CN112903873A - Free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and preparation method thereof - Google Patents
Free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and a preparation method thereof. Feeding animals to obtain a pig urine raw material containing the concentration ranges of free-state salbutamol and conjugated-state salbutamol, and freeze-drying after setting a value to obtain a standard substance containing the free-state salbutamol and conjugated-state salbutamol in pig urine freeze-dried powder with the uniformity and stability meeting the requirement; adding water to redissolve and reduce into liquid, pretreating by a mixed biological enzyme enzymolysis method, and determining the salbutamol content 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 reflects the real condition of the salbutamol standard substance in the pig urine after being metabolized in the animal body, has more accurate fixed value and good uniformity and stability, and can be widely applied to various fields such as salbutamol residue detection in animal urine, quality verification of rapid screening products, instrument detection method evaluation, quality control in laboratories and laboratory capacity verification ratio, etc.
Description
Technical Field
The invention relates to the technical field of preparation of veterinary drug residue detection standards, and particularly relates to a free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and a preparation method thereof.
Background
Salbutamol is beta2One of the receptor agonists, also known as "clenbuterol", is an early-appearing, most typical, toxic and harmful compound, which is characterized by low cost, fast action, and serious risk to human health and public health safety due to illegal and banned use in animal breeding after multiple exposures. Salbutamol is always listed as an important monitoring parameter of animal food, most existing methods for measuring salbutamol residue in edible animal tissues such as pork, pork liver and the like and pig urine are adopted, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are usually adopted as instrument methods, and enzyme-linked immunosorbent assay (ELISA) methods and colloidal gold test strip methods are generally adopted as rapid screening methods.
However, since products such as pork and pork liver are monitored after entering the market, the products have strong hysteresis, so that the products are difficult to trace and retrieve in time, the products with problems are difficult to treat and destroy, 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, the work of rapidly screening and detecting salbutamol in pig urine by an ELISA method, a test paper strip method and the like commonly used in farms and slaughterhouses, or the work of confirming and detecting salbutamol in pig urine by an instrument method such as LC-MS/MS and the like used in laboratories is realized by temporarily adding a salbutamol standard solution into so-called 'blank' pig urine with a long storage time and simulating and preparing a pig urine positive sample containing salbutamol as a quality control sample or a matrix standard sample. However, if the sample obtained by 'temporary preparation' is added by artificial external sources, if the biological enzyme pretreatment is not carried out on the urine to be detected, the detection effect is often inaccurate by adopting a conventional kit or test paper strip on site, and a false negative result is caused. Therefore, the so-called swine urine positive sample obtained by 'temporary preparation' and simulating the salbutamol-containing sample can seriously affect the accuracy, reliability and comparability of a detection result in food safety monitoring work, especially detection under the field environment that experimental conditions cannot meet strict requirements. The on-site detection result shows that the urine sample which does not exceed the standard possibly exceeds the standard after being pretreated by an enzymolysis method in a laboratory and then being tested by a liquid chromatography-tandem mass spectrometry.
For on-site rapid screening detection, under the condition that pretreatment by enzymolysis cannot be used, in order to meet the requirements of on-site detection, reduce the detection cost and the detection time and improve the accuracy of a detection result, a more accurate and more applicable urine matrix standard product is urgently needed to be provided for correction detection and quality control, so that the accuracy of on-site screening of the content of salbutamol in pig urine can be greatly improved, the accurate and effective monitoring of salbutamol in food safety monitoring work is further enhanced, the verification of market products in a rapid screening detection method is standardized, the evaluation of instrument and method in the detection is confirmed by scientific judgment, and the internal quality control and the external capability comparison level in a laboratory are improved.
Disclosure of Invention
The invention aims to provide a standard substance which truly reflects the pig urine matrix after animal metabolism, has good uniformity and stability, and contains salbutamol in free state and conjugated state in pig urine freeze-dried powder.
In long-term detection and screening, the invention discovers that if the same urine sample is subjected to calibration and value determination by temporarily adding prepared standard substances, 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 part of the sample shows that salbutamol 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, salbutamol 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 the positive urine contains salbutamol in free state and conjugated state, the existing commonly used standard substance is salbutamol in pig urine obtained by artificially and externally adding temporary preparation, and the temporarily prepared standard substance is not subjected to the circulating metabolic process in animal bodies, so that the salbutamol in the standard substance is completely in the free state. In the detection of high performance liquid chromatography-tandem mass spectrometry in a laboratory, because the enzyme pretreatment is used, salbutamol in a sample is completely in a free state, 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 field rapid screening test, but a salbutamol standard substance containing both free state and conjugated state should be used to accurately determine the value in the field rapid screening test, otherwise, false negative samples can flow into the market, and the food safety hazard can be caused.
In view of the above, the present invention considers that pig urine containing salbutamol in free state and conjugated state obtained after artificial feeding of animal (pig) before slaughter is metabolized is used as a standard substance for correction standard substance for salbutamol on-site rapid detection. After a large amount of groping and data comparison, the swine urine containing the salbutamol in free state and conjugated state obtained by animal metabolism by the method is used as a standard substance, the result is evaluated 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 prepared by temporary addition is avoided, the uniformity and the stability are good, and the joint evaluation of a plurality of laboratories is obtained.
Specifically, according to the preparation method of the swine urine freeze-dried powder containing free-state salbutamol and conjugated-state salbutamol after animal metabolism, the feed containing salbutamol is prepared to feed live pigs, urine fed for 72-96h is collected and uniformly mixed; the concentration of the salbutamol in the feed containing the salbutamol is 2-15 mg/kg. The inventor finds out through long-term experiments that due to differences of individual pigs and different metabolism degrees, the pig urine matrix containing too low albuterol concentration (less than 3ng/mL) or too high albuterol concentration (more than 50ng/mL) can be obtained by the feed with too low or too high concentration. The pig urine with the excessively low salbutamol concentration loses the quality evaluation effect on the colloidal gold test strip in the rapid screening detection; the pig urine with excessively high salbutamol 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 can be influenced.
Preferably, urine is collected between 72 and 96 hours of feeding.
Preferably, the concentration of salbutamol in the feed containing salbutamol is 7 mg/kg. The number of live pigs for preparing the standard substance 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 salbutamol is present in the pig urine after 3 hours of feeding; the concentration of salbutamol in the pig urine is in a continuous rising period during 3-72 hours; 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; the salbutamol content in the pig urine was in a slow decline and plateau after 48 h. According to the analysis and research on salbutamol content data obtained by continuously detecting 12 parallel samples, after continuously feeding feed with salbutamol with a certain concentration, urine obtained after 72 hours is uniformly mixed, and the content range is 3-30 ng/mL. The salbutamol content concentration in this range is for two reasons. Firstly, the lowest detection lower limit of the existing colloidal gold test strip for rapidly screening and detecting salbutamol in pig urine is 3 ng/mL; secondly, tests prove that the salbutamol concentration in the pig urine after metabolism of the live pigs is detected to be between 10 and 30ng/mL by an LC-MS/MS method within 2 to 4 weeks after the medicine feeding is stopped and before the live pigs are slaughtered in the market. Therefore, the concentration range obtained after the metabolism in the live pigs is most suitable for directly determining the value and preparing the urine matrix standard substance, is convenient to be used for the rapid screening detection of a colloidal gold test strip and can also be used for the LC-MS/MS method detection of laboratory confirmation
In the preparation method of the standard substance containing the salbutamol in free state and conjugated state in the pig urine freeze-dried powder after animal metabolism, after the urine is obtained, the uniformly mixed urine is filtered and centrifuged, the supernatant is taken and transferred into a brown sample bottle, and the mixture is frozen and dried until the water content is less than 2 percent. 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.
The freeze drying process is carried out by a specific auxiliary freeze drying means.
1. And (3) freezing control, wherein the temperature of the first stage is-25 ℃, the time required for adjusting the temperature from room temperature to the set temperature is 60min, and the time for keeping the temperature is 90 min.
2. The refrigeration set temperature of the water catcher is-50 ℃ and the duration time is 20 min.
3. The pre-vacuum is 0.2mbar, the alarm vacuum is 0.8mbar, and the alarm vacuum duration is 120 min.
4. The temperature of the first stage of primary drying was 5 ℃, the time required to adjust from the last temperature to this set temperature was 360min, the duration was 360min, and a vacuum of 0.7mbar was set. The temperature was set at 20 ℃ in the second stage of primary drying, the time required to adjust from the last temperature to this set temperature was 60min, the duration was 180min and the vacuum was set at 0.7 mbar.
5. The set temperature for the analysis drying was 40 ℃, the time required for the adjustment from the previous temperature to the set temperature was 240 minutes, and the duration was 80 minutes. The total time for lyophilization was approximately 26 hours.
The invention provides a freeze-dried powder of pig urine containing free and conjugated salbutamol standard substances after animal metabolism prepared by the preparation method. The standard substance has a quantitative value of 16.63-19.13ng and an average value of 17.61ng, wherein the mass ratio of salbutamol in a free state to the total amount of salbutamol is about 90-95%, and the mass ratio of salbutamol in a conjugated state to the total amount of salbutamol is about 5-10%. Through uniformity test, the characteristic values of salbutamol in the pig urine are tested by F, which shows that 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 salbutamol content in pig urine on site, which takes the standard substance containing the free-state salbutamol and conjugated-state salbutamol pig urine freeze-dried powder as a standard substance for detecting a fixed value, does not need an enzymolysis pretreatment step on the urine to be detected, and is suitable for rapidly detecting the salbutamol content in the pig urine by using a site kit or a test strip.
Furthermore, the invention provides application of the free-state and conjugated-state salbutamol standard substance contained in the animal metabolized pig urine freeze-dried powder in pork food safety supervision.
The invention provides application of a free-state and conjugated-state salbutamol standard substance contained in the prepared pig urine freeze-dried powder after animal metabolism in detecting the salbutamol content in a pig to be born.
When the swine urine freeze-dried powder after animal metabolism contains free-state and conjugated-state salbutamol standard substances for use, pure water is added for dissolution, vortex oscillation is carried out, standing is carried out for 3-8min after ultrasonic treatment, the swine urine freeze-dried powder is used as a standard substance containing fixed-value salbutamol swine urine, and when urine to be detected is detected, the step of carrying out enzymolysis pretreatment on 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 human external source and is in a free state, the prepared swine urine freeze-dried powder after animal metabolism contains the free state and the conjugated state salbutamol standard substance, can more truly reflect that the salbutamol is in an actual matrix state containing the free state and the conjugated state in the swine urine after being metabolized in the animal body (tests prove that the obtained numerical value has about 5 to 10 percent difference under the two treatment conditions of enzymolysis and non-enzymolysis), has good uniformity and stability, fills the blank that the swine urine freeze-dried powder after animal metabolism contains the free state and the conjugated state salbutamol standard substance in China, is mainly applied to the fields of detection of salbutamol residue in animal urine, quality verification of rapid screening products, evaluation of instrument confirmation detection methods, quality control in laboratories, capability comparison in laboratories and the like, has excellent social benefit and economic benefit and good application prospect.
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 pigs, 12 representative normal fattening period live pigs (4-5 months old, 2-4 weeks before marketing, body weight of about 120 kg) were randomly selected as representative samples of the parallel test. After preparing the feed containing salbutamol with certain concentration, the live pigs are fed regularly. Meanwhile, continuously collecting the pig urine in a clean sample bottle, and determining the content of the salbutamol. The pig urine sample obtained by 12 samples is continuously detected, the following general rule is found, and the salbutamol content in the pig urine can be detected after the pig urine is fed for 3 hours; the concentration of salbutamol in the pig urine is in a continuous rising period during 3-72 hours; 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; the salbutamol content in the pig urine was in a slow decline and plateau after 48 h.
According to the analysis and research on salbutamol content data obtained by continuously detecting 12 parallel samples, after continuously feeding feed with salbutamol with a certain concentration, urine obtained after 72 hours is uniformly mixed, and the content range is within the range of 3-30 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 live pig compound feed containing 2-15mg/kg salbutamol, feeding live pigs on an empty stomach after the live pigs get up every morning, collecting urine after feeding for 72h, uniformly mixing, filtering, centrifuging, accurately taking 1.00mL of supernate, transferring into a 5mL brown sample bottle, and freeze-drying until the water content is less than 2%. The freeze drying process is carried out by a specific auxiliary freeze drying method as follows:
1. and (3) freezing control, wherein the temperature of the first stage is-25 ℃, the time required for adjusting the temperature from room temperature to the set temperature is 60min, and the time for keeping the temperature is 90 min.
2. The refrigeration set temperature of the water catcher is-50 ℃ and the duration time is 20 min.
3. The pre-vacuum is 0.2mbar, the alarm vacuum is 0.8mbar, and the alarm vacuum duration is 120 min.
4. The temperature of the first stage of primary drying was 5 ℃, the time required to adjust from the last temperature to this set temperature was 360min, the duration was 360min, and a vacuum of 0.7mbar was set. The temperature of the second stage of primary drying was set at 20 ℃, the time required to adjust from the last temperature to this set temperature was 60min, the duration was 180min, and a vacuum of 0.7mbar was set.
5. The set temperature for the analysis drying was 40 ℃, the time required for the adjustment from the previous temperature to the set temperature was 240 minutes, and the duration was 80 minutes. The total time for lyophilization was approximately 26 hours.
And (4) immediately transferring to-20 ℃ for freezing and storing after the bottle cap is sealed, thus obtaining the salbutamol-containing swine urine freeze-dried powder matrix standard substance.
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 sample can be used as a standard substance containing definite value salbutamol and pig urine.
EXAMPLE 2 homogeneity test
Randomly sampling 11 packaging units before, during and after the whole packaging process, numbering from 1 to 11 randomly sampled samples, and taking 3 sub-samples in parallel from each randomly sampled unit, wherein the numbers are recorded as 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 value of salbutamol in 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 salbutamol
The experimental data show that the characteristic values of salbutamol in pig urine pass F test, and the matrix standard substance has good uniformity and meets the technical specification requirements. In addition, the sampling volume for uniformity test in this experiment is 1.00mL, therefore, 1.00mL 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 results of the long-term stability of the salbutamol-containing pig urine lyophilized powder standard substance are shown in table 2, and the stability test results are statistically analyzed by adopting a trend analysis method by fitting a straight line with the detection time and the results.
TABLE 2 Long-term stability monitoring results (ng) for salbutamol-containing lyophilized pig urine matrix standard
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 project optimizes sample pretreatment and instrument methods by adopting a stable isotope internal standard-based mode, 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 ten-thousandth balance and METTLER AL104 one ten-thousandth of a day on average have been calibrated by the institute of metrology and inspection science, Beijing; 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 (N-EVAPTM 111, OA-SYSTM water bath heating device, U.S.A.).
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 salbutamol purity standard substance is a national second-grade certified standard substance, is numbered as 100328, has the purity of 99.3 percent and the uncertainty of 0.6 percent (k is 2), and is purchased from a national standard substance research center; salbutamol-D3: the purity was 99.0%, Witega laboratories, Germany.
3. Preparation of Standard solutions
(1) Salbutamol standard stock solution
Accurately weighing 10.0mg (accurate to 0.1mg) of salbutamol solid standard, fully dissolving with methanol, fixing the volume in a 10mL brown volumetric flask, preparing salbutamol standard stock solution with the concentration of 1mg/mL, storing at-20 ℃ and having the validity period of 12 months.
(2) Salbutamol-D3 internal standard stock solution
Accurately weighing 1.0mg (accurate to 0.1mg) of salbutamol-D3 isotope solid standard, fully dissolving with methanol, fixing the volume in a 10mL brown volumetric flask, preparing salbutamol-D3 standard stock solution with the concentration of 100mg/L, storing at-20 ℃ and having the validity period of 12 months.
(3) Salbutamol standard intermediate liquid
Accurately measuring 1.0mL of salbutamol standard stock solution, diluting with methanol, and making into a 100mL brown bottle, preparing salbutamol standard intermediate solution with concentration of 10mg/L, storing at-20 deg.C, and having validity period of 6 months.
(4) salbutamol-D3 internal standard intermediate liquid
Accurately measuring 1.0mL of salbutamol-D3 internal standard stock solution, diluting with methanol, fixing in a 100mL brown bottle, preparing 1mg/L salbutamol-D3 standard intermediate solution, storing at-20 ℃ and having an effective period of 6 months.
(5) Standard working fluid
Respectively and accurately measuring a certain amount of salbutamol standard intermediate solution, and respectively diluting with a mobile phase to prepare salbutamol standard working solution with the concentration of 1, 2, 10, 20 and 50 mug/L, wherein the salbutamol standard working solution is prepared on site when in use.
(6) Isotope labeled working fluid
Accurately measuring a certain amount of salbutamol-D3 standard intermediate solution, diluting with mobile phase to prepare salbutamol-D3 standard working solution with concentration of 200 μ g/L, and preparing the working solution as it is.
(7) Mixing calibration solutions
Accurately measuring a certain amount of salbutamol standard working solution and salbutamol-D3 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 solution is used for single-point calibration and is prepared at present 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 size 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: 0.2 mL/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: 3.5 kV;
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 salbutamol 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 salbutamol in pig urine by adopting a single-point calculation method. The calculation formula is as follows:
C1-mass concentration to analyte in pig urine (ng/mL);
R1-the ratio of peak areas of the analyte and the isotopic label in the swine urine sample solution as measured by the instrument;
R2-the ratio of the peak areas of the analyte and the isotopic label in the standard working solution measured by the instrument;
M1' -the mass of isotope label added to the pig urine sample (mg);
M2' -mass of isotope label added to standard working solution (mg);
M2-mass of standard substance (mg) added to standard working solution;
MS-volume on pig urine sample (mL);
PCRMpurity of standard substance.
7. Joint setting value for multiple laboratories
The project develops a salbutamol-containing swine urine freeze-dried powder standard substance, adopts a liquid chromatogram-isotope dilution-tandem mass spectrometry (LC-ID-MSMS) as a fixed value measurement method, and combines fixed values in 8 laboratories.
TABLE 4 results of cooperative quantification of salbutamol-containing lyophilized pig urine standard (ng)
In conclusion, the result of the quantitative determination of the characteristic quantity of the salbutamol-containing swine urine lyophilized powder standard substance is 17.61ng 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 instrumental detection of salbutamol standard substance in pig urine lyophilized powder after animal metabolism and temporarily prepared salbutamol standard substance in pig urine
The salbutamol pig urine freeze-dried powder standard substance (the fixed value of salbutamol is 3ng) 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 3ng/mL, and the calibration solution is used as calibration standard solution 1.
Taking blank pig urine by the prior art, temporarily adding a salbutamol standard solution to prepare a solution with the concentration of 3ng/mL, and taking the solution as a calibration standard solution 2.
The same swine urine sample (the mass concentration of salbutamol measured by LC-MS/MS is 3ng/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 solutions
(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 microplate 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 are shown in the following tables 5 and 6 after the calibration standard 1 and the calibration standard 2 were evaluated, respectively.
TABLE 5 comparison of results of sample evaluation after method 1 (ng/mL)
| Parallel sample | Calibration standard solution 1 | Deviation value 1 | Calibration standard solution 2 | Deviation value 2 |
| 1 | 3.02 | +0.02 | 3.08 | +0.08 |
| 2 | 3.05 | +0.05 | 3.10 | +0.10 |
| 3 | 3.03 | +0.03 | 3.07 | +0.07 |
| 4 | 3.06 | +0.06 | 3.06 | +0.06 |
| 5 | 3.04 | +0.04 | 3.11 | +0.11 |
| 6 | 3.01 | +0.01 | 3.05 | +0.05 |
| 7 | 3.07 | +0.07 | 3.09 | +0.09 |
| Mean value of | 3.04 | 0.04 | 3.08 | 0.08 |
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 | 3.07 | +0.07 | 2.79 | -0.21 |
| 2 | 3.05 | +0.05 | 2.78 | -0.22 |
| 3 | 3.04 | +0.04 | 2.76 | -0.24 |
| 4 | 3.05 | +0.05 | 2.81 | -0.19 |
| 5 | 3.10 | +0.10 | 2.75 | -0.25 |
| 6 | 3.06 | +0.06 | 2.77 | -0.23 |
| 7 | 3.08 | +0.08 | 2.80 | -0.20 |
| Mean value of | 3.06 | +0.06 | 2.78 | -0.22 |
As is evident by comparing the data in table 5 and table 6:
in the case of method 1 (laboratory instrumental method), the sample was pretreated by the enzymatic method, and salbutamol in the conjugated state in urine was finally free, so that in the case of method 1 (laboratory instrumental method), the final result of the measurement sample was not much different (<0.08ng/mL) from the actual value of the sample (3ng/mL) using calibration standard solution 1 prepared in this study and calibration standard solution 2 prepared temporarily.
However, in case of method 2 (rapid on-site screening method), since no pretreatment by an enzymatic method can be performed on the sample, and part of salbutamol in a conjugated state in the sample cannot be detected, the results obtained by calibration measurement of the actual sample with the calibration standard solution 1 prepared in this study are very close to those obtained by method 1 and the actual value (3ng/mL) (+0.06 ng/mL); the results of calibration measurements on the actual samples with calibration standard solution 2 prepared by extemporaneous addition were much lower (-0.22ng/mL) than those obtained by method 1 and the actual values of the samples (3 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 calibrated by the 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 (3ng/mL) of salbutamol in pig urine is judged according to the current colloidal gold test strip, and samples with actual content larger than the detection limit (3ng/mL) are easy to cause negative (3ng/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 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 (10)
1. A method for preparing a standard substance containing salbutamol in free state and conjugated state in pig urine lyophilized powder after animal metabolism is characterized in that feed containing salbutamol is prepared for feeding live pigs, urine for feeding between 72h and 96h is collected and mixed uniformly; the concentration of the salbutamol in the feed containing the salbutamol is 2-15 mg/kg.
2. The method of claim 1, wherein the urine is collected between 72 and 96 hours of feeding.
3. 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.
4. The preparation method according to any one of claims 1 to 3, wherein 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 carrying out uniformity detection and stability detection on the standard substance of the pig urine freeze-dried powder, and carrying out definite value and uncertainty evaluation.
5. The standard substance of swine urine lyophilized powder containing salbutamol in free state and conjugated state after being metabolized by animals prepared by the preparation method of any one of claims 1 to 4.
6. The salbutamol-containing pig urine lyophilized powder standard substance in free form and in conjugated form according to claim 5, wherein the mass ratio of salbutamol in free form to salbutamol in total amount is 90% -95%, the mass ratio of salbutamol in conjugated form to salbutamol in total amount is 5% -10%, and the quantitative value of the standard substance is 16.63-19.13 ng.
7. A detection method suitable for rapidly detecting the salbutamol content in pig urine on site, which is characterized in that the standard substance containing free-state and conjugated salbutamol pig urine freeze-dried powder of claim 5 or 6 is used as a standard substance for detection of a fixed value without carrying out an enzymolysis pretreatment step on the urine to be detected.
8. A method for improving the accuracy of on-site rapid detection of salbutamol content in swine urine, which is characterized in that the standard substance containing free-state and conjugated salbutamol swine urine lyophilized powder of claim 5 or 6 is used as a standard substance for detection of a fixed value, and a detection kit or a test strip is used for detection without performing an enzymolysis pretreatment step on the urine to be detected so as to detect the salbutamol content in the swine urine to be detected.
9. Use of the free and conjugated salbutamol-containing pig urine lyophilized powder standard substance of claim 5 or 6 for pork food safety supervision or for detecting the salbutamol content in pigs to be slaughtered.
10. The application of claim 9, wherein when the animal-metabolized pig urine freeze-dried powder contains free-state and conjugated-state salbutamol standard substances for use, pure water is added for dissolution, vortex oscillation is performed, standing is performed for 3-8min after ultrasonic treatment, the pig urine freeze-dried powder is used as a standard substance containing fixed-value salbutamol pig urine, and when urine to be detected is detected, an enzymolysis pretreatment step for the urine to be detected is not needed.
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