LC-MS / MS method for detection of β-stimulant drugs in animal tissues

The Ministry of Health has recently published the "List of Non-edible Substances and Food Additives that May Be Illegally Added in Foods". From the published black list, statistics have been found that include melamine, Sudan red, malachite green, synthetic pigments, etc. In. In response to these shocking food additives, it also provides strong technical support and guarantee for food safety inspection and supervision. The relevant personnel have developed convenient, fast and accurate detection methods. LC-MS / MS is now used to detect animal tissues The method of β-stimulant drugs in China is published as follows:

1. Scope of application

It is suitable for the detection of clenbuterol and ractopamine hydrochloride in animal tissues.

2. Extract

Place 5 g of animal tissue, 15 mL of ethyl acetate, and 3 mL of 10% sodium carbonate solution in a 50 mL centrifuge tube, and homogenize at 10000 rpm for 2 min. Centrifuge at 6000 rpm for 2 min, and transfer the supernatant to another centrifuge tube. The extraction was repeated once with 15 mL of ethyl acetate, and the extracts were combined.

Add 5 mL of 0.1 mol / L hydrochloric acid solution to the ethyl acetate extract, vortex for 1 min, and then centrifuge at 6000 rpm for 1 min to collect the lower aqueous phase. The extraction was repeated once, the lower aqueous phases were combined, and the pH was adjusted to 5.2 with 2.5 mol / L NaOH solution to be purified.

3. Purification

Activation / Equilibrium: Add 3 mL methanol, 3 mL water and 3 mL 30 mmol / L hydrochloric acid solution to Welchrom ® P-SCX (60mg / 3mL) (provided by Shanghai Yuexu) in turn, and discard the effluent;

Loading: Add the above extract to the column and discard the effluent;

Rinse: Rinse with 3 mL of water and 3 mL of methanol in turn, discard the eluent, and drain the cartridge;

Elution: elute with 2 × 2 mL of 5% ammoniated methanol, collect the eluent, blow at 40 ° C with nitrogen, reconstitute with 50% acetonitrile, pass 0.22μm filter membrane, to be analyzed

4. Chromatographic mass spectrometry conditions

4.1 Chromatographic conditions

Column: Ultimate XB-C18 liquid chromatography column (2.1mm * 100 mm, 3 μm) (provided by Shanghai Yuexu)

Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 3 95 5 5 45 55 10 45 55 10.1 95 5 15.0 95 5

Mobile phase:

A: 0.1% formic acid aqueous solution

B: Acetonitrile

Gradient elution

Flow rate: 0.2 mL / min

Injection volume: 10 μL

Column temperature: 35 ° C

Running time: 12 min

4.2 Mass spectrometry conditions

Scanning mode: MRM

Ion source: ESI +

Capillary ionization voltage: 3.20 Kv

Hole voltage (cone): 45 V

Ionization source temperature: 100 ℃

Desolvation temperature: 300 ℃

Cone hole gas flow rate: 50 L / Hr

Desolvation gas flow rate: 450 L / Hr

Name precursor ion product ion pore voltage (V) collision voltage (eV) residence time (ms) quantitative ion pair qualitative ion pair clenbuterol 277.2 167.8 20 28 100-+ 203.0 20 16 200 +-ractopamine 302.17 106.8 18 28 100-+ 164.1 18 14 200 +-Clenbuterol hydrochloride-D9 288.2 209.2 22 146 100 + ractopamine-D3 305.2 167.1 18 14 100 +

5. Results

5.1 Recovery rate and reproducibility experiment

After the preliminary treatment of the pig muscle blank sample by liquid-liquid extraction, a certain amount of standard solution was added, and the concentrations were 0.5μg / kg, 1μg / kg, 5μg / kg, and the same concentration within each batch was tested 3 times in parallel There are 3 batches in total (see the attached figure for typical recovery chromatogram).

Addition level (μg / kg) Analyte 0.1 0.5 1.0 Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Clenbuterol hydrochloride 93.8 5.82 89.7 5.92 96.1 3.62 Lake dopamine 95.3 7.43 103.5 3.42 97.4 4.55

5.2 Chromatogram

Left picture EIC spectrum of 0.1 mg / kg clenbuterol hydrochloride added to pig muscle Right picture EIC spectrum of 0.1 mg / kg ractopamine added to pig muscle

Consumables used in this method:

Solid phase extraction cartridge Welchrom ® P-SCX 60mg / 3mL (P / N: WSP020306)

Column: Ultimate XB-18, 50 * 2.1mm, 3μm (P / N: Ult3B18205)

13mm, 0.22μm nylon pinhole filter membrane (P / N: WEL-SFNY213022)

12-well solid phase extraction device (WEL-Vac-12)

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