Introduction of lean meat and its detection method

In our country, what is commonly referred to as "clenbuterol" refers to Clenbuterol. It was once used as a medicine to treat bronchial asthma and was later banned due to its side effects.

Clenbuterol is a type of animal drug, and several drugs are called clenbuterol, such as Ractopamine and Clenbuterol. Adding lean meat to the feed can increase the lean meat content of the animal, reduce the use of the feed, bring the meat to the market, and reduce the cost. However, because of the side effects that are caused to the human body, the standards for open use in various countries vary.

Other such similar drugs, such as salbutamol and terbutaline, can also play a role of "lean meat", but they are too harmful to human health, thus posing a safety hazard. They are thus banned globally.
English name:
Clenbuterol; Spiropent; Planipart; NAB365; 4-Amino-3,5-dichloro-alpha-(((1,1-dimethylethyl)amino)methyl)benzenemethanol
CAS No.: 37148-27-9

Clenbuterol structure simple formula: C12-H18-Cl2-N2-O

Physical and chemical properties White or white-like crystalline powder, odorless, bitter, melting point 161 ° C, soluble in water, ethanol, slightly soluble in acetone, insoluble in ether.

Detection method
GB/T 5009.192-2003 Determination of Clenbuterol Residues in Animal Foods
Gas chromatography-mass spectrometry (GC-MS)
The GC-MS method combines the efficient and rapid separation of chromatograms with the qualitative analysis of high-sensitivity mass spectrometry. It can qualitatively and quantitatively analyze a specific residue in the presence of multiple residues. Higher detection limits. Fente C. A et al. used GC-MS to detect CLB residues in bovine hair with a minimum detection limit of 5 ng/g [6]; Pteer Batioens applied gas chromatography-tandem mass spectrometry (GC-MS-MS) to cattle, sheep and pigs. The CLB content in the tissue was detected. The minimum detection limit was 2 ng/g [7]; Liu Qi et al. used GC-MS (EI ion source) to detect CLB in pig urine with a detection limit of 0.5 ng/mL; VanRhijin et al. Trimethylsilyl or 2-dimethylsilylmorpholine derivatives detect CLB in urine extracts, and the derivatives are scanned by electrical pulse or chemical ionization to produce higher sensitivity. In addition, the GC-MS method has higher detection sensitivity and lower false positive rate than the HPLC method. Therefore, China has legalized GC-MS as a confirmatory method for detecting CL B (NY/T468~2001).
High performance liquid chromatography (HPLC)
HPLC is suitable for the determination of thermally unstable and highly polar β-agonists and their metabolites. Furthermore, HPLC can be combined with pre-column extraction, purification, post-column fluorescence derivatization and mass spectrometry (MS) systems for easy analysis. Automation of the process. Huang Shixin et al (1995) applied UV detector to detect CLB residues in pig liver and pork at λ=243nm, column: shimpackCLC-ODS150×6.0mn, flow rate: 1mL/min, column temperature: room temperature 30°C. The minimum detection limit is 2 ng/g [4]. In foreign countries, HPLC (diode array detector) was used to determine CLB residues in animal foods. The minimum detection limit was 1.26 ng/g, and the recovery rate was 98.9% [5]. At present, HPLC has been used as a semi-positive method for detecting CLB residues in China. The minimum detection limit is 1-15 ng/g, which has the advantages of good specificity, strong selectivity, high detection accuracy, and low false positive rate. The disadvantage is that the sample processing time is long, the detection process is cumbersome, difficult to operate, and requires expensive instruments, which is subject to certain limitations in practical applications.
Enzyme-linked immunosorbent assay (ELISA) ThermoMK3 FC microplate reader
The plant horseradish peroxidase (HRP) is chemically combined with clenbuterol (CL) to form an enzyme-coupled clenbuterol using immunological antigen-antibody specific binding and high-efficiency catalysis of the enzyme. The coated antibody (goat anti-rabbit IgG antibody) on the solid phase carrier is bound to the specific anti-Clenbuter antibody, and then the Clenbuterol and the enzyme-conjugated Clenbuter are added, which are competitive with The clenbuterol antibody binds, and after washing, a substrate is added, and the amount of clenbuterol to be measured is measured according to the change of the colored substance. If the Clenbuterol is to be tested, the bound enzyme is less coupled with clenbuterol, and the amount of colored matter is less. The Clenbuterol content in the sample is determined by visual or colorimetric methods. The optimal wavelength for colorimetric is 450 nm and the reference wavelength should be greater than 600 nm.
Liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS)
See SN/T 1924-2007 for the detection of residues of clenbuterol, ractopamine, salbutamol and terbutaline in imported and exported animal foods.
This standard applies to the detection of residues of clenbuterol, ractopamine, salbutamol and terbutaline in muscle and viscera of animal-derived foods.
The drug residue in the sample was extracted with ammonium acetate buffer of pH 5.2, and β-hydroglucose hydrazide-aryl thioesterase was added for enzymatic hydrolysis. The extract was purified by C18 and SCX double SPE column. Chromatography-mass spectrometry was performed and quantified by internal standard method.

Research history
Clenbuterol is a beta-agonist that can be added to feed to increase the lean rate of several livestock, including pigs.
Clenbuter is well absorbed in livestock and humans, and its bioavailability is high compared to other beta-agonists, resulting in the poisoning of pork containing Clenbuterol. Zimmer (1976) confirmed that humans took 20 μg of 14C-Clenbuterol at a peak concentration, and the plasma clenbuterol drug accounted for 75% of the total 14C-Clenbuterol. The fact that Clenbuter has a high bioavailability is important for estimating the amount of Clenbuter obtained by consumers who consume Clenbuterol. The amount of clenbuterol-like drugs in animal tissues that cause human poisoning varies widely. Martinez-Navarro (1990) reported that the residual amount of clenbuterol in yak liver was 161-291 ppb. Pulce et al. (1991) reported that the residual amount of clenbuterol in yak liver was 375-500 ppb, Salleras. Et al. (1995) reported that the residual amount of clenbuterol in yak liver was 19-5395 ppb. In addition, Maistro et al. (1995) reported that the residual amount of clenbuterol in calf muscle was 500 ppb.
1 ppm of Clenbuterol is added to pig feed for growth promotion, and human consumption of pig liver or pig lung is sufficient to cause poisoning. There is no formal institution in the world that approves Clenbuterol as a feed additive for animal growth.

Introduction to Toxicology Clenbuterol is able to agonize β2-receptors, has a stimulating effect on the heart, and has a strong and long-lasting expansion effect on bronchial smooth muscle. After oral administration, it is easier to be absorbed through the gastrointestinal tract. As an asthmatic drug, oral adult 20-40μg/time, 3 times/day; children over 5 years old 5-20μg/time, 3 times/day. Human (female) oral TDLo: 4600 ng/kg. Mouse vein LD50: 27600 ug/kg.
Clinical manifestations 1. Acute poisoning has palpitations, facial and neck muscles, muscle vibrations, hand shaking or even standing, dizziness, fatigue, patients with arrhythmia are more likely to respond, tachycardia, ventricular premature beats, ECG ST segment The depression is inverted with the T wave.
2. Patients with pre-existing sympathetic hyperactivity, such as hypertension, coronary heart disease, hyperthyroidism, the above symptoms are more likely to occur.
3. In combination with glucocorticoids can cause hypokalemia, resulting in arrhythmia.
4. Repeated use will produce tolerance, weakened bronchodilation and shortened duration. Although the toxic effects of clenbuterol remain mild, FDA studies have shown that those who use sympathomimetic or who are allergic to prodrugs are more likely to respond to clenbuterol than normal healthy individuals.
The FDA is concerned that the illegal use of Clenbuter can cause illness or death in the production workers of this drug. The FDA noted that the cardiovascular effects of occupational inhalation of clenbuterol may be more harmful than food intake, but further confirmation is needed.
First-aid treatment 1. After oral administration, gastric lavage and infusion will be carried out to promote the discharge of poison.
2. Under the ECG monitoring and electrolyte measurement, use a protective heart drug such as fructose 6-diphosphate (FDP) and β1-receptor blocker Betaloc.

Identification and prevention
Identification method (1) To see if the pork has fat (lard), if the pork is lean or only a small amount of fat under the skin, the pork may contain "clenbuterol".
(2) The lean meat fed the “Clenbuterol” is particularly bright red, the back hip is larger, the fiber is looser, and the pork cut into two or three fingers is softer and can not stand on the case. There is a yellow liquid between lean meat and fat. Outflow, fat is particularly thin; and generally healthy lean pork is light red, meat is elastic, and there is no liquid flowing between lean meat and fat.
(3) At the time of purchase, it must be seen whether the pork is covered with a quarantine seal and a quarantine certificate.

Prevention method 1. Control the source, strengthen the promotion of regulations, and prohibit the incorporation of lean meat in the feed.
2. Strengthen the inspection of listed pork.
3. When consumers buy pork, they should pick some fat (1-2cm) meat, the color should not be too bright red, and the pig's internal organs should not be eaten because of the residual amount of lean meat.
Other related dosages dictate that many chemicals are called clenbuterol, and that Palin (retopaamine, ractopamine, Ractopamine) is extremely toxic and has rapid metabolism (no accumulation), so it is allowed to be added to pig feed by countries such as the United States. Japan also allows the import of pork from Ractopamine. At present, there are 24 countries in the world that open to use Palin, and more than 160 countries are still banned.