Tetracyclines (TCs) antibiotics are produced by actinomycetes and all belong to hydrogenated and tetrabenzene ring derivatives in their chemical structure and have similar physicochemical properties. Source: Food Safety Monitoring Technology (Chemical Industry Press)

Operation method

High Performance Liquid Chromatography

Materials and Instruments

Animal Liver Kidney Muscle Tissue Fish Products Milk
Polypropylene Disodium ethylenediaminetetraacetate Methanol-water Methanol-ethyl acetate Acetonitrile Trifluoroacetic acid
Polypropylene centrifuge tubes Solid phase extraction columns High performance liquid chromatography Tandem mass spectrometry

Move

1. Extraction

Animal liver, kidney, muscle tissue, aquatic products: weigh 5 g of homogenized sample (accurate to 0.01 g), placed in a 50 mL polypropylene centrifuge tube, respectively, with about 20 mL, 20 mL, 1O mL, 0.1 mol/L EDTA-Mcllvaine buffer solution (0.1 mol/L. Weighing 60.5 g of disodium ethylene diamine tetraacetic acid in 1625mL Mcllvaine buffer solution to dissolve, shake well) low temperature ultrasonic extraction three times, 3000 r/min, 5 min centrifugation (temperature below 15 ℃), combined with the supernatant (pay attention to the temperature below 15 ℃). Mcllvaine buffer solution to dissolve, shake well) low-temperature ultrasonic extraction three times, 3000 r/min, 5 min centrifugation (temperature lower than 15 ℃), combined supernatant (pay attention to the control of the volume of the total extract does not exceed 50 mL), and fixed volume to 50 mL, mixing, 5000 r/min, centrifugation for 10 min (temperature lower than 15 ℃), filtration, and to be purified.

Milk: Weigh 5 g of mixed sample (accurate to 0.01 g), put it in a 50 mL polypropylene centrifuge tube, dissolve it with 0.1 mol/L EDTA-Mcllvaine buffer solution and make it volume to 50 mL, vortex mixing for 1 min, low-temperature ultrasonication for 10 min, cool it to 4 ℃, 5000 r/min, centrifugation for 10 min (the temperature is lower than 15 ℃), and then filter it to be purified. ℃), filter, and wait for purification. The sample was then filtered and allowed to be purified.

2. Purification

Accurately aspirate 10 mL of the extract at a rate of 1 drop/s through the Waters Oasis HLB (60 mg, 3 mL) solid phase extraction column, after the supernatant completely flowed out, the supernatant was drenched with 5 mL of water and 5 mL of methanol-water (5 + 95) in turn, and all the effluent was discarded. The column was dried under reduced pressure for 5 min and finally eluted with 10 mL of methanol-ethyl acetate (10 + 90). The eluate was blown nitrogen and concentrated to dryness (temperature less than 30 ℃), and the residue was dissolved with 1.0 mL of standard diluent, sonicated at low temperature for 5 min, and then passed through a 0.45 um filter membrane for determination by high performance liquid chromatography/tandem mass spectrometry (HPLC/MS).

3. 5HPLC measurement conditions

Column: Inertsi1 C8-3, 5 um, 150 mm×2.1 mm;

Mobile phase: methanol, acetonitrile, 0.01 mol/L trifluoroacetic acid, elution gradient is shown in Table 3-11 (column equilibration time 5 min);



Flow rate: 1.5 mL/min;

Column temperature: 30 ℃;

Injection volume: 30 uL.

4. HPLC-MS/MS measurement conditions

Column: Inertsil C8-3, 50 m, 150 mm × 2.1 mm;

Flow rate: 0.3 mL/ min;

Column temperature: 30 ℃;

Injection volume: 300 L;

Mobile phase: methanol + 0.01 mol/L trifluoroacetic acid.

Ionization mode: ESI+ ;

Mass spectral resolution: unit resolution;

Nebulizing gas (NEB): 6.0 L/min;

Curtain gas (CUR): 10.0 L/min;

Spraying voltage (IS): 4500 V;

Desolventization temperature (TEM): 500 Temperature of desolventization (TEM): 500 ℃ ;

Desolventizing airflow: 700 L/min;

Collision air (CAD): 6.0 L/min (nitrogen).

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