1. Experimental purpose
The residual amount of oxytetracycline, tetracycline and chlortetracycline in livestock and poultry meat was determined according to the standard GB/T14931.1-94 . The low detection concentrations of zui are: 0.15 , 0.20 , 0.65mg/kg .
2. Principle of experiment
The sample was extracted, directly filtered by microporous membrane filtration, separated by reversed- phase liquid chromatography , detected by ultraviolet detector, and compared with the standard, the order of the peak was oxytetracycline, tetracycline and chlortetracycline. Standard addition is quantified.
3. Reagents
3.1 Acetonitrile ( analytical grade ) .
3.2 　 0.01mol/L sodium dihydrogen phosphate solution: Weigh 1.56 g ( ± 0.01g ) of sodium dihydrogen phosphate (NaH 2 PH 4 .2H 2 O) dissolved in distilled water, dilute to 100 mL , filter through filter (select micro filter holes is 0.45 μ m), the standby.
3.3 oxytetracycline (OTC) standard solution: Weigh oxytetracycline 0.0100g (± 0.0001g), with 0.1mol / L hydrochloric acid solution containing oxytetracycline per ml 1mg.
3.4 Tetracycline (TC) standard solution: Weigh 0.0100 g ( ± 0.001 g ) of tetracycline , dissolve it with 0.01 mol/L hydrochloric acid solution and make up to 10.00 mL . This solution contains 1 mg of tetracycline per ml .
3.5 chlortetracycline (CTC) standard solution: Weigh chlortetracycline 0.0100g (± 0.0001g), was dissolved in distilled water to a volume 10.00mL, this gold per ml neomycin 1mg. The above standard products are converted at 1000 units /mg . 3.3 ~ 3.5 solution should be stored below 4 °C , can be used for 1 week.
3.6 Mixing standard solution: Take 1.00 mL of each of 3.3 and 3.4 standard solutions , take 2.00 mL of 3.5 standard solution , place in a 10 mL volumetric flask, and add distilled water to the mark. This solution contains 0.1 mg of oxytetracycline and tetracycline per liter, and 0.2 mg of chlortetracycline .
3.7 　 5 % perchloric acid solution.
4. Instrument
4.1 Â Â Â Â Â Â Â High Performance Liquid Chromatograph (HPLC) : with UV detector.
4.2 Â Â Â Â Â Â Â Oscillator: Tianjin Hengao Technology Development Co., Ltd.
4.3 Â Â Â Â Â Â Â Filter: Tianjin Hengao Technology Development Co., Ltd.
4.4 Â Â Â Â Â Â Â Ultrasound: Tianjin Hengao Technology Development Co., Ltd.
5. Chromatographic conditions
5.1 LC column: ODS-C18 (5 μ m) 　 6.2mm × 15 cm .
5.2 Detection wavelength: 355nm .
5.3 Sensitivity: 0.002AUFS .
5.4 column temperature: room temperature.
5.5 Flow rate: 1.0 mL/min .
5.6 Injection volume: 10 μ L.
5.7 Mobile phase: acetonitrile / 0.01 mol / L sodium dihydrogen phosphate solution ( pH 2.5 with 30 % (V / V) nitric acid solution ) , 35 : 65 (V / V) , degassed with ultrasonic waves for 10 min before use .
6. Method of operation
6.1 Sample determination: Weigh 5.00 g ( ± 0.01 g ) of minced meat ( < 5 mm ) , place in a 50 mL Erlenmeyer flask, add 25.0 mL of 5 % perchloric acid , shake on a shaker for 10 min , and move in. to a centrifuge tube, at 2000 r / min centrifugation 3min, the supernatant through 0.45 μ m membrane filter, take the solution 10 μ L sample, recording the peak height, from the working curve Richard content.
6.2 Calibration: Weigh 7 parts of chopped meat samples, each 5.00 g (± 0.01 g), were mixed standard solution was added 0, 25, 50, 100, 150, 200, 250 μ L ( containing oxytetracycline tetracycline are each 0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 μ g, neomycin gold 0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 μ g), by the method of operation 6.1, peak height On the ordinate, the working curve is plotted with the antibiotic content as the abscissa.
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