China Education Equipment Purchasing Network News: Since CCTV exposed Shuanghui Clenbuterol pork on March 15th, it has attracted widespread attention. "Clenbuterol" has become a popular term for online search, and it has also been a people's livelihood issue.

Clenbuterol is a kind of animal medicine. Adding Clenbuterol to the feed can increase the amount of lean meat of the animal, reduce the use of feed, make the meat market early, and reduce the cost. In China, the commonly referred to as "clenbuterol" refers to Clenbuterol, which was used as a medicine to treat bronchial asthma, and was subsequently banned because of its side effects. At present, the substance that can achieve this function is a class of drugs called beta-agonists, such as ractopamine and salbutamol, which can also play the role of "lean meat", but it is too harmful to human health, thus causing safety Hidden dangers. They are therefore banned globally. Molecular formula: C12-H18-Cl2-N2-O. White or off-white crystalline powder, odorless, bitter, melting point 161 ℃, soluble in water, ethanol, slightly soluble in acetone, insoluble in ether.

Clenbuterol was tested according to GB / T 5009.192-2003 Determination of Clenbuterol Residues in Animal Food. Common detection methods are as follows:

Gas chromatography-mass spectrometry (GC-MS)

The advantage of GC-MS method is to combine the high-efficiency and rapid separation effect of chromatography and the high-sensitivity qualitative analysis of mass spectrometry. It can qualitatively and quantitatively analyze a specific residue in the presence of multiple residues. Higher detection limit. In addition, GC-MS method has higher detection sensitivity and lower false positive rate than HPLC method. Therefore, China has established GC-MS as the confirmatory method for detecting CLB (NY / T468 ~ 2001).

Shimadzu Shanghai Analysis Center launched the Clenbuterol detection scheme based on the GC-MS method. GC-MS method has the characteristics of high sensitivity and low false positive rate, and is often used to confirm positive samples after screening. According to the chemical properties of clenbuterol hydrochloride, this scheme established a two-dimensional extraction method of C18 small column and poly (methacrylic acid-ethylene glycol dimethacrylate) monolithic column. After the sample is extracted, it is further enriched and purified using a C18 small column and a poly (methacrylic acid-ethylene glycol dimethacrylate) monolithic column, which is derivatized by N, O-bistrimethylsilane trifluoroacetamide (BSTFA) Select ion monitoring mode for gas chromatography mass spectrometry. The method uses Clenbuterol-D9 as the internal standard and the internal standard method for quantification. The detection limit of clenbuterol in pork is 0.13 μg / kg, which has a good linear relationship in the concentration range of 0.5-50 μg / kg, r is greater than 0.999. The relative standard deviation within and within the day is not higher than 20%, and the recovery rate of the standard addition is greater than 75%. The results show that the method is simple, rapid, highly sensitive and reproducible, and is suitable for the determination of clenbuterol in pork.

High performance liquid chromatography (HPLC)

HPLC is suitable for the determination of thermally unstable and highly polar β-agonists and their metabolites, and HPLC can be used in conjunction with pre-column extraction, purification and post-column fluorescence derivatization reactions and mass spectrometry (MS) systems for easy analysis Process automation. At present, China has adopted HPLC as a semi-confirmed method for the detection of CLB residues. The minimum detection limit is 1-15 ng / g. Its advantages are good specificity, strong selectivity, high detection accuracy, and low false positive rate. The disadvantage is that the sample processing time is long, and detection.

Hegong Scientific Instruments launched STI5000 series liquid chromatograph (HPLC) to detect Clenbuterol in meat products. The detection limit of this method in pig muscle and liver tissues is 0.001ug / g 'and the recovery range is 80% ± 20%.

Enzyme-linked immunosorbent assay (ELISA)

Using immunological antigen-antibody specific binding and efficient catalytic action of enzymes, plant horseradish peroxidase (HRP) and clenbuterol (CL) are chemically combined to form an enzyme-coupled clenbuterol. Combine the coated antibody (goat anti-rabbit IgG antibody) on the solid phase carrier with the specific anti-Clenbuterol antibody, and then add the test Clenbuterol and enzyme-coupled Clenbuterol. The clenbuterol antibody is combined, the substrate is added after washing, and the amount of clenbuterol to be measured is measured according to the change of the colored substance. If Clenbuterol is to be tested, less enzyme-coupled Clenbuterol is bound and the amount of colored matter is less. Determine the content of clenbuterol in the sample by visual inspection or colorimetry. The best wavelength for colorimetry is 450 nm, and the reference wavelength should be greater than 600 nm. The enzyme-linked immunosorbent assay (ELISA) has the characteristics of fast detection speed and high sensitivity, and is suitable for qualitative and rapid detection on site.

Microplate reader model suitable for Clenbuterol testing—Biochrom Anthos zenyth 340rt microplate reader

Recommended reason:

Wavelength range: 340-750nm --- the wavelength range that meets the measurement needs

Reading range: 0.000-4.000 OD—meet the reading result range

Plate reading speed: 18 seconds / 96 holes—fast detection speed

Temperature control range: room temperature + 4-45 ℃-the best reaction temperature of the kit is 25 ℃, the microplate reader with temperature control function can make the reading result more accurate

Shock mode: 3 speeds-meet the needs of shock to make the reaction proceed more fully and the results are more accurate

Standard filter: 340, 405, 450, 492, 620nm-meet the measurement wavelength of 450nm and the reference wavelength of 620nm, UV detection can also be carried out

Built-in software: ADAP Basic software-can store and output results to meet data processing

Colloidal gold immunochromatography

Using competitive colloidal gold immunochromatography technology, Clen in the detection solution combines with the gold label antibody on the gold label pad to form a complex. If the concentration of Clen in the detection solution is lower than the sensitivity value, the unbound gold label antibody flows to the T zone At the time, the Clen-BSA conjugate fixed on the membrane binds and gradually condenses into a visible T line; if the Clen concentration is higher than the sensitivity value, the gold-labeled antibodies all form a complex and will no longer bind to the C line at the T line. BSA conjugates combine to form visible T lines. Unfixed compound flows through the T zone and is captured by the secondary antibody in the C zone and forms a visible C line. The appearance of line C indicates that immunochromatography has occurred, that is, the test strip is effective.

Clenbuterol hydrochloride (clenbuterol) rapid test card (convenient and quick operation, the public can operate it) uses the principle of competitive suppression immunochromatography to determine clenbuterol hydrochloride in animal urine, tissues and feed. The reagent contains a clenbuterol hydrochloride conjugate pre-immobilized on the membrane (T) and an anti-clenbuterol monoclonal antibody labeled with colloidal gold. During detection, the sample and labeled antibody compete for binding to the conjugate. The detection limit was 3ng / ml (3ppb).

Liquid chromatography-mass spectrometry / mass spectrometry (HPLC-MS / MS)

Refer to SN / T 1924-2007 for the detection methods of residues of clenbuterol, ractopamine, salbutamol, terbutaline in foods of animal origin for import and export.

This standard is applicable to the determination of residues of clenbuterol, ractopamine, salbutamol, terbutaline in animal-derived muscle and internal organs.

The drug residue in the sample was extracted with ammonium acetate buffer solution at pH 5.2, and β-glucuronidase-arylthioesterase was added for enzymatic hydrolysis. Chromatography-mass spectrometry, internal standard method for quantification.

The process is cumbersome, difficult to operate, and requires expensive instruments, which are subject to certain restrictions in practical applications.

Recently, Shimadzu launched a method for the determination of clenbuterol, ractopamine, and salbutamol in pork by combining ultra-high performance liquid chromatography LC-30A and triple quadrupole mass spectrometer LCMS-8030.

Method for detecting clenbuterol, ractopamine and salbutamol in pork using Shimadzu ultra high performance liquid chromatography tandem mass spectrometry. First, after the sample is extracted, it is rapidly separated by ultra high performance liquid chromatography LC-30A The rod mass spectrometer LCMS-8030 was used for quantitative analysis. This method has fast analysis speed, good precision, wide linear range (0.05 ～ 100 ng / mL), and the correlation coefficient of the calibration curve is above 0.999. The quantitation limit of the method meets the requirements of the national standard of 0.5 μg / kg, and the quantitation limit of the detection method of clenbuterol in pork can reach 0.05 μg / kg, and the detection sensitivity fully meets the requirements of the Japanese positive list.

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