Clenbuterol detection method - from on-site rapid screening to laboratory confirmation

Clenbuterol, commonly known as clenbuterol, is a beta-stimulant because it can increase lean meat percentage, reduce fat deposition, and promote animal growth. It is illegally used by some livestock husbandry companies as breeders. After humans consume livestock products produced by animals fed clenbuterol as an additive, residual clenbuterol can cause muscle tremors, palpitations, nervousness, headaches, dizziness, nausea, vomiting, fever, and tremors. , causing great harm to the health of consumers. Therefore, many countries in the world have now been banned from adding clenbuterol to feeds to increase the lean meat percentage of animals.

The Ministry of Agriculture of the People's Republic of China stipulates that the residual standards for clenbuterol hydrochloride in animals are: horse and cattle muscle 0.1 ug/kg, liver, kidney 0.5 ug/kg milk 0.05 ug/kg

1, on-site rapid screening products

Clenbuterol (Clenbuterol Hydrochloride) colloidal gold test card for qualitative detection of clenbuterol residues in animal urine (such as pig urine, cow urine, sheep urine, etc.), the entire detection process takes only 5-10 minutes Left and right, the sensitivity is 3 ng/ml (3 ppb). Fast and convenient, suitable for on-site rapid detection in slaughterhouses, farmers' markets, and supermarkets.

Order No. Product Name Specimen Sensitivity Colloidal Gold Test Card HQ09001 Clenbuterol Hydrochloride (Urinary) 50 Bars/Box 3 ppb HQ09001-1 Clenbuterol Hydrochloride (Organization) 50 Bars/Box 1 ppb

2, rapid detection of products

The Clenbuterol (Clenbuterol Hydrochloride) ELISA Kit uses the enzyme-linked immunosorbent assay kit to detect clenbuterol hydrochloride in urine, muscle, liver, and feed. It was also used to process 96 samples with an operating time of less than 45 minutes and a sensitivity of 0.025-0.1 ng/m (ppb). With economic, rapid and other advantages. Qualitative semi-quantitative, more accurate than colloidal gold test results, suitable for rapid laboratory testing.

ELISA Rapid Detection Kit Order Code Product Name Specification Sensitivity HE09002 Clenbuterol Hydrochloride 96-well 0.1ppb HE09002-1 Clenbuterol Hydrochloride 96-well 0.05ppb HE09002-2 Clenbuterol Hydrochloride 96-well 0.025ppb

3, quasi-deterministic quantitative products

The use of SPE-GC/MS or SPE-LC/MS can quantitatively quantify Clenbuterol in animal tissue, urine, liver, feed, animal products, etc., and provide confirmation data. Suitable for laboratory confirmation experiments or issue legal basis reports.

Solid phase extraction column: Cleanert PCX 150mg/6mL (P/N: CX1506) GC column: DA-5MS, 30m × 0.25mm × 0.25μm (P/N: 1525-3002) Reference configuration: 12-bit SPE vacuum device ( P/N: VM12)

Detection method is as follows

Experimental material

1.1 Solid phase extraction cartridge: Cleanert PCX (150mg/6mL) P/N: CX1506

1.2 Four kinds of β-agonist drugs: 4 kinds of β-agonist drugs such as clenbuterol, salbutamol, cimaterol, ractopamine.

2. Preparation of samples

Accurately weigh 10g of minced tissue sample into a 50mL stoppered centrifuge tube. Add 20mL/L ammonium acetate buffer solution 20mL and homogenize for 5min. Add 50uL of β-glucuronidone HCl solution, shake, sonicate for 15min, and digest at 37°C for 18h. Centrifuge at 3000r/min for 5 min, filter the supernatant and collect the filtrate.

3. Purification

The solid phase extraction cartridge was washed with 5 mL of methanol, 5 mL of water and 5 mL of hydrochloric acid (5 mmol/L) successively. The above stock solution was passed through the column, sequentially rinsed with 5 mL of water and 5 mL of methanol, vacuum-evacuated and eluted with 4% ammoniated methanol (5 mL). The PCX cartridge was collected and the eluate was collected in a stoppered glass tube and dried with nitrogen at 50°C. The flow rate was controlled at about 1 mL/min during the passage of the sample solution and elution.

4. Derivatization and detection

The stoppered, stoppered glass test tube was placed in a 50° C. oven and heated for a while to remove water. Then 100 mL of toluene and 100 mL of bistrimethylsilyltrifluoroacetamide (BSTFA) were added and vortexed for 20 seconds to seal the glass stopper. , placed in a constant temperature oven at 80°C for 1 hour, cooled and then added 300 mL of toluene as a sample solution for gas chromatography-mass spectrometry (Column: DA-5MS, 30m × 0.25mm × 0.25μm, P/N: 1525-3002).

5. Results

5.1 Recovery Experiments (Precision and Accuracy)

After a preliminary treatment of the liver samples with liquid-liquid extraction, a certain amount of standard solutions were added to prepare sample solutions of 5 μg/L, 2 μg/L, 5 μg/L, 10 μg/L, and 100 μg/L each. Perform 5 parallel experiments at the same concentration in the batch, for a total of 4 batches (see the attached figure for typical recovery chromatograms).

The experimental results in pig liver are as follows:

Addition concentration (μg/L) Recovery concentration (μg/L) Average recovery value (μg/L) Average recovery (%) Relative standard deviation (%) 1 0.75 0.72 72.40 5.93 0.67 0.72 0.70 0.78 2 1.62 1.63 81.30 1.23 1.66 1.60 1.61 1.64 5 4.02 4.24 84.80 4.16 4.10 4.27 4.38 4.43 10 8.24 8.45 84.45 2.81 8.35 8.77 8.62 8.25 100 90.24 9.12 91.15 2.86 87.15 91.77 92.62 93.95

5.2 Repeatability Experiment (Interassay Error Experiment):

The experimental results in pig liver are as follows:

Batch Addition Concentration (μg/L) 1 2 5 10 100 Mean Recovery % RSD % Mean Recovery % RSD % Mean Recovery % RSD % Mean Recovery % RSD % Mean Recovery % RSD % 1 72.40 5.93 81.30 3.49 84.80 6.16 84.45 3.59 91.15 2.86 2 75.37 6.12 80.47 5.37 84.74 7.55 87.46 4.68 90.05 3.86 3 70.09 7.85 80.80 6.57 83.10 8.17 83.21 5.39 89.53 4.16 4 76.73 4.90 78.50 8.35 82.90 5.11 85.95 5.72 88.27 5.93 Average 73.65 6.20 80.25 5.95 83.88 6.75 85.27 4.84 89.75 4.20 RSD% 12.95 10.79 9.43 7.00 5.75

Figure: Total ion chromatogram (TIC) representation at six concentrations in pig liver at 0.5, 1, 2, 5, 10, and 100 μg/L.

Related detection instruments: field microclimate automatic observer, hand-held meteorological station, temperature and humidity self-detector

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