What are the methods for detecting clenbuterol

Clenbuterol was originally used to treat bronchial asthma, but its side effects on the human body are so severe that it has long been banned worldwide. However, many meat merchants use it in feed to reduce costs. If humans consume meat with this residue for a long time, it will cause adverse effects on the cardiovascular system.

Thereby inducing malignant tumors. Therefore, the detection of clenbuterol in meat products must be controlled from the source. Currently, there are several detection methods:

Gas chromatography-mass spectrometry (GC-MS)

The advantage of the GC-MS method is that it combines the efficient and rapid separation effect of chromatography with the highly sensitive qualitative analysis of mass spectrometry. It can perform qualitative and quantitative analysis on a specific residue when multiple residues exist at the same time, and has a higher detection limit. Fente C. A et al. used GC-MS to detect the residues of CLB in cattle hair, with a minimum detection limit of 5 ng/g; Pteer Batioens used gas chromatography-tandem mass spectrometry (GC-MS-MS) to detect the CLB content in cattle, sheep and pig tissues, with a minimum detection limit of 2 ng/g; 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; Van Rhijin et al. used trimethylsilyl or 2-dimethylsilylmorpholine derivatives to detect CLB in urine extracts. The derivatives were scanned using electric pulses or chemical ionization, which would produce higher sensitivity. In addition, compared with the HPLC method, the GC-MS method has higher detection sensitivity and lower false positive rate. Therefore, my country has legally established GC-MS as a confirmatory method for detecting CLB (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. Moreover, HPLC can be combined with pre-column extraction, purification, post-column fluorescence derivatization reaction and mass spectrometry (MS) systems to easily automate the analysis process. Huang Shixin et al. (1995) used an ultraviolet detector to detect CLB residues in pig liver and pork at λ=243nm, chromatographic column: shimpack CLC-ODS150×6.0mn, flow rate: 1mL/min, column temperature: room temperature 30℃, and the minimum detection limit could reach 2ng/g. Some people abroad used HPLC (diode array detector) to determine CLB residues in animal foods, and the minimum detection limit was 1.26ng/g, with a recovery rate of 98.9%. At present, my country has used HPLC as a semi-confirmatory method for detecting CLB residues, with a minimum detection limit ranging from 1 to 15 ng/g. Its advantages are good specificity, strong selectivity, high detection accuracy, and low false positive rate; its disadvantages are long sample processing time, cumbersome and difficult detection process, and the need for expensive instruments, which are subject to certain limitations in practical applications.

Enzyme-linked immunosorbent assay (ELISA)

By utilizing the specific binding of immunological antigens and antibodies and the efficient catalytic effect of enzymes, plant horseradish peroxidase (HRP) is combined with clenbuterol (CL) through a chemical method to form enzyme-conjugated clenbuterol. The antibody (sheep anti-rabbit IgG antibody) coated on the solid phase carrier is combined with the specific anti-clenbuterol antibody, and then the clenbuterol to be tested and the enzyme-coupled clenbuterol are added. They competitively bind to the clenbuterol antibody. After washing, the substrate is added and the amount of clenbuterol to be tested is measured according to the change of the colored substance. If there is more clenbuterol to be tested, there will be less enzyme-coupled clenbuterol and the amount of colored substance will be less. The clenbuterol content in the sample was determined by visual inspection or colorimetry. The optimal wavelength for colorimetry was 450 nm and the reference wavelength should be greater than 600 nm.

Colloidal gold immunochromatography

Using competitive colloidal gold immunochromatography technology, Clen in the test solution combines with the gold-labeled antibody on the gold-labeled pad to form a complex. If the concentration of Clen in the test solution is lower than the sensitivity value, the unbound gold-labeled antibody flows to the T zone and is bound by the Clen-BSA conjugate fixed on the membrane, gradually agglomerating into a visible T line; if the Clen concentration is higher than the sensitivity value, all the gold-labeled antibodies form a complex and will no longer combine with the Clen-BSA conjugate at the T line to form a visible T line. The unfixed complex flows through the T zone and is captured by the secondary antibody in the C zone, forming a visible C line. The appearance of line C indicates that immunochromatography has occurred, which means that the test paper is effective.

Folding test method

1. Read the instruction manual completely before testing, and return the reagent plate and sample solution to room temperature before use.

2. Tear open the aluminum-plastic bag and take out the test paper.

3. Operate according to the model (urine must not directly soak the observation area).

3.1 Dip the white end of the test paper into the urine (the liquid level must not exceed the horizontal line) and keep it for 5 seconds;

3.2 Use a dropper to absorb the urine sample to be tested and slowly add 3 drops of sample into the sample well.

4. Lay the test strip flat for 1 minute and wait for the red strip to appear.

5. Read the result within 3 to 8 minutes. The result will be invalid after 10 minutes.

6. Results can be obtained within 10 minutes

Folding result interpretation

Positive (+): Only a purple-red band appears in the quality control area (C). No purple-red bands appeared in the test area (T).

Negative (-): Two purple-red bands appear. One is located in the test area (T) and the other is located in the quality control area (C).

Invalid: No purple-red band appears in the quality control area (C), indicating that the test has failed or the test paper has expired.

Note: The purple-red strip in the test area (T) may show different shades of color. However, within the prescribed observation time, regardless of the color depth of the band, even a very weak band should be judged as a negative result.

Folding Notes

1. Please use the test card once within the shelf life.

2. Avoid direct sunlight and electric fans during testing.

3. Try not to touch the white film surface in the center of the test card.

4. Urine sample droppers should not be mixed to avoid cross contamination.

5. If there is precipitation or turbidity in the urine sample, please centrifuge it before testing.

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

See SN/T

1924-2007 Detection method for residues of clenbuterol, ractopamine, salbutamol and terbutaline in foods of animal origin for import and export.

This standard applies to the detection of clenbuterol, ractopamine, salbutamol and terbutaline residues in muscle and viscera of animal-derived foods.

The drug residues in the sample were extracted with ammonium acetate buffer at pH 5.2, and β-glucuronidase-aryl thioesterase was added for enzymatic hydrolysis. The extract was purified by C18 and SCX double SPE columns and determined by liquid chromatography-mass spectrometry and quantified by the internal standard method.