflect the coexistence condition of the conjugated state and the free state of the ractopamine standard substance in the pig urine after being metabolized in the animal body, has more accurate fixed value and good uniformity and stability in on-site rapid detection compared with the standard substance prepared by temporary addition, and can be widely applied to the fields of ractopamine residue detection in animal urine, quality verification of rapid screening products, evaluation of instrument detection methods, internal quality control in laboratories and the like.
Description
Technical Field
The invention relates to the technical field of preparation of veterinary drug residue detection standards, and particularly relates to a ractopamine standard substance with coexisting conjugated state and free state in pig urine freeze-dried powder after animal metabolism and a preparation method thereof.
Background
Ractopamine is beta 2 One of the receptor agonists, also known as "Clenbuterol, a poisonous and harmful compound which appears earlier in substances and is one of the most typical representatives, is characterized by low cost and quick response, and brings serious risks to human health and public health safety because the clenbuterol is illegally and illegally used in animal breeding after being exposed for many times. Ractopamine is always listed as an important monitoring parameter of animal food, currently, methods for measuring ractopamine residues in edible animal tissues such as pork and pork liver and pig urine are more, an instrument method generally adopts a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and a rapid screening method generally adopts an enzyme-linked immunosorbent assay (ELISA) method and a colloidal gold test strip method.
However, as products such as pork and pork liver entering the market are monitored, the hysteresis quality is high, so that the products are difficult to trace and retrieve in time, difficult to treat and destroy problematic products, and great waste is caused. Therefore, aiming at monitoring the quality safety of animal products, the animal urine is a monitoring target substance with the outstanding advantages of convenience, rapidness, directness, low cost, early warning and the like, whether the animal uses prohibited substances or not can be effectively monitored in time on the premise of not slaughtering the animal, thereby more effectively improving the supervision capability of food safety, avoiding the problem products from flowing into the market, eliminating residues through methods such as animal metabolism and the like, reducing unnecessary loss and having extremely strong economic benefit.
At present, whether the rapid screening detection such as an ELISA method and a test strip method which are commonly used in a farm and a slaughter house or the confirmation and detection work of ractopamine in pig urine by using an instrument method such as LC-MS/MS and the like in a laboratory, a pig urine positive sample containing ractopamine is simulated and prepared as a quality control sample or a matrix standard sample by temporarily adding a ractopamine standard solution into the so-called blank pig urine which is preserved for a long time. However, a large number of experiments and documents at home and abroad prove that a sample obtained by artificially and externally adding the temporary preparation is greatly different from a sample obtained by metabolizing in an animal body in terms of authenticity and representativeness of the sample and influence of a sample matrix on the existence state of a target object. The ractopamine in the pig urine obtained by artificially and externally adding temporary preparation is completely in a free state because the ractopamine does not pass through the circulatory metabolism process in an animal body; a great deal of experiments and documents at home and abroad at the early stage show that the ractopamine in the pig urine after being metabolized by animals is in a conjugate state in a certain proportion (about 5-15%). Therefore, the accuracy, reliability and comparability of the detection result can be seriously influenced in the food safety monitoring work by simulating the so-called swine urine positive sample containing ractopamine through 'temporary preparation'. In view of this, in order to further enhance accurate and effective monitoring of ractopamine in food safety monitoring work, standardize verification of market products in a rapid screening detection method, scientifically judge and confirm instrumental and method evaluation in detection, and improve comparison levels of internal quality control and external capacity in a laboratory, a ractopamine standard substance which has good uniformity and stability and coexists in a conjugated state and a free state in pig urine after animal metabolism is urgently needed, and the ractopamine standard substance plays an indispensable role.
Disclosure of Invention
The invention aims to provide a ractopamine standard substance which truly reflects the matrix of pig urine after animal metabolism, has good uniformity and stability, and coexists in a conjugated state and a free state in pig urine freeze-dried powder.
In long-term detection and screening, the invention finds that if the same urine sample is subjected to calibration and value determination by temporarily adding the prepared ractopamine standard substance, the field rapid detection is compared with the detection by adopting the laboratory high performance liquid chromatography-tandem mass spectrometry, the detection result of the sample has larger deviation, which shows that the part of the sample shows that the ractopamine is negative under the field rapid detection condition, and if the same urine sample is simultaneously detected by the laboratory high performance liquid chromatography-tandem mass spectrometry, the sample shows that the ractopamine is positive. The inventor also tries to carry out on-site rapid kit method detection in a laboratory according to the positive urine which is subjected to enzyme treatment in advance in the high performance liquid chromatography-tandem mass spectrometry method, and finds that the results of the two methods are consistent. The step of adding enzyme to pretreat the urine to be detected in the high performance liquid chromatography-tandem mass spectrometry is a key factor for ensuring the consistency of the detection result of the on-site rapid detection and the detection result of the high performance liquid chromatography-tandem mass spectrometry in a laboratory. The invention further researches show that positive urine contains ractopamine in a free state and a conjugated state, while the existing commonly used standard substance for detection is ractopamine in pig urine obtained by artificially and externally adding temporary preparation, and the temporarily prepared standard substance is not subjected to the circulation and metabolism process in an animal body, so that all the ractopamine in the temporarily prepared standard substance is in the free state. In the detection of the high performance liquid chromatography-tandem mass spectrometry in the laboratory, the ractopamine in the sample is completely in a free state due to the use of enzyme pretreatment, so that the detection result is not influenced; however, in the field rapid detection, because no enzyme pretreatment is used, the field rapid detection result is lower than the laboratory detection result, and false negative results are easy to occur.
Therefore, the 'temporarily prepared' standard substance cannot be used in the on-site rapid screening test, but a ractopamine standard substance coexisting in a conjugated state and a free state is used to accurately determine the value in the on-site rapid screening test, otherwise, a false negative sample flows into the market, and the food safety hazard is caused.
In view of this, the present invention considers pig urine obtained by artificially feeding an animal (pig) before slaughter after metabolism of ractopamine in a conjugated state and a free state as a calibration standard for rapid on-site detection of ractopamine. After a large amount of groping and data comparison, the invention finds that the ractopamine pig urine which is obtained by the method and coexists in a conjugated state and a free state after animal metabolism is used as a standard substance, the result is fixed in the field rapid detection and is consistent with the detection result of a laboratory instrument method, the false negative problem caused by using the standard substance which is temporarily added and prepared is avoided, the uniformity and the stability are good, and the joint fixed value of a plurality of laboratories is obtained.
Specifically, the preparation method of ractopamine coexisting in a conjugated state and a free state in the lyophilized powder of pig urine after animal metabolism, provided by the invention, comprises the steps of preparing a feed containing ractopamine to feed live pigs, collecting urine fed for 72-96h, and uniformly mixing; the concentration of the ractopamine in the feed containing the ractopamine is 5-20mg/kg. The reason for this concentration range is that it has been shown by experiments that, due to differences in individual pigs and differences in metabolic levels, with too low or too high a concentration of feed, it is possible to obtain a pig urine matrix with too low a ractopamine concentration (less than 5 ng/mL) or too high a ractopamine concentration (more than 50 ng/mL). The pig urine with too low ractopamine concentration loses the quality evaluation effect on the colloidal gold test strip in the rapid screening detection; the pig urine with excessively high ractopamine concentration needs to be diluted by blank urine in the preparation of the later standard substance, and the real proportion of the urine obtained after animal metabolism is influenced.
Preferably, urine is collected between 72-96 hours of feeding.
Preferably, the concentration of ractopamine in the feed containing ractopamine is 10mg/kg. The number of live pigs for preparing standard substances and collecting urine is not less than 10; the live pigs are 2-4 weeks before marketing.
In one embodiment of the invention, the applicant has found that by continuously and uninterruptedly testing 12 samples of pig urine, it is generally possible to determine that the pig urine contains ractopamine after 3 hours of feeding; during 3-72 hours, the concentration of ractopamine in the pig urine is in a continuous rising period; the content was at a plateau after 72 h. After feeding is stopped, the feed is in a rapid decline period within 3-48 h; after 48h the ractopamine content of the pig urine was in a slow decline and plateau. Analysis and research on ractopamine content data obtained by continuously detecting 12 parallel samples show that after a feed with certain concentration of ractopamine is continuously fed, urine obtained after 72 hours is uniformly mixed, and the content range of the urine is within 5-30 ng/mL. The lowest detection lower limit of the existing colloidal gold test strip for rapidly screening and detecting ractopamine in pig urine is 5ng/mL; and tests prove that the ractopamine concentration in the pig urine after metabolism of the live pig is detected to be between 10 and 30ng/mL by an LC-MS/MS method within 2 to 4 weeks after feeding and stopping medicine and before the live pig is slaughtered on the market. Therefore, the ractopamine obtained after metabolism in the live pigs ranges from 5ng/mL to 30ng/mL, is most suitable for directly determining values and preparing urine matrix standard substances, is convenient to be used for rapid screening detection of colloidal gold test strips, and can also be used for LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) method detection of laboratory confirmation
The preparation method of the ractopamine standard substance containing the conjugation state and the free state coexisting in the swine urine freeze-dried powder after animal metabolism comprises the steps of obtaining urine, filtering and centrifuging the uniformly mixed urine, taking supernatant, transferring the supernatant into a brown sample bottle, and freeze-drying until the water content is less than 2%. The preparation method of the invention also comprises the steps of carrying out uniformity detection and stability detection on the standard substance, and carrying out definite value and uncertainty evaluation.
During the freeze drying process, a special auxiliary freeze drying means is carried out.
1. Freezing control, the temperature of the first stage is-25 ℃, the set time is 60 minutes, and the duration is 90 minutes.
2. The refrigeration set temperature of the water catcher is-50 ℃ and the duration is 20 minutes.
3. The pre-evacuation was 0.2mbar, the alarm vacuum was 0.8mbar, and the duration of the alarm vacuum was 120 minutes.
4. The temperature of the first stage of primary drying was 5 degrees, set for 360 minutes, duration 360 minutes, and vacuum 0.7mbar. The second stage of primary drying was set at 20 degrees, for 60 minutes, duration 180 minutes and vacuum 0.7mbar.
5. The set temperature for the analytical drying was 40 degrees, the set time was 240 minutes, and the duration was 9999 minutes. The total time for lyophilization was approximately 26 hours.
The invention provides a ractopamine standard substance which coexists in a conjugated state and a free state in pig urine freeze-dried powder after animal metabolism, which is prepared by the preparation method. The quantitative value of the standard substance is 20.55-23.39 ng, the average value is 22.21ng, wherein the free state mass ratio of the ractopamine accounts for 85% -95% of the total amount of the ractopamine, and the mass ratio of the conjugated ractopamine accounts for 5% -15% of the total amount of the ractopamine. Through uniformity test, the characteristic values of ractopamine in pig urine are tested by F, and the uniformity of the matrix standard substance is good; according to the long-term stability monitoring result, the pig urine freeze-dried powder standard substance has good stability within 12 months.
The invention provides a detection method suitable for rapidly detecting the content of ractopamine in pig urine on site, which takes the free-state and conjugated-state ractopamine-containing freeze-dried pig urine powder standard substance as a detection standard substance without carrying out an enzymolysis pretreatment step on the urine to be detected.
Further, the invention provides application of the ractopamine standard substance coexisting in a conjugated state and a free state in the pig urine freeze-dried powder after animal metabolism in pork food safety supervision.
The invention provides application of a ractopamine standard substance which coexists in a conjugated state and a free state in the prepared pig urine freeze-dried powder after animal metabolism in detecting the ractopamine content in a pig to be born.
When the ractopamine standard substance coexisting in the conjugated state and the free state in the pig urine freeze-dried powder after animal metabolism is used, pure water is added for dissolving, vortex oscillation is carried out, standing is carried out for 3-8min after ultrasonic treatment, the obtained product is used as a standard substance containing definite value ractopamine pig urine, and when urine to be detected is detected, an enzymolysis pretreatment step of the urine to be detected is not needed.
The invention has the beneficial effects that: compared with the 'simulation standard substance' which is temporarily prepared by adding an external source and in which ractopamine is in a free state, the ractopamine standard substance which coexists in a conjugated state and a free state in the pig urine freeze-dried powder after animal metabolism can more truly reflect the actual matrix state in which ractopamine coexists in a conjugated state and a free state in pig urine after being metabolized in an animal body (tests prove that the obtained numerical value has a difference of about 5-15% under the conditions of enzymolysis and non-enzymolysis), has good uniformity and stability, fills the domestic field of no ractopamine standard substance which coexists in a conjugated state and a free state in the pig urine freeze-dried powder after animal metabolism, is mainly applied to the blank fields of ractopamine residue detection in animal urine, quality verification of rapid screening products, evaluation of instrument confirmation detection methods, quality control in laboratories, capability comparison and the like, has excellent social benefits and economic benefits, and has good application prospects.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 1 Standard substance candidate preparation
In a live pig farm of about 1000-size, 12 representative live pigs (4-5 months old, 2-4 weeks before slaughtering, weight of about 120 kg) in a common fattening period are randomly selected as representative samples of parallel tests (the live pigs in the time period are selected as test samples, because the lakedopamine is illegally added in actual breeding and used in the time period before slaughtering, and the live pigs in the time period are selected, so that the urine matrix of the pigs prepared by the method is close to the actual condition). After preparing the feed containing ractopamine with a certain concentration, the live pigs are fed regularly. Meanwhile, continuously collecting the pig urine in a clean sample bottle, and determining the content of ractopamine. Through continuous and uninterrupted detection of 12 samples of the obtained pig urine sample, the following general rule is found, and the content of ractopamine in the pig urine can be detected after the pig urine is fed for 3 hours; during 3-72 hours, the concentration of ractopamine in the pig urine is in a continuous rising period; the content was at a plateau after 72 h. After feeding is stopped, the feed is in a rapid decline period within 3-48 h; after 48h the ractopamine content of the pig urine was in a slow decline and plateau.
Analysis and research on ractopamine content data obtained by continuously detecting 12 parallel samples show that after a feed with certain concentration of ractopamine is continuously fed, urine obtained after 72 hours is uniformly mixed, and the content range of the urine is within the range of 5-30 ng/mL. Therefore, the concentration range obtained after the in-vivo metabolism of the live pigs is most suitable for directly determining the value and preparing the urine matrix standard substance.
The specific process is as follows: preparing a compound feed containing 5mg/kg-20mg/kg ractopamine for live pigs, getting up the live pigs every morning, feeding the live pigs on an empty stomach, collecting urine fed for 72h, uniformly mixing, filtering, centrifuging, accurately taking 1.00mL of supernatant, transferring into a 5mL brown sample bottle for freeze drying until the water content is less than 2%, sealing a bottle cap, and immediately transferring to-20 ℃ for freeze storage to obtain the standard substance containing the ractopamine pig urine freeze-dried powder matrix.
Before use, 1.00mL of pure water is accurately added into a brown sample bottle, vortex for 20s, ultrasonic treatment is carried out for 1min, and standing is carried out for 5min, so that the standard substance containing definite value ractopamine for pig urine can be used.
EXAMPLE 2 homogeneity test
Randomly extracting 11 packaging units in front, middle and back of the whole packaging process, numbering randomly extracted samples from 1 to 11, and parallelly extracting 3 subsamples from each randomly extracted unit, wherein the numbers are 1-1, 1-2, 1-3,2-1, 2-2, 2-3, … …,11-1, 11-2 and 11-3. The uniformity test method adopts the liquid chromatogram-isotope dilution mass spectrometry to determine the result (the specific method parameters are shown in the standard substance fixed value part), the result adopts the variance analysis method to carry out statistical analysis, and the judgment is carried out by comparing the F test value with the F critical value. The results of the uniformity test and measurement of the characteristic quantity values of the ractopamine component in the pig urine and the results of the data statistical analysis are shown in table 1.
TABLE 1 homogeneity test results (ng/mL) for pig urine matrix standard containing ractopamine
The experimental data show that the characteristic values of ractopamine in pig urine pass through F test, and that the uniformity of the matrix standard substance is good and meets the technical specification requirements. In addition, the sampling volume for uniformity test in this experiment is 1.0mL, therefore, 1.0mL is used as the minimum sampling volume for the preparation of matrix standard substance in this item.
Example 3 stability
Long-term stability monitoring studies were conducted at months 0, 1, 3, 6, 9, and 12, respectively. 3 packaging units are randomly taken each time, each unit is parallelly measured three times, and the measuring method is the same as that adopted by uniformity test and is liquid chromatogram-isotope dilution chromatography-mass spectrometry. And taking the average value of the measurement results of the three packaging units as the long-term stability monitoring result, adopting a trend analysis method for result analysis, fitting a straight line by using the monitoring time and the result, and carrying out statistical analysis on the result. The monitoring result of the long-term stability of the standard substance of the pig urine freeze-dried powder containing ractopamine is shown in table 2, a straight line is fitted according to the detection time and the result, and a trend analysis method is adopted to carry out statistical analysis on the stability test result.
TABLE 2 Long-term stability monitoring results (ng) of lyophilized porcine urine matrix standard containing ractopamine
EXAMPLE 4 valuing
1. Measurement method selection
According to the technical specification requirements of JJF1006-1994 first-level standard substances, the first-level standard substance can be simultaneously set by two methods with different principles or a method of jointly setting values in multiple laboratories. However, complex matrix standard substances are generally difficult to simultaneously define values by two different principle methods, and an absolute measurement method, namely Isotope Dilution Mass Spectrometry (IDMS) and a multi-laboratory combined value defining method is generally adopted. The method adopts a mode based on stable isotope internal standard to optimize sample pretreatment and instrument method, and adopts a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the value.
2. Laboratory apparatus and reagent
Liquid chromatography of the agent 1290 (agent, usa); an agent Tripe Auad MS 6495 Mass Spectrometry (Agilent Corp., USA); the METTLER XS105 one-tenth-ten-thousandth balance and METTLER AL104 one ten-thousandth of a day have been calibrated by the scientific research institute of metrological testing in Beijing on average; high speed refrigerated centrifuge (Thermo corporation, usa); vortex mixer model Vortex-Genie 2 Vortex mixer (Scientific Industries, USA); BAKERBOND SOLID-PHASE EXTRACTOR (Waters, USA); a water bath nitrogen blower (U.S. organization, N-EVAPTM 111, OA-SYSTM water bath heating device).
Methanol, acetonitrile (LC-MS grade, fisher, usa); formic acid (LC-MS grade, sigma, germany); beta-glucuronidase hydrochloride/arylsulfatase (dima, 300U), the laboratory water being deionized water filtered through a Milli-Q water purification system (0.22 μm filter membrane); the ractopamine purity standard substance is a national second-grade certified standard substance, is numbered as CCAD300394, has the purity of 99.0 percent and the uncertainty of 0.7 percent (k = 2), and is purchased from a national standard substance research center; ractopamine-D6: the purity was 98.0%, witega laboratories, germany.
3. Preparation of Standard solutions
(1) Ractopamine standard stock solution
Accurately weighing 10.0mg (accurate to 0.1 mg) of ractopamine solid standard, fully dissolving with methanol, fixing the volume in a 10mL brown volumetric flask, preparing 1mg/mL ractopamine standard stock solution, storing at-20 ℃ and having the validity period of 12 months.
(2) Ractopamine-D6 internal standard stock solution
Accurately weighing 1.0mg (accurate to 0.1 mg) of ractopamine-D6 isotope solid standard, fully dissolving with methanol, fixing the volume in a 10mL brown volumetric flask, preparing ractopamine-D6 standard stock solution with the concentration of 100mg/L, storing at-20 ℃ and having the validity period of 12 months.
(3) Ractopamine standard intermediate liquid
Accurately measuring 1.0mL of ractopamine standard stock solution, diluting with methanol, and making into a 100mL brown bottle, preparing a 10mg/L ractopamine standard intermediate solution, storing at-20 deg.C, and having a validity period of 6 months.
(4) Ractopamine-D6 internal standard intermediate liquid
Accurately measuring 1.0mL of ractopamine-D6 internal standard stock solution, diluting with methanol, quantitatively accommodating in a 100mL brown bottle, preparing 1mg/L ractopamine-D6 standard intermediate solution, storing at-20 deg.C, and having expiration date of 6 months.
(5) Standard working fluid
Respectively and accurately measuring a certain amount of ractopamine standard intermediate solution, and respectively diluting with a mobile phase to prepare ractopamine standard working solution with the concentrations of 1, 2, 10, 20 and 50 mu g/L, wherein the ractopamine standard working solution is prepared as it is.
(6) Isotope labeled working fluid
Accurately measuring a certain amount of ractopamine-D6 standard intermediate solution, diluting with a mobile phase to prepare 200 mug/L ractopamine-D6 standard working solution, and preparing the working solution on site.
(7) Mixing calibration solutions
Accurately measuring a certain amount of ractopamine standard working solution and ractopamine-D6 standard working solution, mixing, and determining the volume by using a mobile phase, wherein the concentration is as close as possible to the concentration on a sample computer, and the method is used for single-point calibration and is prepared in situ when in use.
4. Sample pretreatment
Extraction: taking a freeze-dried matrix standard sample, adding 1.0mL of water, fully vortexing (for a liquid sample, 1.0mL of water is absorbed and placed in a 5mL test tube), adding a proper amount of isotope labeling internal standard working solution, adding 0.25mL of 2mol/L ammonium acetate buffer solution (pH 5.2), vortexing and oscillating for 1min, adding 40 mu L of beta-glucuronidase hydrochloride/arylsulfatase, and oscillating for 16h in a dark water bath at 37 ℃. Adding 10mL ethyl acetate, mixing uniformly by vortex for 1min, centrifuging at 5000r/min for 5min, transferring the supernatant into a 15mL centrifuge tube, blowing nitrogen to dry at 40 ℃, dissolving with 1.0mL 0.1% formic acid water solution, filtering with a 0.22 μm filter membrane, and measuring on a computer.
5. LC-MSMS measuring method
Chromatographic conditions are as follows:
a chromatographic column: c18 column (100 mm. Times.2.1 mm, particle diameter 1.8 μm) or equivalent;
mobile phase a (0.1% aqueous formic acid): mobile phase B (0.1% methanoic acid in methanol) =1:1;
flow rate: 0.2mL/min; the sample injection volume is 5 mu L; the column temperature was 30 ℃.
Mass spectrum conditions:
electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode;
ion source temperature: 110 ℃; capillary voltage: 3.5kV;
temperature of the drying gas: 350 ℃;
dry gas (nitrogen) flow rate: 450L/h;
collision gas (argon) flow rate: 50L/h.
Other mass spectral parameters are shown in table 3.
TABLE 3 Mass Spectrometry MRM monitoring of Ractopamine ion-pair information
6. Measurement calculation
The LC-MS/MS computer-on sequence is as follows: blank sample-calibration solution-standard substance sample …, and finally calculating the mass concentration of ractopamine in the pig urine by adopting a single-point calculation method. The calculation formula is as follows:
C 1 -mass concentration to analyte in pig urine (ng/mL);
R 1 -the ratio of peak areas of the analyte and the isotopic label in the swine urine sample solution as measured by the instrument;
R 2 -the ratio of the peak areas of the analyte and the isotopic label in the standard working solution measured by the instrument;
M 1 ' -the mass of isotope label added to the pig urine sample (mg);
M 2 ' -mass of isotope label added to standard working solution (mg);
M 2 -mass of standard substance (mg) added to standard working solution;
M S -volume on pig urine sample (mL);
P CRM -standard ofThe purity of the material.
7. Joint setting value for multiple laboratories
The project develops a standard substance containing ractopamine pig urine freeze-dried powder, adopts a liquid chromatogram-isotope dilution-tandem mass spectrometry (LC-ID-MSMS) as a fixed value measuring method, and combines fixed values in 8 laboratories.
TABLE 4 Cooperation-quantification results (ng) for the content of standard substance of lyophilized powder of pig urine containing ractopamine
In conclusion, the result of the quantitative determination of the characteristic quantity of the standard substance containing the ractopamine-containing pig urine lyophilized powder is 22.21ng of the total average value of 8 quantitative laboratories, namely the standard value.
Example 5 comparison of results of on-site rapid screening and detection and laboratory instrument detection of ractopamine standard substance in pig urine lyophilized powder after animal metabolism and ractopamine standard substance in temporarily prepared pig urine
The ractopamine pig urine lyophilized powder standard substance (the fixed value of ractopamine is 5 ng) metabolized by animals, prepared by the method, is added with 1.0mL of water for redissolution to prepare a calibration solution with the concentration of 5ng/mL, and the calibration solution is used as calibration standard solution 1.
Taking blank pig urine by the prior art, temporarily adding ractopamine standard solution to prepare solution with the concentration of 5ng/mL, and taking the solution as calibration standard solution 2.
The same swine urine sample (the mass concentration of ractopamine measured by LC-MS/MS is 5 ng/mL) collected after animal metabolism is subjected to calibration measurement by using a calibration standard solution 1 and a calibration standard solution 2 respectively, which is specifically as follows.
1. Measurement method selection
(1) The method comprises the following steps: a laboratory detection method adopts stable isotope as an internal standard, carries out enzymolysis pretreatment on a sample, and carries out value setting by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
(2) The method 2 comprises the following steps: the on-site rapid screening detection method adopts a common enzyme linked immunosorbent assay kit (ELISA) method according to the on-site rapid screening detection condition, and directly measures the sample without enzymolysis because of on-site detection, and quantifies the sample by using a standard solution enzyme-linked immunosorbent assay curve carried by the kit.
2. Laboratory apparatus and reagent
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: enzyme-linked immunosorbent assay (made in China with curve quantitative software); a printer; a micropipette; and a liquid transferring groove.
3. Preparation of Standard solution
(1) The method comprises the following steps: calibration standard solution 1 and calibration standard solution 2.
(2) The method 2 comprises the following steps: calibration standard solution 1 and calibration standard solution 2.
4. Sample pretreatment
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: directly sucking to measure.
5. Measurement method
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: quantitation of elisa reader curves.
6. Measurement calculation
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: the microplate reader reads directly.
7. Two methods of constant value comparison
The same samples were subjected to the parallel measurement 7 times by the methods 1 and 2, respectively, and the results after the values were fixed by the calibration standard solution 1 and the calibration standard solution 2, respectively, are shown in the following tables 5 and 6.
TABLE 5 comparison of results of sample evaluation after method 1 (ng/mL)
TABLE 6 comparison of results of sample evaluation by method 2 (ng/mL)
| Parallel sample | Calibration standard solution 1 | Deviation value 1 | Calibration standard solution 2 | Deviation value 2 |
| 1 | 5.03 | +0.03 | 4.71 | -0.29 |
| 2 | 5.01 | +0.01 | 4.74 | -0.26 |
| 3 | 5.04 | +0.04 | 4.72 | -0.28 |
| 4 | 5.02 | +0.02 | 4.75 | -0.25 |
| 5 | 5.04 | +0.04 | 4.77 | -0.23 |
| 6 | 5.06 | +0.06 | 4.71 | -0.29 |
| 7 | 5.07 | +0.07 | 4.73 | -0.27 |
| Mean value of | 5.04 | +0.04 | 4.73 | -0.27 |
As is evident by comparing the data in table 5 and table 6:
in the case of method 1 (laboratory instrumental method), since the samples were all pretreated by the enzymatic method, and ractopamine in the conjugated state in urine was finally also in the free state, in the case of method 1 (laboratory instrumental method), the final results of the samples were not much different (< 0.05 ng/mL) from the actual values of the samples (5 ng/mL) using calibration standard solution 1 prepared in this study and calibration standard solution 2 prepared temporarily.
However, for method 2 (on-site rapid screening method), since no pretreatment by an enzymatic method can be performed on the sample, and part of ractopamine in a conjugated state in the sample cannot be detected, the results of the calibration measurement of the calibration standard solution 1 prepared in the present study on the actual sample are very close to those of method 1 and the actual value (5 ng/mL) of the sample (+ 0.04 ng/mL); the results of calibration measurements on the actual samples with calibration standard solution 2 prepared by the temporary addition were much lower (-0.27 ng/mL) than those of method 1 and the actual values of the samples (5 ng/mL).
Thus, in the case of method 2 (on-site rapid screening method), actual samples obtained after metabolism by the animal would yield results much lower than the actual values if they were calibrated using a extemporaneous preparation method. Particularly, under the condition that the colloidal gold test strip which is most used in slaughterhouses and farms at present is used for on-site rapid screening detection, the detection limit (5 ng/mL) of ractopamine in pig urine is judged according to the current colloidal gold test strip, and samples with the actual content of ractopamine in pig urine larger than the detection limit (5 ng/mL) are easy to cause negative (less than 5 ng/mL), so that 'fish missing' under the false negative condition is caused, harmful animal products flow into the market, the health of human bodies is harmed, and the risk potential is caused to the food safety.
The following conclusions are drawn therefrom:
1. if the standard product (the temporary preparation method of adding the standard solution to the blank sample) in the prior art is used as the correction solution, the result of the on-site detection is lower than the result of the laboratory instrument detection, and the false negative result of the on-site detection is easily caused.
2. The standard substance after the value setting of the invention is used as the correction fluid, the field detection result and the laboratory result are very close, thereby showing that the standard substance of the invention plays an accurate role and can realize accurate detection without enzyme treatment. The effect difference of the field detection method and the laboratory detection method is small. The standard product of the invention has more practical application value.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A preparation method of ractopamine standard substance coexisting in a conjugated state and a free state in pig urine freeze-dried powder after animal metabolism is characterized in that feed containing ractopamine is prepared to feed live pigs, urine fed for 72-96h is collected and uniformly mixed; the concentration of the ractopamine in the feed containing ractopamine is 5-20 mg/kg; the free state mass ratio of ractopamine in the ractopamine standard substance coexisting in the conjugated state and the free state in the pig urine freeze-dried powder after animal metabolism accounts for 85-95% of the total amount of ractopamine, the mass ratio of conjugated state ractopamine accounts for 5-15% of the total amount of ractopamine, and the quantitative value of the standard substance is 20.55-23.39 ng.
2. The method of claim 1, wherein the number of live pigs is not less than 10, and the live pigs are 2-4 weeks before marketing.
3. The preparation method according to any one of claims 1-2, characterized in that the mixed urine is filtered and centrifuged, the supernatant is taken and transferred into a brown sample bottle, and the mixture is freeze-dried until the water content is less than 2%; and/or performing uniformity detection and stability detection on the standard substance of the pig urine freeze-dried powder, and performing definite value and uncertainty evaluation.
4. The standard substance of the freeze-dried powder of ractopamine pig urine prepared by the preparation method of any one of claims 1 to 3, wherein the ractopamine pig urine standard substance coexists in a conjugated state and a free state after being metabolized by animals.
5. A detection method suitable for rapidly detecting the content of ractopamine in pig urine on site is characterized in that the ractopamine freeze-dried powder standard substance coexisting in the conjugated state and the free state of claim 4 is used as a standard substance for detecting a fixed value, and an enzymolysis pretreatment step is not required for the urine to be detected.
6. A method for improving the accuracy of on-site rapid detection of the ractopamine content in pig urine, which is characterized in that the ractopamine freeze-dried powder standard substance coexisting in the conjugated state and the free state in claim 4 is used as a standard substance for detection of a fixed value, an enzymolysis pretreatment step is not required for the urine to be detected, and a detection kit or a test strip is used for detection so as to detect the ractopamine content in the pig urine to be detected.
7. The use of the ractopamine pig urine lyophilized powder standard substance coexisting in the conjugated state and the free state as defined in claim 4 in pork food safety supervision or in detecting the ractopamine content in pigs to be slaughtered.
8. The application of claim 7, wherein when the ractopamine standard substance coexisting in the conjugated state and the free state in the animal-metabolized pig urine freeze-dried powder is used, pure water is added to dissolve the ractopamine standard substance, vortex oscillation is carried out, the obtained product stands for 3-8min after ultrasonic treatment, the obtained product is used as a standard substance containing fixed value ractopamine pig urine, and when urine to be detected is detected, an enzymolysis pretreatment step on the urine to be detected is not needed.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4172049A (en) * | 1977-05-13 | 1979-10-23 | Behringwerke Aktiengesellschaft | Control-solution for diagnostic detection methods for substances contained in the urine |
| CN203455348U (en) * | 2013-09-09 | 2014-02-26 | 杭州南开日新生物技术有限公司 | Immune colloidal gold combined reagent plate for detecting clenbuterol hydrochloride, ractopamine and salbutamol in swine urine |
| CN104155162A (en) * | 2014-08-22 | 2014-11-19 | 威海出入境检验检疫局检验检疫技术中心 | Preparation method of freeze-dried powder standard sample of animal-sourced substrate drug and metabolite residue thereof |
| CN104330512A (en) * | 2014-10-28 | 2015-02-04 | 中国农业科学院农业质量标准与检测技术研究所 | Online SPE-based method for rapidly measuring conjugation state beta-receptor stimulant in urine |
| CN104360052A (en) * | 2014-11-05 | 2015-02-18 | 中国农业科学院农业质量标准与检测技术研究所 | Animal urine dry powder for detecting and controlling quality of clenbuterol as well as preparation method and application of animal urine dry powder |
| CN106872600A (en) * | 2016-04-06 | 2017-06-20 | 中国计量科学研究院 | A kind of powder of pork standard substance preparation method containing Clenbuterol |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512984B (en) * | 2013-10-21 | 2015-07-08 | 中国动物卫生与流行病学中心 | Sample pretreatment method for detecting different clenbuterol residuals |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4172049A (en) * | 1977-05-13 | 1979-10-23 | Behringwerke Aktiengesellschaft | Control-solution for diagnostic detection methods for substances contained in the urine |
| CN203455348U (en) * | 2013-09-09 | 2014-02-26 | 杭州南开日新生物技术有限公司 | Immune colloidal gold combined reagent plate for detecting clenbuterol hydrochloride, ractopamine and salbutamol in swine urine |
| CN104155162A (en) * | 2014-08-22 | 2014-11-19 | 威海出入境检验检疫局检验检疫技术中心 | Preparation method of freeze-dried powder standard sample of animal-sourced substrate drug and metabolite residue thereof |
| CN104330512A (en) * | 2014-10-28 | 2015-02-04 | 中国农业科学院农业质量标准与检测技术研究所 | Online SPE-based method for rapidly measuring conjugation state beta-receptor stimulant in urine |
| CN104360052A (en) * | 2014-11-05 | 2015-02-18 | 中国农业科学院农业质量标准与检测技术研究所 | Animal urine dry powder for detecting and controlling quality of clenbuterol as well as preparation method and application of animal urine dry powder |
| CN106872600A (en) * | 2016-04-06 | 2017-06-20 | 中国计量科学研究院 | A kind of powder of pork standard substance preparation method containing Clenbuterol |
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