Determination of Clenbuterol (Clenbuterol) Residues in Animal Foods (1)

Clenbuterol is a potent and selective β-receptor agonist, which can significantly promote animal growth and increase the rate of lean meat, so it is commonly known as "Clenbuterol"
.

For the determination of clenbuterol residues in animal foods , GB/T5009.
192-2003 stipulates three methods: gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) and enzyme-linked immunoassay Method (ELISA screening method)
.

This training uses high performance liquid chromatography (HPLC) (according to the second method of GB/T5009.
192-2003)
.

1.
Purpose requirements

(1) Understand the working principle of high performance liquid chromatograph and the selection method of working conditions
.

(2) Master the principle and method of determining clenbuterol residues in animal food by high performance liquid chromatography
.

(3) Learn to use high performance liquid chromatograph and learn to recognize chromatograms
.

2.
Principles of training

The solid sample was cut into small pieces, homogenized with perchloric acid solution, and the liquid sample was added with perchloric acid solution.
After ultrasonic heating and extraction, it was extracted with isopropanol + ethyl acetate (40+60).
The organic phase was concentrated and subjected to weakening.
The cation exchange column is used for separation, eluted with ethanol +concentrated ammonia (98+2) solution, the eluent is concentrated, the mobile phase is fixed in volume and then measured on a high performance liquid chromatograph, and quantified by external standard method
.

3.
Training supplies

(1) High performance liquid chromatograph
.

(2) Water bath ultrasonic cleaner
.

(3) Ground glass centrifuge tube: 11.
5cm (length)×3.
5cm (inner diameter), with stopper
.

(4) 5mL glass centrifuge tube or equivalent

(5) Acidity meter

(6) Centrifuge

(7) Oscillator

(8) Rotary evaporator

(9) Vortex mixer

(10) Microporous filter membrane (0.

(11) Weak cation exchange column (LC-WCX) 3mL or equivalent

(12) N ₂ -Evaporator

(13) Homogenizer

(14) Clenbuterol hydrochloride, purity ≥99.

(15) Ultra-pure water

(16) Sodium dihydrogen phosphate (NaH ₂ PO ₄ ·2H ₂ O): analytically pure
.

(17) Sodium hydroxide : analytically pure
.

(18) Sodium chloride : analytically pure
.

(19) Perchloric acid : analytically pure, with a purity of 70%
.

(20) Concentrated ammonia: analytically pure
.

(21) Isopropanol : HPLC grade
.

(22) Ethyl acetate : HPLC grade
.

(23) Methanol: HPLC grade
.

(24) Ethanol: HPLC grade
.

(25) Perchloric acid solution (0.
1mol/L): accurately weigh 14.
35g of perchloric acid (analytical purity, 70%), dilute with ultrapure water and dilute to 1L, shake well
.

(26) Sodium hydroxide solution (1mol/L): Weigh 40g of sodium hydroxide accurately , dissolve it with ultrapure water and dilute to 1L, shake it well
.

(27) Sodium dihydrogen phosphate buffer (0.
1 mol/L, pH=6.
0): accurately weigh 15.
6 g of sodium dihydrogen phosphate , dissolve it with ultrapure water and dilute to 1L, and shake it well
.

(28) Isopropanol + ethyl acetate (40+60)
.

(29) Ethanol + concentrated ammonia (98+2)
.

(30) Methanol+water (45+55)
.

(31) Clenbuterol standard solution: accurately weigh the Clenbuterol standard and use methanol to prepare a standard stock solution with a concentration of 250mg/L, and store it in the refrigerator (0～4℃); dilute it with methanol when using it.
The 0.
5mg/L clenbuterol standard solution was further diluted appropriately with methanol+water (45+55)
.

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