1. Analyze the target compound: Oxytetracycline, chlortetracycline, tetracycline 2. Equipment: High-performance liquid chromatograph with fluorescence detector, 3. Reagents: In addition to the following reagents, use the reagents listed in Appendix 2, Imidazole: Premium Imidazole buffer solution: dissolve 68.08g imidazole, 0.37g EDTA and 10.72g magnesium acetate in 800mL water, adjust the pH to 7.2 with acetic acid, add water to 1,000mL, Citric acid buffer solution containing ethylenediaminetetraacetic acid: Solution 1: Dissolve 21.0 g of citric acid in water to 1,000 mL, Solution 2: Dissolve 71.6 g of disodium hydrogen phosphate in water to 1,000 mL, at 1.86 g Add 307mL of the first solution and 193mL of the second solution to diaminetetraacetic acid to dissolve, Styrene divinylbenzene copolymer small column (265mg): a polyethylene tube with an inner diameter of 8-9mm is filled with 265mg styrene divinylbenzene copolymer for column chromatography or a substance with equivalent separation characteristics, 4. Standard products: Oxytetracycline hydrochloride: This product 1.000mg contains oxytetracycline 0.850mg or more potency, the decomposition point is 190 ℃ ～ 194 ℃, Chlortetracycline hydrochloride: 1.000mg of this product contains chlortetracycline hydrochloride with a titer above 0.900mg, the decomposition point is above 210 ℃, Tetracycline hydrochloride: 1.000mg of this product contains more than 0.900mg tetracycline hydrochloride, the decomposition point is above 214 ℃, 5. Preparation of test solution: A. Extraction method: ①Muscle, liver and kidney Muscle: Remove the fat layer as much as possible, after crushing and mixing, weigh it 5.00g, Liver and kidney: After mixing and mixing, weigh 5.00g, Add 30mL of citric acid buffer solution containing ethylenediaminetetraacetic acid, stir for 1 minute, centrifuge at 3000 rpm for 10 minutes, move the water layer into a 100mL separatory funnel, add 20mL of ethylenedichloride to the residue of the centrifuge tube The citric acid buffer solution of amine tetraacetic acid was vigorously shaken with a shaker for 1 minute, and then centrifuged according to the same conditions as above. Centrifuge at 3000 rpm for 10 minutes to separate the water layer, ② fat Fat: Remove the muscle layer as much as possible, after crushing and mixing, weigh 20.0g, Add 200mL of n-hexane, and after stirring for 1 minute, add 40mL of citric acid buffer solution containing ethylenediaminetetraacetic acid, and after stirring for another minute, centrifuge at 3000 rpm for 10 minutes, separate the water layer, and add 20mL to the n-hexane layer The citric acid buffer solution containing ethylenediaminetetraacetic acid was vigorously shaken with a shaker for 1 minute, then centrifuged according to the same conditions as above, and the water layer was separated and combined into the first water layer. ③ milk Weigh 5.00g of sample, add 30mL of citric acid buffer solution containing ethylenediaminetetraacetic acid and 20mL of n-hexane, shake vigorously with a shaker for 5 minutes, centrifuge at 3000 rpm for 10 minutes, and separate the water layer. ④ Egg After removing the eggshell and fully homogenizing, weigh 5.00g, add 30mL of citric acid buffer solution containing ethylenediaminetetraacetic acid, after homogenization, add 100mL of n-hexane, stir for 1 minute, centrifuge at 3000 rpm for 10 minutes , The water layer was transferred into a 100mL separatory funnel, and 20mL of citric acid buffer solution containing ethylenediaminetetraacetic acid was added to the residue of the centrifuge tube. Add 20 mL of n-hexane to the separatory funnel, shake it with a shaker for 5 minutes, centrifuge at 3000 rpm for 10 minutes, and separate the water layer. ⑤Fish and shellfish Shellfish: After removing the shell, stir and mix well, weigh 5.00g, Other fish and shellfish: After crushing and mixing, weigh 5.00g, Add 30mL of citric acid buffer solution containing ethylenediaminetetraacetic acid, stir for 1 minute, centrifuge at 3000 revolutions per minute for 10 minutes, transfer the water layer into a 100mL separatory funnel, add 20mL of ethylenediamine to the residue of the centrifuge tube The citric acid buffer solution of tetraacetic acid was vigorously shaken with a shaker for 1 minute, then centrifuged according to the same conditions as above, and the combined aqueous layer was added to a separatory funnel, 20 mL of n-hexane was added, and shaken vigorously with a shaker for 5 minutes, every minute Centrifuge at 3000 rpm for 10 minutes, separate the water layer, B. Purification method: In a small column of styrene divinylbenzene copolymer (265 mg), inject 10 mL of methanol, 10 mL of water and 5 mL of saturated EDTA solution sequentially, discard the effluent, and after injecting the solution obtained by the extraction method into the column, inject 10 mL of water and discard Remove the effluent, inject 10 mL of methanol, collect the effluent in a mill-mouth vacuum concentrator, remove methanol below 40 ° C, add 1.0 mL of 1.36% potassium dihydrogen phosphate solution to the residue to dissolve, this is the test solution, 6. Operation method: A. Qualitative test Perform the test according to the following operating conditions, the test results must be consistent with the standard product, Operating conditions: Column packing: octadecyl silylated silica gel (particle size 5μm), Column: inner diameter 4.0 ～ 6.0mm, length 150mm stainless steel tube, Column temperature: 40 ℃ Detector: excitation wavelength 380nm, emission wavelength 520nm, Mobile phase: Oxytetracycline and tetracycline test, use a mixed solution of imidazole buffer solution: methanol (17: 3), adjust the flow rate to make oxytetracycline flow out for about 5 minutes, In the chlortetracycline test, use a mixed solution of imidazole buffer solution: methanol (3: 1), adjust the flow rate to allow chlortetracycline to flow out for about 7 minutes, B. Quantitative test According to the test results obtained under the same operating conditions as the qualitative test of A, the peak height method or peak area method is used to quantify, 7. Limit of quantification: A. Muscle, liver, kidney, milk, egg and fish and shellfish Oxytetracycline: 0.02mg / kg Chlortetracycline: 0.03mg / kg Tetracycline: 0.02mg / kg B. Fat Oxytetracycline: 0.005 mg / kg 8. Matters needing attention: (1) Preparation of test solution: ①Keep the centrifugal separation temperature at room temperature, ②When the filtrate is cloudy after suction filtration, the filtrate is centrifuged again. ③ In column chromatography of styrene / divinylbenzene copolymer, after injecting the test solution, 30 mL of water is added in portions to wash the residual ethylenediaminetetraacetic acid in the column. (2) Preparation of standard solution: ①Weigh a standard product equivalent to 10.0mg oxytetracycline, dissolve in methanol to 10mL, as the standard stock solution of oxytetracycline (oxytetracycline 1,000mg (potency) / L, 1,000mg / L), this standard stock solution Store at -20 ℃, stable within 1 year, ② Weigh a standard product equivalent to 10.0mg chlortetracycline, dissolve in methanol to 10mL, as the standard stock solution of chlortetracycline (chlortetracycline 1,076mg (potency) / L, 1,000mg / L), this standard stock solution Store at -20 ℃, stable within 1 year, ③ Weigh a standard product equivalent to 10.0mg tetracycline, dissolve in methanol to 10mL, as the standard stock solution of tetracycline (tetracycline 1,082mg (potency) / L, 1,000mg / L), the standard stock solution is stored at -20 ℃, 1 Stable during the year, ④Diluting each standard stock solution of oxytetracycline, chlortetracycline and tetracycline with 1.36% potassium dihydrogen phosphate solution as the standard solution for making the standard curve, (3) Others: ①When the screening test method of many sample tests is carried out at the same time to improve the judgment efficiency, if the screening method of oxytetracycline, chlortetracycline and tetracycline (refer to page 117) is positive, it may be possible to carry out the test using this test method. ②When using this detection method to detect oxytetracycline, chlortetracycline and tetracycline, it is best to confirm with high-performance liquid chromatography with a mass detector. 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Detection methods of oxytetracycline, chlortetracycline and tetracycline