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

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

A kind of standard substance of ractopamine coexisting in conjugated state and free state in pig urine matrix Its preparation method and application

technical field

The invention relates to the technical field of preparation of veterinary drug residue detection standard substances, in particular to a ractopamine standard substance co-existing in a conjugated state and a free state in pig urine freeze-dried powder after animal metabolism and a preparation method thereof.

Background technique

Ractopamine is one of the β ₂ -receptor agonists, and it is also a poisonous and harmful compound that appeared earlier and is one of the most typical representatives of substances commonly known as "clenbuterol". It is characterized by low cost, It takes effect quickly and has been exposed to illegal and prohibited use in animal breeding many times, posing serious risks to human health and public health safety. Ractopamine has been included in the key monitoring parameters of animal food. At present, there are many methods for the determination of ractopamine residues in edible animal tissues such as pork and pig liver and pig urine. The instrument method is usually liquid chromatography-tandem mass spectrometry (LC-MS). /MS) method, and rapid screening methods generally use enzyme-linked immunosorbent immunoassay (ELISA) method and colloidal gold test strip method.

However, due to the monitoring of products such as pork and pork liver after entering the market, there is a strong lag, not only difficult to trace and recover in time, but also difficult to process and destroy problematic products, and at the same time cause great waste. Therefore, for the monitoring of the quality and safety of animal products, animal urine is a monitoring target substance with outstanding advantages such as convenience, quickness, directness, low cost, and early warning. Prohibited substances, so as to more effectively improve food safety supervision capabilities, prevent problematic products from entering the market, and eliminate residues through animal metabolism and other methods to reduce unnecessary losses, which has strong economic benefits.

So far, no matter whether it is the rapid screening test such as ELISA method and test strip method commonly used in farms and slaughterhouses, or the use of LC-MS/MS and other instrument methods in the laboratory to confirm the detection of ractopamine in pig urine, Both are by temporarily adding ractopamine standard solution to the so-called "blank" pig urine that has been stored for a long time to simulate the preparation of a positive sample of pig urine containing ractopamine as a quality control sample or matrix standard sample. However, a large number of experiments and documents at home and abroad can prove that the samples obtained through artificial exogenous addition "temporary preparation" and the samples obtained through animal metabolism, no matter from the authenticity and representativeness of the samples, or from the sample matrix itself. There is a big difference in the influence of the state of existence of objects. The ractopamine in pig urine obtained by artificially adding "temporary preparation" from exogenous sources is in a free state because it has not undergone the circulation and metabolism process in the animal body. A certain proportion (about 5%-15%) of ractopamine is in the conjugated state. Therefore, the so-called positive samples of pig urine simulated to contain ractopamine obtained through "temporary preparation" will seriously affect the accuracy, reliability and comparability of the test results in the food safety monitoring work. In view of this, in order to further strengthen the accurate and effective monitoring of ractopamine in the food safety monitoring work, standardize the verification of market products in the rapid screening test method, scientifically evaluate the instrument method evaluation in the confirmation test, and improve the internal quality control of the laboratory and the external To compare the level of ability, there is an urgent need for a ractopamine standard substance with good uniformity and stability, which coexists in conjugated and free states in pig urine after animal metabolism, which will play an indispensable role.

Contents of the invention

The purpose of the present invention is to provide a ractopamine standard substance that 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 the freeze-dried pig urine powder.

The present invention finds in the long-term detection and screening that if the same urine sample is calibrated and determined by temporarily adding the prepared ractopamine standard, the on-site rapid detection is compared with the detection by laboratory high performance liquid chromatography-tandem mass spectrometry , The test results of the samples will have a large deviation, which is manifested in the fact that some samples that are negative for ractopamine under the conditions of on-site rapid testing will show positive for ractopamine if they are tested by laboratory high performance liquid chromatography-tandem mass spectrometry at the same time. The inventor also tried in the laboratory to detect the positive urine after pre-enzyme treatment in the high performance liquid chromatography-tandem mass spectrometry method, and found that the results of the two methods tended to be consistent. This shows that the step of adding enzymes to pre-treat the urine to be tested in the high-performance liquid chromatography-tandem mass spectrometry method is a key factor to ensure that the on-site rapid detection is consistent with the accuracy of the high-performance liquid chromatography-tandem mass spectrometry detection results in the laboratory. After further research, the present invention finds that the positive urine contains both free and conjugated ractopamine, and the existing commonly used standard substance for detection is ractopamine in pig urine obtained by artificial exogenous addition of "temporary preparation". In this temporarily prepared standard product, all the ractopamine in it is in a free state because it has not undergone the circulating metabolic process in the animal body. In laboratory detection by high performance liquid chromatography-tandem mass spectrometry, all ractopamine in the sample is in a free state because of the use of enzyme pretreatment, so it has no effect on the detection results; but in the rapid on-site detection, because no enzyme is used The pretreatment resulted in lower on-site rapid test results than laboratory test results, and false negative results were prone to occur.

Therefore, it is no longer possible to use "temporarily prepared" standard substances in the on-site rapid screening test, but a ractopamine standard substance that coexists in a conjugated state and a free state should be used to accurately determine the value in the on-site rapid screening test. Otherwise, it will cause false negative samples to flow into the market, causing food safety hazards.

In view of this, the present invention considers the pig urine obtained by artificially feeding pre-slaughter animals (pigs) after metabolism of ractopamine in a conjugated state and in a free state, as a calibration standard for on-site rapid detection of ractopamine as a standard substance. After a lot of groping and data comparison, it was found that the ractopamine pig urine obtained through the method of the present invention, which coexists in the conjugated state and the free state after animal metabolism, was used as a standard substance, and the value was determined in the on-site rapid detection, which was compared with the laboratory instrument method. The results are consistent, avoiding the false negative problem caused by the use of temporarily added standard products. Its uniformity and stability are good, and it has obtained the joint determination of many laboratories.

Specifically, in the method for preparing ractopamine that coexists in a conjugated state and a free state in pig urine freeze-dried powder after animal metabolism provided by the present invention, feed pigs by preparing feed containing ractopamine, and collect and feed the ractopamine between 72 and 96 hours. The urine of the ractopamine is mixed uniformly; the concentration of ractopamine in the feed containing ractopamine is 5-20mg/kg. The reason for choosing this range of concentrations is that, due to the differences in individual pigs and the degree of metabolism, feeds with too low or too high a concentration may result in feeds containing too low a concentration of ractopamine (less than 5 ng/mL) or too high a concentration of ractopamine. Pig urine matrix with high ractopamine concentration (greater than 50 ng/mL). Pig urine with too low ractopamine concentration will lose its quality evaluation effect on colloidal gold test strips in rapid screening tests; pig urine with too high ractopamine concentration needs to be diluted with blank urine in the preparation of later standard substances , will affect the real ratio of urine obtained after animal metabolism.

Preferably, urine is collected between 72-96 hours of feeding.

Preferably, the concentration of ractopamine in the feed containing ractopamine is 10 mg/kg. The number of live pigs used to prepare standard substances and collect urine is not less than 10; the live pigs are live pigs 2-4 weeks before slaughter.

In one embodiment of the present invention, the applicant has found the following general rules through continuous and uninterrupted detection of pig urine samples obtained from 12 samples. After feeding for 3 hours, the pig urine can be detected to contain Lac Dopamine: During the period of 3-72 hours, the concentration of ractopamine in pig urine was in a continuous rising period; after 72 hours, the content was stable. After stopping feeding, it was in a rapid decline period within 3-48 hours; after 48 hours, the content of ractopamine in pig urine was in a slow decline and stable period. Through the analysis of ractopamine content data obtained by continuous detection of 12 parallel samples, it was found that after continuous feeding of a certain concentration of ractopamine feed, the urine obtained after 72 hours was mixed, and the content range was in the range of 5-30ng/mL . The existing colloidal gold test strips for rapid screening and detection of ractopamine in pig urine have a lower limit of detection of 5 ng/mL; and it has been proved by experiments that within 2-4 weeks after the withdrawal of feeding, before the slaughter of pigs on the market, The concentration of ractopamine in pig urine after metabolism by live pigs is between 10-30 ng/mL detected by LC-MS/MS method. Therefore, the range of ractopamine obtained after metabolism in pigs is 5-30ng/mL, which is most suitable for direct determination and preparation of urine matrix standard substances, and is convenient for rapid screening and detection of colloidal gold test strips. LC-MS/MS method detection for laboratory confirmation

In the preparation method of the present invention containing the ractopamine standard substance that coexists in the conjugated state and the free state in the pig urine freeze-dried powder after animal metabolism, after the urine is obtained, the mixed urine is filtered and centrifuged, and the supernatant is taken liquid, transferred into a brown sample bottle, and freeze-dried until the water content was less than 2%. In the preparation method of the present invention, it also includes performing homogeneity detection and stability detection on standard substances, and carrying out fixed value and uncertainty evaluation.

During the freeze-drying process, specific auxiliary freeze-drying means are carried out.

1. Freezing control, the first stage temperature is -25 degrees, the set time is 60 minutes, and the duration is 90 minutes.

2. The set temperature of the water trap refrigeration is -50 degrees, and the duration is 20 minutes.

3. The pre-vacuum is 0.2mbar, the alarm vacuum is 0.8mbar, and the alarm vacuum duration is 120 minutes.

4. The temperature of the first stage of primary drying is 5 degrees, the set time is 360 minutes, the duration is 360 minutes, and the set vacuum is 0.7mbar. In the second stage of primary drying, the set temperature is 20 degrees, the set time is 60 minutes, the duration is 180 minutes, and the set vacuum is 0.7mbar.

5. The set temperature of analytical drying is 40 degrees, the set time is 240 minutes, and the duration is 9999 minutes. The total lyophilization time was about 26 hours.

The invention provides a ractopamine standard substance that coexists in a conjugated state and a free state in the pig urine freeze-dried powder after animal metabolism prepared by the above preparation method. The quantitative value of the standard substance is 20.55-23.39 ng, and the average value is 22.21ng, wherein the free state mass ratio of ractopamine accounts for 85%-95% of the total amount of ractopamine, and the mass ratio of conjugated state ractopamine accounts for the total amount of ractopamine. 5%-15% of the amount. Through the homogeneity test, the characteristic values of ractopamine in pig urine all passed the F test, indicating that the matrix standard substance has good uniformity; the long-term stability monitoring results show that the porcine urine freeze-dried powder standard substance has good stability within 12 months.

The present invention provides a detection method suitable for rapid on-site detection of ractopamine content in pig urine, using the above-mentioned standard substance of freeze-dried pig urine powder containing free and conjugated ractopamine as the standard substance for detection, without the need for urine to be detected. Enzymatic pretreatment step.

Further, the present invention provides the application of the ractopamine standard substance in the pig urine freeze-dried powder metabolized by animals in pork food safety supervision.

The invention provides the application of the obtained ractopamine standard substance co-existing in the conjugated state and the free state in the freeze-dried powder of pig urine after animal metabolism in detecting the content of ractopamine in the pig body to be born.

When using the ractopamine standard substance that coexists in the condensed state and free state in the pig urine freeze-dried powder after animal metabolism, it is dissolved in pure water, vortexed, and left to stand for 3-8 minutes after ultrasonication, which is used as the standard substance containing a fixed value of ractopamine. The standard product of pig urine is used. When the urine to be tested is tested, there is no need to perform enzymatic hydrolysis pretreatment steps on the urine to be tested.

The beneficial effect of the present invention is that: the ractopamine standard substance that coexists in the conjugated state and the free state in the pig urine freeze-dried powder after animal metabolism prepared by the present invention can be compared with the "analog standard substance" that is temporarily prepared by artificially added exogenous sources and the ractopamine is in a free state. Compared with "substance", it can more truly reflect the actual matrix state of ractopamine in the urine of pigs after being metabolized in animals in a conjugated state and in a free state (tests have shown that under the conditions of enzymatic hydrolysis and non-enzymatic hydrolysis, the obtained There is a gap of about 5%-15% in the numerical value), and the uniformity and stability are good. It fills the blank of ractopamine standard substance that coexists in condensed state and free state in pig urine freeze-dried powder after animal metabolism, mainly It is used in the detection of ractopamine residues in animal urine, the quality verification of rapid screening products, the evaluation of testing methods for instrument confirmation, the internal quality control of laboratories and the comparison of laboratory capabilities, and has excellent social and economic benefits. The outlook is good.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Standard substance candidate preparation

In a pig farm with a scale of about 1000 pigs, 12 representative pigs during the fattening period (4-5 months old, 2-4 weeks before slaughter, weighing about 120kg) were randomly selected as representative samples of parallel experiments (selected The live pigs in this time period are used as test samples because the illegal addition of ractopamine in actual breeding is used in the time period before slaughter, and the selection of live pigs in this time period can make the pig urine matrix prepared by this method closer to actual status). After formulating feed containing a certain concentration of ractopamine, feed pigs regularly. At the same time, pig urine was continuously collected in a clean sample bottle, and the content of ractopamine was determined. Through the continuous and uninterrupted detection of pig urine samples obtained from 12 samples, it was found that the following general rules were found. After 3 hours of feeding, ractopamine could be detected in pig urine; during 3-72 hours, pig urine The concentration of ractopamine was in a continuous rising period; the content was stable after 72h. After stopping feeding, it was in a rapid decline period within 3-48 hours; after 48 hours, the content of ractopamine in pig urine was in a slow decline and stable period.

Through the analysis and research of the ractopamine content data obtained by continuous detection of 12 parallel samples, it was found that after continuous feeding of a certain concentration of ractopamine feed, the urine obtained after 72 hours was mixed, and the content range was in the range of 5-30ng/mL Inside. Therefore, the concentration range obtained after metabolism in pigs is most suitable for direct determination and preparation of urine matrix standard substances.

The specific process is as follows: prepare pig compound feed containing 5mg/kg-20mg/kg ractopamine, feed the pigs on an empty stomach every morning after the pigs get up, collect the urine after feeding for 72 hours, mix it, filter it, and centrifuge it accurately. Take 1.00mL of the supernatant, transfer it into a 5mL brown sample bottle, and freeze-dry until the water content is less than 2%. After the bottle cap is sealed, immediately transfer it to -20°C for cryopreservation, and then you can obtain the pig urine freeze-dried powder matrix standard containing ractopamine substance.

Before use, accurately add 1.00mL of pure water to the brown sample bottle, vortex for 20s, sonicate for 1min, and let it stand for 5min, then it can be used as a pig urine standard substance containing a fixed value of ractopamine.

Embodiment 2 homogeneity test

Randomly select 11 packaging units according to the whole packaging process before, during and after, and the randomly selected samples are numbered from 1 to 11, and each randomly selected unit takes 3 sub-samples in parallel, and the record numbers are 1-1, 1-2 , 1-3, 2-1, 2-2, 2-3, ..., 11-1, 11-2, 11-3. The homogeneity test method adopts liquid chromatography-isotope dilution mass spectrometry to measure the results (see the standard substance determination part for specific method parameters), and the results are statistically analyzed by analysis of variance, and judged by comparing the F test value with the F critical value. See Table 1 for the results of the homogeneity test and statistical analysis of the ractopamine components in pig urine.

Table 1 Pig urine matrix standard substance homogeneity test result containing ractopamine (ng/mL)

The above experimental data show that the characteristic values of ractopamine in pig urine all pass the F test, indicating that the matrix standard substance has good uniformity and meets the requirements of technical specifications. In addition, the sampling volume for the uniformity test in this experiment is 1.0mL, therefore, 1.0mL is used as the minimum sampling volume for the preparation of matrix reference materials in this project.

Embodiment 3 Stability

Long-term stability monitoring studies were carried out at 0, 1, 3, 6, 9, and 12 months, respectively. Three packaging units are randomly selected each time, and each unit is measured in parallel three times. The measurement method is the same as that used in the homogeneity test, both of which are liquid chromatography-isotope dilution chromatography mass spectrometry. The average value of the measurement results of the three packaging units is taken as the long-term stability monitoring result. The trend analysis method is used to analyze the results, and the monitoring time and the results are fitted with a straight line, and the results are statistically analyzed. The long-term stability monitoring results of the pig urine lyophilized powder standard substance containing ractopamine are shown in Table 2. A straight line was fitted with the detection time and the results, and the trend analysis method was used to statistically analyze the stability test results.

Table 2 Long-term stability monitoring results of pig urine freeze-dried powder matrix standard substance containing ractopamine (ng)

Embodiment 4 fixed value

1. Selection of measurement method

According to the technical specifications of JJF1006-1994 primary standard substance, two methods with different principles can be used to determine the value at the same time for the primary standard substance, or one method can be used to jointly determine the value of multiple laboratories. However, it is generally difficult for complex matrix standard substances to meet the simultaneous determination of two different principles and methods, and an absolute measurement method-isotope dilution mass spectrometry (IDMS) is usually used to jointly determine the value of multiple laboratories. This project adopts the method based on stable isotope internal standard to optimize the sample pretreatment and instrument method, and adopts high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for value.

2. Experimental instruments and reagents

Aglient 1290 Liquid Chromatography (Aglient Corporation, USA); Aglient Tripe Auad MS 6495 Mass Spectrometry (Aglient Corporation, USA); METTLER XS105 1/100,000 Balance and METTLER AL104 1/10000 Day Average have passed Beijing Institute of Metrology and Testing Science Standard; high-speed refrigerated centrifuge (Thermo Company, U.S.); Vortex-Genie2 type vortex mixer (Scientific Industries Company, U.S.); BAKERBOND solid-phase extraction instrument (Waters Company, U.S.); water bath nitrogen blowing instrument (Organomation Company, N-EVAPTM, U.S. 111, OA-SYSTM water bath heating device).

Methanol, acetonitrile (LC-MS grade, Fisher Company, USA); formic acid (LC-MS grade, Sigma Company, Germany); β-glucuronidase hydrochloride/arylsulfatase (Dima Company, 300U), laboratory water It is deionized water filtered by the Milli-Q water purification system (0.22 μm filter membrane); the standard substance of ractopamine purity is a national secondary certified standard substance, the number is CCAD300394, the purity is 99.0%, and the uncertainty is 0.7% ( k=2), purchased from the National Center for Standard Materials; Ractopamine-D6: 98.0% pure, from Witega Laboratories, Germany.

3. Standard solution preparation

(1) Ractopamine standard stock solution

Accurately weigh 10.0mg (accurate to 0.1mg) of solid standard ractopamine, fully dissolve it with methanol and dilute it in a 10mL brown volumetric flask, prepare a standard stock solution of ractopamine with a concentration of 1mg/mL, and store it at -20°C Save, valid for 12 months.

(2) Ractopamine-D6 internal standard stock solution

Accurately weigh 1.0mg (accurate to 0.1mg) ractopamine-D6 isotope solid standard, fully dissolve it with methanol and settle it in a 10mL brown volumetric flask to prepare a ractopamine-D6 standard stock solution with a concentration of 100mg/L. Stored at -20°C, the validity period is 12 months.

(3) Ractopamine standard intermediate solution

Accurately measure 1.0mL ractopamine standard stock solution, dilute it with methanol to a 100mL brown bottle, prepare ractopamine standard intermediate solution with a concentration of 10mg/L, store it at -20°C, and have a validity period of 6 months.

(4) Ractopamine-D6 internal standard intermediate solution

Accurately measure 1.0mL ractopamine-D6 internal standard standard stock solution, dilute it with methanol to a 100mL brown bottle, prepare a ractopamine-D6 standard intermediate solution with a concentration of 1mg/L, and store it at -20°C. 6 months.

(5) Standard working fluid

Accurately measure a certain amount of ractopamine standard intermediate solution, respectively dilute with mobile phase to prepare ractopamine standard working solutions with concentrations of 1, 2, 10, 20, and 50 μg/L, and prepare them immediately for use.

(6) Isotope labeled working solution

Accurately measure a certain amount of ractopamine-D6 standard intermediate solution, dilute it with mobile phase to prepare a ractopamine-D6 standard working solution with a concentration of 200 μg/L, and prepare it immediately for use.

(7) mixed calibration solution

Accurately measure a certain amount of ractopamine standard working solution and ractopamine-D6 standard working solution. After mixing, use the mobile phase to make up the volume. The concentration should be as close as possible to the concentration of the sample on the machine. It is used for single-point calibration and is ready for use.

4. Sample pretreatment

Extraction: Take the freeze-dried matrix standard sample, add 1.0mL water and vortex fully (for liquid samples, pipette 1.0mL and place it in a 5mL test tube), add an appropriate amount of isotope-labeled internal standard working solution, and then add 2mol/L Ammonium acetate buffer (pH 5.2) 0.25 mL, vortexed for 1 min, added 40 μL of β-glucuronidase hydrochloride/arylsulfatase, and shaken in a water bath at 37 ° C for 16 h in the dark. Add 10 mL of ethyl acetate, vortex and mix for 1 min, centrifuge at 5000 r/min for 5 min, transfer the supernatant to a 15 mL centrifuge tube, blow to dryness with nitrogen at 40 °C, dissolve with 1.0 mL of 0.1% formic acid aqueous solution, pass through a 0.22 μm filter membrane, On-board measurement.

5. LC-MSMS determination method

Chromatographic conditions:

Chromatographic column: C18 column (100mm×2.1mm, particle size 1.8μm) or equivalent;

Mobile phase A (0.1% formic acid aqueous solution): mobile phase B (0.1% formic acid methanol solution) = 1:1;

Flow rate: 0.2mL/min; injection volume: 5μL; column temperature: 30°C.

Mass Spectrometry Conditions:

Electrospray ion source (ESI); Positive ion scanning mode; Multiple reaction monitoring (MRM) mode;

Ion source temperature: 110°C; capillary voltage: 3.5kV;

Drying gas temperature: 350°C;

Dry gas (nitrogen) flow rate: 450L/h;

Collision gas (argon) flow rate: 50L/h.

Other mass spectrometry parameters are listed in Table 3.

Table 3 Mass spectrometry MRM monitoring ractopamine ion pair information

6. Measurement result calculation

The sequence of LC-MS/MS on the machine is: blank sample—calibration solution—standard substance sample—calibration solution—standard substance sample…, and finally the mass concentration of ractopamine in pig urine was calculated using the single-point calculation method. Calculated as follows:

C ₁ - the mass concentration (ng/mL) of the analyte in pig urine;

R ₁ - the peak area ratio of the analyte and the isotope marker in the pig urine sample solution measured by the instrument;

R ₂ - the peak area ratio of the analyte and the isotope label in the standard working solution measured by the instrument;

M ₁ '-is added to the quality (mg) of the isotope label in the pig urine sample;

M ₂ '-the quality (mg) of the isotope label added in the standard working solution;

M ₂ - the standard substance mass (mg) added in the standard working solution;

M _S - the volume (mL) of pig urine sample;

P _CRM - Purity of reference material.

7. Joint valuation by multiple laboratories

In this project, liquid chromatography-isotope dilution-tandem mass spectrometry (LC-ID-MSMS) was used as the measurement method for the determination of ractopamine-containing freeze-dried porcine urine powder reference material, and 8 laboratories jointly determined the value.

Table 4 is the cooperative determination result (ng) of the standard substance content of the pig urine freeze-dried powder containing ractopamine

To sum up, the quantitative value of the standard substance of freeze-dried pig urine powder containing ractopamine was determined, and the result was the total average value of 22.21ng from 8 laboratories, which was the standard value.

Example 5 Comparison of Ractopamine Standard Substance in Pig Urine Freeze-dried Powder and Temporarily Prepared Ractopamine Standard Substance in Pig Urine After Metabolism by Animals on-site Rapid Screening Detection and Laboratory Instrument Detection

Use the ractopamine porcine urine freeze-dried powder standard substance after animal metabolism prepared by the present invention (the value of ractopamine is 5 ng), redissolve after adding 1.0 mL of water, and prepare a calibration solution with a concentration of 5 ng/mL as a calibration solution. Standard solution 1.

Use the existing technology to take blank pig urine, temporarily add ractopamine standard solution, and prepare a solution with a concentration of 5 ng/mL as calibration standard solution 2.

Calibration standard solution 1 and calibration standard solution 2 were used to perform calibration determination on the same pig urine sample collected after animal metabolism (the mass concentration of ractopamine determined by LC-MS/MS was 5 ng/mL), as follows.

1. Selection of measurement method

(1) Method 1: Laboratory detection method, using stable isotope as internal standard, enzymatic hydrolysis pretreatment of samples, and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for value.

(2) Method 2: On-site rapid screening and detection method. According to the situation of on-site rapid screening and detection, the common enzyme-linked immunosorbent assay kit (ELISA) method is used. Because of on-site detection, the sample is not enzymatically hydrolyzed, and the sample is directly measured. The box itself carries the standard solution microplate reader curve quantification.

2. Experimental instruments and reagents

(1) Method 1: Same as above-mentioned embodiment 4.

(2) Method 2: microplate reader (made in China, with curve quantitative software); printer; micropipette; pipette tank.

3. Standard solution preparation

(1) Method 1: calibration standard solution 1 and calibration standard solution 2.

(2) Method 2: calibration standard solution 1 and calibration standard solution 2.

4. Sample pretreatment

(1) Method 1: Same as above-mentioned embodiment 4.

(2) Method 2: direct suction measurement.

5. Determination method

(1) Method 1: Same as above-mentioned embodiment 4.

(2) Method 2: quantification by microplate reader curve.

6. Measurement result calculation

(1) Method 1: Same as above-mentioned embodiment 4.

(2) Method 2: direct reading with microplate reader.

7. Comparison of two methods for fixed value

Use method 1 and method 2 to measure the same sample in parallel 7 times, and use calibration standard solution 1 and calibration standard solution 2 to determine the value, the results are shown in Table 5 and Table 6 below.

Table 5 Comparison of the determination results of samples after method 1 (ng/mL)

Table 6 Comparison of the determination results of samples after method 2 (ng/mL)

Sample Parallel Calibration standard solution 1 Deviation 1 Calibration standard solution 2 Deviation 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 average value 5.04 +0.04 4.73 -0.27

By comparing the data in Table 5 and Table 6, it can be clearly seen that:

In the case of method 1 (laboratory instrument method detection), because the samples were pre-treated by enzymatic hydrolysis, the conjugated ractopamine in the urine finally became free, so in method 1 (laboratory In the case of instrument method detection), using the calibration standard solution 1 prepared in this study and the temporarily prepared calibration standard solution 2, there is little difference between the final result of the measured sample and the actual value (5ng/mL) of the sample (<0.05ng /mL).

However, for method 2 (on-site rapid screening method), the samples cannot be pretreated by enzymatic hydrolysis, and part of the ractopamine in the sample cannot be detected. The calibration standard solution 1 prepared in this study is used for the actual The calibration determination of the sample is very close (+0.04ng/mL) to the result of method 1 and the actual value (5ng/mL) of the sample; while the calibration measurement result of the actual sample with the calibration standard solution 2 prepared temporarily is better than that of method 1 and The actual value of the sample (5ng/mL) was much lower (-0.27ng/mL).

Therefore, in the case of method 2 (on-site rapid screening method), if the temporary preparation method is used for calibration of the actual sample obtained after animal metabolism, the result will be much lower than the actual value. Especially when the colloidal gold test strips currently used most in slaughterhouses and farms are used for on-site rapid screening and detection, according to the current detection limit (5ng/mL) of colloidal gold test strips for ractopamine in pig urine It is judged that for samples whose actual content is greater than the detection limit (5ng/mL), it is easy to cause negative results (<5ng/mL), resulting in false negative "fish that slipped through the net", thus causing harmful animal products to enter the market, It is harmful to human health and poses risks to food safety.

From this we draw the following conclusions:

1. If the standard product of the prior art (temporarily prepared by adding the standard solution to the blank sample) is used as the calibration solution, the result of the on-site test will be lower than the test result of the laboratory instrument, which will easily cause a false negative result of the on-site test .

2, use the standard substance after the present invention's fixed value as calibration liquid, then the spot test result and the laboratory result are both very close, thereby explaining that the standard substance of the present invention has brought into play an accurate effect, under the situation that does not need enzyme treatment, can all achieve accurate detection. There was little difference in the effect of the two detection methods in the field and in the laboratory. Illustrate that the standard product of the present invention has more practical application value.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (8)

1. A method for preparing a ractopamine standard substance that coexists in a conjugated state and a free state in the pig urine freeze-dried powder after animal metabolism, it is characterized in that the feed containing ractopamine is prepared and fed to pigs, collected and fed for 72-96 hours Mix the urine between them; the concentration of ractopamine in the feed containing ractopamine is 5-20 mg/kg; the ractopamine standard substance that coexists in conjugated state and free state in pig urine freeze-dried powder after animal metabolism The free state mass ratio of ractopamine accounts for 85%-95% of the total amount of ractopamine, the mass ratio of condensed 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 preparation method according to claim 1, characterized in that the number of said live pigs is not less than 10, and said live pigs are live pigs 2-4 weeks before slaughter. 3. according to the arbitrary described preparation method of claim 1-2, it is characterized in that, the urine after mixing is filtered, centrifuged, get supernatant, move in the brown sample bottle, freeze-dry to moisture less than 2%; And/or it also includes homogeneity testing, stability testing, value determination and uncertainty assessment of the pig urine freeze-dried powder standard substance. 4. The ractopamine porcine urine lyophilized powder standard substance that coexists in a conjugated state and a free state after being metabolized by an animal, prepared by any one of claims 1-3. 5. A detection method applicable to on-site rapid detection of ractopamine content in pig urine, characterized in that, the ractopamine pig urine freeze-dried powder standard substance of condensed state and free state coexistence as claimed in claim 4 is used as a detection value standard, without the need for an enzymatic pretreatment step on the urine to be tested. 6. A method for improving the accuracy rate of ractopamine content in pig urine rapidly detected on the spot, characterized in that, the ractopamine pig urine freeze-dried powder standard substance of condensed state and free state coexistence as claimed in claim 4 is used as the detection value The standard product does not need to be subjected to an enzymatic hydrolysis pretreatment step for the urine to be tested, and is tested with a detection kit or test strip to detect the content of ractopamine in the urine of the pig to be tested. 7. The application of the ractopamine porcine urine freeze-dried powder standard substance of condensed state and free state coexisting in claim 4 in the supervision of pork food safety or in the detection of ractopamine content in pigs to be slaughtered. 8. The application according to claim 7, characterized in that, when the ractopamine standard substance in the condensed state and free state coexists in the pig urine freeze-dried powder after animal metabolism is used, pure water is added for dissolution, vortex oscillation, Let it stand for 3-8 minutes after ultrasound, and then use it as a standard product containing pig urine with a fixed value of ractopamine. When detecting the urine to be tested, it is not necessary to perform an enzymatic hydrolysis pretreatment step on the urine to be tested.