| Name | trenbolone--dea schedule iii item |
| Synonyms | Trembolona Trienolone Trenbolone Trenbolonum 17-beta-Trenbolone 4,9,11-Estratrien-17b-ol-3-one trenbolone--dea schedule iii item 19-Norandrosta-4,9,11-trien-17b-ol-3-one (17beta)-17-Hydroxyestra-4,9,11-trien-3-one 17b-Hydroxy-19-norandrosta-4,9,11-trien-3-one estra-4,9,11-trien-3-one, 17-hydroxy-, (17beta)- Estra-4,9,11-trien-3-one, 17-hydroxy-, (17-beta)- (9CI) |
| CAS | 10161-33-8 |
| EINECS | 600-229-1 |
| InChI | InChI=1/C18H22O2/c1-18-9-8-14-13-5-3-12(19)10-11(13)2-4-15(14)16(18)6-7-17(18)20/h8-10,15-17,20H,2-7H2,1H3/t15-,16+,17+,18+/m1/s1 |
| InChIKey | MEHHPFQKXOUFFV-OWSLCNJRSA-N |
| Molecular Formula | C18H22O2 |
| Molar Mass | 270.366 |
| Density | 1.19g/cm3 |
| Melting Point | 170℃ |
| Boling Point | 490.8°C at 760 mmHg |
| Flash Point | 208.2°C |
| Vapor Presure | 1.09E-11mmHg at 25°C |
| Appearance | neat |
| Storage Condition | 2-8°C |
| Refractive Index | 1.605 |
| Use | Androgens, anabolic hormones |
| Risk Codes | R60 - May impair fertility R36 - Irritating to the eyes R20/21/22 - Harmful by inhalation, in contact with skin and if swallowed. R11 - Highly Flammable |
| Safety Description | S53 - Avoid exposure - obtain special instructions before use. S22 - Do not breathe dust. S36/37/39 - Wear suitable protective clothing, gloves and eye/face protection. S45 - In case of accident or if you feel unwell, seek medical advice immediately (show the label whenever possible.) S36/37 - Wear suitable protective clothing and gloves. S26 - In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S16 - Keep away from sources of ignition. |
| UN IDs | UN 1648 3 / PGII |
| WGK Germany | 3 |
| RTECS | KG7752000 |
| Raw Materials | Acetic anhydride ETHYLENEDIAMINE TETRAKIS(PROPOXYLATE-BLOCK-ETHOXYLATE) TETROL Sodium tert-pentoxide Methanol Acetic acid |
| Downstream Products | trendione |
The use of Trenbolone can accelerate the aging of children's bones, the development of male breasts, and may even cause bones to stop growing. Many synthetic anabolic steroids are designed to produce more male hormones instead of feminizing them. Side effects include high blood pressure, increased cholesterol, skin acne, sexual dysfunction, and testicular atrophy. Male users may have breast enlargement. Female users have increased body hair, lower voice, smaller breasts, enlarged clitoris, and irregular menstrual periods. Some side effects are inevitable. Long-term users can also cause severe damage to liver function.
due to the androgen effect of trenbolone, long-term application in women can cause water and sodium retention and slight masculinization in women. Sometimes cause cholestasis of intrahepatic bile ducts and jaundice. Patients with nephritis, heart failure and liver dysfunction should be used with caution, and pregnant women and prostate cancer patients should be contraindicated.
Trenbolone, which has a strong role in promoting protein synthesis, is often used as an animal growth promoter for animal breeding. Trenbolone accumulates in the cells, tissues or organs of animals in the form of prototype or intermediate metabolites (β-demethylandrotrienolone, α-demethylandrotrienolone).
The hazards of trenbolone residues include the hazards to the animals themselves and the hazards to the human body due to the residues of trenbolone in animal-derived foods. Trenbolone, as an animal growth promoter, will remain in the liver, kidney and muscle of eating animals. Trenbolone residue refers to the use of Trenbolone in the process of animal breeding. Trenbolone accumulates in animal cells, tissues or organs with its original shape or intermediate metabolites. Once eaten by humans, it can produce a series of hormone-like effects, such as destroying the hormone balance of the human body, interfering with human endocrine function, affecting fertility, and has potential carcinogenicity, developmental toxicity (precocious puberty in children) and female masculinization, Large doses can cause liver dysfunction. The European Union has banned the use of growth hormone in the breeding of food animals since 1998. In 2004, the European Union put forward 18 types of veterinary drug residues and antibiotic detection and monitoring requirements for my country's livestock and poultry products, which clearly pointed out that in animal-derived food drug residues, Qunbolone cannot be detected. Announcement No. 193 issued by the Ministry of Agriculture of my country in April 2002, "List of Veterinary Drugs and Other Compounds Prohibited for Food Animals" also clearly stated that sex hormone raw materials and their unilateral and compound preparation products are not allowed to resist stress, increase feed remuneration, and promote The growth of animals is used in the breeding process of all food animals. The International Codex Alimentarius Commission (CAC), the United States, Japan and other organizations and countries have strict requirements on the residues of anabolic hormones in animal food. Document No. 235 issued by the Ministry of Agriculture of my country in 2002 stipulates that the maximum residue limit of testosterone propionate, nandrolone phenylpropionate, methyltestosterone, and trenbolone in animal foods shall not be detected in animal foods.
Most anabolic hormones produce effects through testosterone activity, such as anabolism (Anabolism) and masculinization (virilization). Examples of assimilative utility: promote protein biosynthesis in amino acids, promote muscle growth, promote appetite, promote bone and iliac growth, stimulate bone marrow, and promote the production of red blood cells. The effect of promoting synthesis in increasing muscle circumference is stronger than testosterone or vigorous supplement.
Trenbolone is a synthetic triene 19-desteryl compound with complex properties. Its anti-estrogen and anti-progesterone activity can be strong or weak. Acting on the lower part of the thalamus. The pituitary axis reduces the release of gonadotropin, and the peak before ovulation disappears, thus inhibiting ovulation. Animal experiments show that it can inhibit the secretion of progesterone, and also has the effect of progesterone on the endometrium, which inactivates and degenerates the cells of the endometrium and ectopic lesions, which leads to the atrophy of the ectopic lesions. Its anti-fertility effect may be to inhibit ovulation and inhibit endometrial development, change the nature of cervical mucus, affect the running speed of eggs and antagonize endometrial progesterone receptors, thereby interfering with the implantation of pregnant eggs. It is clinically used to treat endometriosis, which shrinks and absorbs it. Because of its strong role in promoting protein synthesis, with the large-scale and intensive development of animal husbandry, a small number of production enterprises, breeders and operators, in order to seek maximum profits, ignore national laws and regulations, abuse or illegally use hormones and other illegal drugs. As an assimilative hormone additive, Trenbolone keeps livestock, which often causes the residue of Trenbolone in animal-derived food to exceed the standard. When people eat animal food with excessive residues, it will accumulate in the human body, resulting in a variety of adverse consequences, directly endangering human health and life.
Method 1: A high-yield synthesis method of normetamethirone acetate:
1) etherification reaction: 80kg of alcohol is put into a 500L reaction tank to cool the temperature to 0 ℃, stir, add 10kg of acetyl chloride dropwise, and control the temperature to 0~10 ℃. After dropping, 10kg of estrogen 4,9 diene 3,17-diketone is put in, and stirred at 10~13 ℃ for 2 hours. After the heat preservation is completed, the material liquid is pumped into the sodium carbonate aqueous solution, the temperature is controlled at 20~25 ℃, the temperature is controlled at 20~25 ℃ and stirred for 30 minutes after dropping, and the material is washed to be neutral with a large amount of water, centrifuged to obtain wet material.
2) reductive hydrolysis dehydrogenation reaction: 100kg of methanol is pumped into a 500L reaction tank, the reaction wet material is put into the previous step, the temperature is reduced to 0 ℃, 5kg of potassium boron hydrogen is added, the temperature is controlled at 0~5 ℃ when added, the addition is completed, and the stirring is kept for 1.5 hours. Point board. After the point board is qualified, add glacial acetic acid to adjust the PH value to neutral, concentrate to the oil under reduced pressure, dissolve the oil with 200kg of dichloromethane, and wash the neutral with 150kg of tap water each time after complete dissolution, about three times, and concentrate to the oil after washing. After concentration, 70kg of methanol is added to the oil, dissolved and completely dissolved, the temperature is reduced to below 10 ℃, 5kg of reagent hydrochloric acid is added, the PH1 ~ 2 is adjusted, the temperature is kept at 10~15 ℃ for 2 hours, and the plate is set. After the point plate is qualified, add NaOH water to adjust the PH value to neutral, adjust it, concentrate to dry, add 200kg dichloromethane to dissolve, and add 150kg of water to wash three times each time after complete dissolution. After washing with water, add 10kg of anhydrous magnesium sulfate for dehydration. After dehydration, anhydrous magnesium sulfate is filtered out, and the filter cake is rinsed with appropriate amount of dichloromethane. After filtering and rinsing, 9kg of DDQ was added at 0~5 ℃ for 3 hours during filtering night. Point plate, point plate after qualified, suction filtration, with appropriate amount of dichloromethane to wash DDQ rinse, dichloromethane layer plus 200kg of 30% sodium carbonate water wash. After stratification, the organic layer is repeated twice, 1000kg of tap water is used, and the organic layer is washed three times. After washing with water, concentrate under reduced pressure to a thick paste, cool to 0~5 ℃, centrifuge, and dry at 90100 ℃ to remove methylandrotrienolone.
3) acylation reaction: in a 300L glass-lined reaction pot, open the vacuum valve and pump in benzene 100 ㎏ and pyridine 10 ㎏. Then open the feeding port and put 10kg of desmetaandrotrienolone (desmetatrienolone),0.5 kg4DMAP, add 10kg of acetyl chloride, control the temperature at 0~10 ℃, control the dripping within 1 hour, and keep the temperature for 0.5 hours. After the plate is qualified, add acid and water to stir for 30 minutes, delaminate, add 100kg of water to wash three times each time, wash with water, and concentrate until dry under reduced pressure. Add 10kg of ethyl acetate to dissolve, and add 25kg of petroleum ether to cool down to 0~5 ℃ for centrifugation.
Method 2: A method for synthesizing desmethylandrotrienolone acetate (desmethylandrotrienolone), comprising the following steps.
step 1) reduction reaction: 4,9 ring-opening substance (I) is dissolved in methanol, potassium boron hydrogen is added in batches at 20~25 ℃, after addition, heat preservation reaction until TLC analysis raw material reaction is complete, and compound (II) is obtained.
step 2) hydrolysis reaction: add dilute acid to the reaction system that completes step 1) reaction to adjust the pH value to neutral, control the temperature by 20~25 ℃, add dilute sulfuric acid dropwise into the above reaction system, finish dropping, stir the reaction until the TLC analysis raw material reaction is complete, add the reaction liquid to sodium carbonate solution for water analysis, stir until the solid is completely precipitated, and control the temperature below 50 ℃ to concentrate under, stir with water, centrifuge and dry to obtain 17 β-hydroxy-estradi-5 (10),9(11)-diene -3-one (III).
step 3) dehydrogenation reaction: compound (III) is dissolved in dichloromethane, DDQ (dichloro dicyanobenzoquinone) is added, the temperature is controlled by 20~25 ℃, the reaction is stirred until the TLC analysis raw material reaction is complete, and the suction is filtered. The filtrate is washed with alkaline reducing agent solution and alkaline solution respectively, the water layer is merged, and the water layer is extracted with dichloromethane; all dichloromethane layers are merged, dehydrated and concentrated under reduced pressure until there is no distillate solution, and ethyl acetate is added to entrain and distillate residual dichloromethane. After distilling all dichloromethane, ethyl acetate is added to cool down and crystallize, and centrifuge. Drying to obtain desmethylandrotrienolone (desmethylandrotrienolone)(IV).
step 4) esterification reaction: demethylandrotrienolone (v) is dissolved in dichloromethane, DMAP (4-dimethylaminopyridine) and acetic anhydride are added, the temperature is controlled at 20~25 ℃, the reaction is stirred until the TLC analysis raw material reaction is complete, the sodium carbonate solution is added to wash to pH8 ~ 9, the water layer is merged, and the water layer is extracted, after concentrating under reduced pressure until there is no distillate, isopropyl ether is added to entrain and distillate the residual dichloromethane, distillate all dichloromethane and then add isopropyl ether, cool down, crystallize, centrifuge, and dry to obtain acetic acid to demethylandrotrienolone (demethylandrotrienolone);
the sample matrix of hormone residue detection is complex, there are many interfering substances, and the concentration is very low (the level is 1 Pg/kg-1mg/kg). the detection of animal-derived food hormone residue requires accurate qualitative or accurate quantification, and the sensitivity, accuracy and specificity of the method are extremely high. At present, detection methods can be roughly divided into two categories: chromatography/mass spectrometry and immunological methods. High performance liquid chromatography (HPLC) and gas chromatography were used to analyze anabolic hormones in animal tissues. Mass spectrometry (GCoMS) and liquid chromatography. Mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) liquid chromatography-tandem mass spectrometry (LC-MS-MS). Immunological methods mainly use the principle of specific binding of antigen and antibody. Labeling different markers can establish corresponding immunoassay methods. Common ones include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (Radio imunoassay,RIA), chemiluminescence immunoassay (Chemiluminescent immnoassay,CLEIA), fluorescence immunoassay technology (F team) and capillary electrophoresis detection technology.
1, chromatography/mass spectrometry
(1) high performance liquid chromatography (HPLC)
The working principle of high-performance liquid chromatography is basically the same as that of gas chromatography. It uses liquid as the mobile phase and uses a high-efficiency stationary phase with extremely fine particles. Column chromatography separation technology. Gas chromatography uses gas as the mobile phase. According to the properties of stationary phase, high liquid chromatography can be divided into adsorption chromatography, bonding chromatography, ion exchange chromatography and size exclusion chromatography. High performance liquid chromatography is not limited by the volatility and thermal stability of the analysis object, so it has a wide range of applicability to the sample.
(2) gas chromatography-mass spectrometry (CC-MS)
gas chromatography. Mass spectrometry is the main use of the International Anti-Doping Center to detect protein assimilation and excitation. The principle process is that when a mixture sample is injected into the inlet of the gas chromatograph, it is separated in the chromatographic column, and each component is different The retention time leaves the chromatographic column outlet. The carrier gas is removed through the molecular separator, and only the component molecules enter the ion source. After ionization, molecular ions and fragment ions are focused into ion beams and sent to the mass filter. By selecting the electric field parameters of the mass filter, ions with different mass-to-charge ratios can sequentially pass through the mass filter, which is received and amplified by the secondary electron multiplier to form a mass spectrum, I .e. the abscissa is the mass-to-charge ratio (m,e), and the ordinate is a graph of the intensity of ion flow, which can be qualitative to the compound. In addition, during the outflow of the compound from the column, the ions of each m/e in each mass spectrum repeatedly scanned can be added to form a total ion flow chromatogram, thereby quantitatively analyzing the compound. The whole working process is controlled by the computer to adjust, operate, data acquisition and processing of the instrument, and the testing process is carried out under vacuum conditions. Suitable for analysis of small molecules, volatile, thermally stable, and vaporizable compounds.
gas chromatography. Mass spectrometry has been widely used in the detection of trenbolone since the 8. 1990 s. Shih.Hsien Hsu is equal to the detection of TR in bovine tissues in 1988, and the detection limit of TR in beef and viscera is 0.5ppb. CaSadmont et al. extracted calf urine by reverse separation column and purified by column. The detection limit of TR reached 0.5ng/mg by GC-MS detection. There are also related reports in China.
(4) liquid chromatography-tandem mass spectrometry (LC-MS-MS)
LC-MS is an important modern separation and analysis technology. It combines a wide range of separation methods with sensitive and exclusive mass spectrometry that can provide relative molecular mass and structural information, but its disadvantage is that the matrix is difficult to eliminate the interference of the components to be tested and the structural information of the components to be tested cannot be fully utilized. The combination of liquid chromatography and tandem mass spectrometry (LC-MS-MS) obtains a strong excimer ion peak of the component to be tested under the condition of first-level mass spectrometry MS, and almost no fragment ions are generated, and the excimer ions can be subjected to multi-stage lysis, And then obtain rich compound fragment information, so as to infer the structure of the compound, confirm the target compound, identify the overlapping chromatographic peaks, and quantify the target compound in the presence of high background or, therefore, it has become a more powerful tool in the study of drug metabolism processes and products, the identification and quantitative determination of a component in complex components, and the study of medicinal plant components. Liu Qing et al. obtained clear chromatograms of trenbolone and budione by selecting ions. the detection limit of trenbolone is O.31ug/kg. Mara Gasparinit et al. used affinity immune technology to extract urine trenbolone metabolites, and the detection limit was between 0.54 and 0.60ug/L by LC-MS-MS detection. F Buiarellia et al. used LC-MS-MS to determine TR in bovine urine and serum. The detection limit (1imit ofdetection,LOD) and quantification limit (1imit of quantification,LOQ) were 350 pg/ml and lng/ml respectively.
2. immunological methods
immunological methods mainly use the principle of specific binding of antigen and antibody. labeling different markers can establish corresponding immunoassay methods. common ones include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (Radio imunoassay,Rta), chemiluminescence immunoassay (Chemiluminescent immnoassay,CLEIA), fluorescence immunoassay (CLEIA), etc. However, RIA is prone to contamination due to the radioactivity of the markers used. In recent years, it has been replaced by other immune labeling methods. At present, ELISA (Germany) is the immunological method used for TR detection abroad. CLEIA, CLEIA and colloidal gold have not been reported for TR detection.
Enzyme linked immunosorbent assay (ELISA) is the result of amplifying the immune response using the high catalytic efficiency of the enzyme, making the assay extremely sensitive. The basic principle is to bind the known antibody or antigen to the solid phase carrier, add the antigen or antibody to be tested, and then add the labeled antibody or antigen or second antibody, and develop color through the developer, according to the color of the reaction product Qualitative or quantitative detection can be carried out. The coating and enzyme markers maintain their immune activity. During the measurement, the sample to be tested and the enzyme-labeled antigen or antibody are reacted with the antibody or antigen adsorbed on the surface of the solid phase carrier according to different steps, and the antigen-antibody complex and free components are removed by washing, and then the enzyme substrate is added to catalyze color development, Qualitative or quantitative determination is carried out according to the color development.
The World Food and Agriculture Organization (FAO) recommends this technology to many countries, and the American Chemical Society also lists ELISA as one of the pillar technologies for pesticide residue analysis. Domestic research in this area has been carried out relatively late, but some progress has also been made. This method can greatly simplify the residue analysis including pre-treatment steps. Compared with chromatography/mass spectrometry, the ELISA method has the advantages of strong specificity, high sensitivity, simple operation, low instrumentation and analysis cost, simple sample pretreatment, short detection time, and large sample capacity. There is no limitation of HPLC or GC on the physical and chemical properties of the measured object such as vapor pressure, thermal stability, halogen, chromophore, etc., which is suitable for on-site monitoring, large-scale samples, and rapid screening of the grassroots, it is one of the most ideal residual screening analysis methods at present.
It is imperative to monitor and enforce the production process of animal-derived products. One is to formulate mandatory standards for the maximum residue limit, and the other is to improve residue analysis methods and technologies, and develop fast, accurate and sensitive quality detection methods. The main content of this study is to prepare TR-BSA conjugates and give TR immunogenicity, which lays a foundation for the preparation of trenbolone monoclonal antibody and the establishment of a rapid detection method for trenbolone residues in food. Establish a rapid and sensitive TR detection method that is convenient for the application of grassroots inspection and quarantine departments to monitor and control the diffusion of such substances from the source.
Trenbolone,TRE) is a widely used steroid anabolic hormone, which is a chemically synthesized derivative similar to the human male hormone testosterone in structure and activity. It can promote protein synthesis, increase appetite, increase muscle growth, and promote calcium and phosphorus in bone tissue. It can be used clinically to treat severe nutritional deficiencies and osteoporosis and other diseases, but it is also the most frequently used type of sports stimulant. In addition, in livestock production, demethylandrotrienolone plays an important role in animal disease control and treatment.