Determination of Clenbuterol (Clenbuterol) Residues in Animal Foods (2)

4.
Safety reminder

(1) Most chemical reagents and drugs are harmful to the human body.
Before the experiment operation, the experimenter should wear disposable gloves and avoid direct contact with their hands
.

(2) All drugs and reagents should be affixed with obvious labels, indicating the name of the drug, quality specifications and the date of delivery, and when necessary, the dangerous nature of the drugs and reagents should be marked with obvious signs
.

(3) Methanol has high volatility and is a nerve poison.
It can enter the body through the respiratory tract, skin, and digestive tract, causing damage to the optic nerve and multiple organs; isopropanol is flammable and explosive; ethyl acetate is flammable and irritating Sexual and allergenic
.
The above reagents should be managed by special laboratory custodians, and users and dosages should be strictly recorded

(4) Turn on the switches of the ventilation facilities in advance, and personnel can enter the experimental area after 20 minutes of ventilation
.

(5) The extraction, purification, and constant volume steps involving the use of harmful or irritating gas generating reagents should be carried out in a fume hood, and the head should not be put into the fume hood
.

5.
Operation steps

(1) Extraction Weigh 10g of muscle, liver or kidney sample (accurate to 0.
01g), homogenize it with 20mL 0.
1mol/L perchloric acid solution, place it in a ground glass centrifuge tube; then place it in an ultrasonic cleaner Ultrasound for 20 minutes, take it out and place it in a water bath at 80°C for 30 minutes, take it out and cool it and centrifuge (4500r/min) for 15 minutes
.
Pour out the supernatant, wash the precipitate with 5mL 0.

(2) Purify the weak cation exchange column with 10 mL ethanol, 3 mL water, 3 mL 0.
1 mol/L sodium dihydrogen phosphate buffer (pH=6.
0), and 3 mL water in sequence, and take an appropriate amount of the extract in (1) to weak cation exchange On the column, discard the effluent, wash the column with 4 mL of water and 4 mL of ethanol, discard the effluent, rinse the column with 6 mL of ethanol + concentrated ammonia (98+2), collect the effluent, and place the effluent on the N2-evaporator Concentrate to dryness
.

(3) Preparation before sample measurement.
Add 100-500uL mobile phase to the purified and blow-dried sample residue, shake it fully on the vortex mixer to dissolve the residue, and use a 0.
45um syringe type when the liquid is turbid.
Filtration with a microporous filter membrane, and the supernatant is to be measured by liquid chromatography
.

(4) Determination

①Reference conditions for liquid chromatography determination:

Chromatographic column: BDS or ODS column, 250mm×4.
6mm, 5um; Mobile phase: methanol+water (45+55); Flow rate: 1mL/min; Injection volume: 20-50uL; Oven temperature: 25℃; UV detection Device: 244nm
.

②Sampling measurement: draw 20-50uL standard calibration solution and sample solution into the liquid chromatograph, qualitatively use retention time, and quantify with external standard single-point or multi-point calibration
.

③High performance liquid chromatogram of Clenbuterol standard (100ug/L): see Figure 6-6
.

Figure 6-6 High performance liquid chromatogram of Clenbuterol standard (100μg/L)

6.
Result calculation

Where X-the content of clenbuterol in the sample, ug/kg or ug/L

m ₁ ——The mass of clenbuterol corresponding to the ratio of the peak area of ​​the sample chromatographic peak to the standard chromatographic peak, ng

f——The dilution factor of the sample

m ₂ ——Sampling amount of the sample, g or mL

The calculation result is expressed to two decimal places
.

7.
Tips

(1) The ideal high performance liquid chromatography water should be 18.
2Ω ultrapure water, and pass through a 0.
22um filter membrane to remove heat sources, organic matter, inorganic ions, CO ₂ and so on
.
Qualified purified water can be purchased, and ultra-pure water machine can be purchased

(2) The standard substance of clenbuterol exists in the form of hydrochloride.

(3) The BDS or ODS chromatographic column is a reverse phase column.

(4) LC-WCX is a weak cation exchange column with a carboxylic acid functional group bonded to silica gel.