GB/T 19684-2026Determination of chlortetracycline in feeds (English PDF)

4.2.6 Na2EDTA-Mclvaine buffer solution (pH=6). Weigh

7.1 g citric acid monohydrate and

17.9 g dihydrogen phosphate dodecahydrate. Sodium,

37.2 g disodium ethylenediaminetetraacetate dihydrate (Na2EDTA·2H2O), dissolved in water, transferred to a 1000 mL volumetric flask, and diluted. Mix well and adjust the pH to 6 with sodium hydroxide solution (4.2.4) or hydrochloric acid solution (4.2.5).

4.2.7 Extraction solution. Take 900 mL of Na2EDTA-Mclvaine buffer solution (4.2.6) and 100 mL of methanol, and mix well.

4.2.8 Oxalic acid solution (10 mmol/L). Weigh

1.26 g of oxalic acid dihydrate, dissolve in water, and dilute to 1000 mL. Mix well.

4.2.9 Standard stock solution (1 mg/mL). Accurately weigh an appropriate amount of chlortetracycline hydrochloride standard (calculated as chlortetracycline, accurate to

0.01 mg). CAS No.. 64-72-2 (purity >=98%) was dissolved in a 10 mL volumetric flask with methanol (4.2.2), diluted to volume, and mixed well. Store below -18°C. Stored, valid for 6 months.

4.2.10 Standard Intermediate Solution (100 µg/mL). Accurately transfer 1 mL of the standard stock solution (4.2.9) into a 10 mL volumetric flask, and use [method name missing] to [previous preparation]. Dilute and bring to volume with alcohol (4.2.2), mix well. Store below -18°C, shelf life 7 days.

4.2.11 Standard series solutions. Accurately transfer an appropriate amount of the standard intermediate solution (4.2.10), dilute it with the extract (4.2.7), and prepare it into a series of solutions with different mass concentrations. Prepare a series of standard solutions with concentrations of 0.4 µg/mL, 1 µg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, and 20 µg/mL. Prepare fresh before use.

4.2.12 Microporous filter membrane. 0.45µm, aqueous system.

4.3 Instruments and Equipment

4.3.1 High-performance liquid chromatograph. equipped with an ultraviolet detector or a diode array detector.

4.3.2 Analytical balance. accuracy 0.1mg, 0.01mg.

4.3.3 Centrifuge. The rotation speed shall not be less than 10,000 r/min.

4.3.4 pH meter. accuracy ±0.1.

4.3.5 Vortex mixer.

4.3.6 Ultrasonic cleaner.

4.4 Samples Prepare the sample according to GB/T 20195, at least.200g, crush it so that it all passes through a 0.425mm test sieve, mix it evenly, and pack it into a dense container. Store in a closed container for later use.

4.5 Test Procedure

4.5.1 Extraction Perform two parallel tests. Weigh 2g of the sample (5g of compound feed), accurate to 0.1mg, and place it in a 50mL centrifuge tube. Accurately add... 20 mL of extraction buffer (4.2.7) was vortexed and shaken for 10 min, followed by low-temperature ultrasonic extraction in an ice-water bath for 10 min, and then centrifuged at 10000 r/min. After 5 minutes, collect the supernatant in a centrifuge tube. Repeat the extraction twice with 15 mL of extract each time (4.2.7). Combine the supernatants (V), mix well, and centrifuge. Filtration was performed using a porous membrane (4.2.12). The filtrate was analyzed within 12 hours.

4.5.2 Measurement

4.5.2.1 Reference conditions for liquid chromatography The reference conditions for liquid chromatography are as follows:

a) Chromatographic column. C18 column, 250 mm in length,

4.6 mm in inner diameter, 5 µm in particle size, or equivalent;

b) Detection wavelength. 375nm;

c) Column temperature. 30°C;

e) Injection volume. 20 µL;

f) Mobile phase. Phase A is oxalic acid solution (4.2.8), Phase B is acetonitrile (4.2.3). methanol (4.2.2) = 3. 2 (volume ratio), gradient elution. The procedure is shown in Table 1.

4.5.2.2 Determination of Standard Series Solutions and Sample Solutions Under optimal instrument conditions, standard series solutions (4.2.11) and sample solutions (4.5.1) were tested using the instrument. Chlortetracycline standard solution... See Appendix A for the liquid chromatogram.

4.5.2.3 Qualitative Under the same test conditions, the retention time of chlortetracycline in the sample solution should be the same as that of chlortetracycline in the standard series solutions (with equivalent mass concentration). The retention time is consistent, with a relative deviation within ±2.5%.

4.5.2.4 Quantitative analysis A standard curve was plotted with the mass concentration of chlortetracycline in the standard series solutions as the x-axis and the peak area as the y-axis. The correlation coefficient was then calculated. The concentration should not be less than 0.99.The concentration of chlortetracycline in the sample solution (4.5.1) should be within the linear range of the standard curve. If it exceeds the range, the sample solution should be... The solution (4.5.1) was diluted with the extraction solution (4.2.7) and re-measured. For single-point calibration quantification, the mass concentration of chlortetracycline in the sample solution (4.5.1) was compared with... The difference in mass concentration of the standard solutions should not exceed 30%.

4.6 Experimental Data Processing The content of chlortetracycline in the sample is expressed as a mass fraction w, with the value expressed in milligrams per kilogram (mg/kg). Multi-point calibration is calculated according to formula (1);

4.7 Precision Under repeatability conditions, the absolute difference between two independent measurements and their arithmetic mean shall not exceed 10% of the arithmetic mean.

5.2.8 Na2EDTA-Mclvaine buffer solution (pH=6). Weigh

7.1 g citric acid monohydrate and

17.9 g dihydrogen phosphate dodecahydrate. Sodium,

37.2 g disodium ethylenediaminetetraacetate dihydrate (Na2EDTA·2H2O), dissolved in water, transferred to a 1000 mL volumetric flask, and diluted. Mix well and adjust the pH to 6 with sodium hydroxide solution (5.2.6) or hydrochloric acid solution (5.2.7). 5.2.9 10% methanol solution. Measure 10 mL of methanol (5.2.2) into a 100 mL volumetric flask, dilute with water, make up to volume, and mix well.

5.2.10 Ethyl acetate in methanol solution. Measure 90 mL of methanol (5.2.2) and 10 mL of ethyl acetate (5.2.5) and mix well. 5.2.11 30% methanol solution. Measure 30 mL of methanol (5.2.2) into a 100 mL volumetric flask, dilute with water, make up to volume, and mix well. 5.2.12 0.1% Formic Acid Solution. Measure 500 mL of water into a 1000 mL volumetric flask, add 1 mL of formic acid (5.2.4), dilute with water, and set aside. Rong, to mix evenly.

5.2.13 Standard stock solution (1 mg/mL). Accurately weigh an appropriate amount of chlortetracycline hydrochloride standard (calculated as chlortetracycline, accurate to

0.01 mg). CAS No.. 64-72-2 (purity >=98%) was dissolved in a 10 mL volumetric flask with methanol (5.2.2), diluted to volume, and mixed well. Store below -18°C. Stored, valid for 6 months.

5.2.14 Standard intermediate solution (10 µg/mL). Accurately transfer 1 mL of the standard stock solution (5.2.13) into a 100 mL volumetric flask, and use [method name missing] to [previous preparation]. Dilute and bring to volume with alcohol (5.2.2), mix well. Store below -18°C, shelf life 7 days.

5.2.15 Standard Series Solutions. Accurately transfer an appropriate amount of the standard intermediate solution (5.2.14) and prepare solutions with methanol (5.2.2) to concentrations of [missing values]. A series of standard solutions with concentrations of 1 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 50 ng/mL. Prepare fresh before use.

5.2.16 Hydrophilic and lipophilic solid phase extraction column. 60mg/3mL, or equivalent.

5.2.17 Microporous filter membrane. 0.22µm, organic system.

5.3 Instruments and Equipment

5.3.1 Liquid Chromatography-Tandem Mass Spectrometry. Electrospray Ionization Source (ESI).

5.3.2 Analytical balance. accuracy

0.1 mg,

0.01 mg.

5.3.3 Centrifuge. The rotation speed shall not be less than 10,000 r/min.

5.3.4 pH meter. accuracy ±0.1.

5.3.5 Vortex mixer.

5.3.6 Ultrasonic cleaner.

5.3.7 Nitrogen blowing device.

5.3.8 Solid phase extraction apparatus.

5.4 Samples Prepare a sample according to GB/T 20195, at least.200g, crush it so that it all passes through a test sieve with a 0.425mm aperture, mix thoroughly, and load it into a container. Store in a sealed container for later use. Select samples of the same type, uniform in composition, where the instrument response value at the retention time of the analyte is less than 30% of the method limit of quantitation. Feed samples were used as blank samples.

5.5 Test Procedure

5.5.1 Extraction Perform two parallel tests. Weigh 2g of the sample (accurate to 0.1mg) into a 50mL centrifuge tube, and accurately add 15mL of [unspecified liquid]. Na2EDTA-Mclvaine buffer solution (5.2.8), vortexed for 10 min, then subjected to low-temperature ultrasonic extraction in an ice-water bath for 10 min. Centrifuge at 10000 rpm for 10 min, and transfer the supernatant to a 50 mL volumetric flask. Dissolve the residue in 15 mL of Na2EDTA-Mclvaine buffer solution. (5.2.8) Repeat the extraction twice, combine the supernatants, and make up to volume (V) with Na2EDTA-Mclvaine buffer solution (5.2.8), mix well, and set aside.

5.5.2 Purification The solid-phase extraction column was activated sequentially with 3 mL of methanol (5.2.2), 3 mL of water, and 3 mL of Na2EDTA-Mclvaine buffer solution (5.2.8). (5.2.16) Accurately transfer 2 mL (V1) of the stock solution (5.5.1) through the column, and rinse with 3 mL of water and 3 mL of 10% methanol solution (5.2.9) respectively. After drying, add 3 mL of ethyl acetate-methanol solution (5.2.10) to elute, collect the eluent and dry it under nitrogen at 40°C. Accurately add 1 mL of... (V2) Dissolve the residue in a 30% methanol solution (5.2.11) by vortexing for 30 seconds, then filter through a microporous membrane (5.2.17). The filtrate should be completely dissolved within 12 hours. Measurement.

5.5.3 Preparation of matrix-matched standard series solutions Take 6 blank samples and process them according to steps

5.5.1 to 5.5.2.After column chromatography, accurately add 1 mL of standard working solution to each of the eluent samples collected. The series of solutions (5.2.15) were dried under nitrogen at 40°C, and the residue was dissolved in 1 mL of 30% methanol solution (5.2.11) to prepare a solution of mass concentration. Matrix-matched standard solutions with concentrations of 1 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 50 ng/mL were mixed. Uniform, filtered through a microporous membrane (5.2.17), to be tested.

5.5.4 Measurement

5.5.4.1 Reference conditions for liquid chromatography The reference conditions for liquid chromatography are as follows:

a) Chromatographic column. C18 column, 50 mm in length,

2.1 mm in inner diameter, 1.7 µm in particle size, or equivalent;

b) Column temperature. 40°C;

0.3 mL/min;

d) Injection volume. 3 µL;

e) Mobile phase. Phase A is 0.1% formic acid solution (5.2.12), Phase B is acetonitrile (5.2.3), and the gradient elution program is shown in Table 2.

5.5.4.2 Mass Spectrometry Reference Conditions The reference conditions for mass spectrometry are as follows:

a) Ionization method. Electrospray ionization, positive ion mode (ESI);

b) Detection method. Multiple reaction monitoring (MRM);

c) Electrospray voltage (IS). 5500V;

d) Ion source temperature (TEM). 550°C. The qualitative ion pairs, quantitative ion pairs, declustering voltage, and collision energy of multiple reaction monitoring (MRM) are shown in Table 3.

5.5.4.3 Determination of matrix-matched standard series solutions and sample solutions Under optimal instrument conditions, chlortetracycline matrix-matched standard series solutions (5.5.3) and sample solutions (5.5.2) were tested on the instrument. The quantitative ion chromatogram of chlortetracycline standard solution is shown in Appendix A.

5.5.4.4 Qualitative Analysis Under the same test conditions, the retention time of chlortetracycline in the sample solution should be the same as that of chlortetracycline in the standard series solutions (with equivalent mass concentration). The retention times were consistent, with relative deviations within ±2.5%. Based on the qualitative ion pairs selected in Table 3, the chlortetracycline monitoring in the sample spectra was compared. The relative ionic abundance of the ion pair is close to the relative ionic abundance of the corresponding monitoring ion pair in a standard series solution with a concentration close to that of the ion pair. If the deviation does not exceed [the specified value], [the remaining information is missing]. If the sample exceeds the range specified in Table 4, it is determined that chlortetracycline is present in the sample. Table