Determination of chlortetracycline in the word material - High performance liquid chromatography GB/T 19684-2005
Determination of chlortetracycline in the word material - High performance liquid chromatography GB/T 19684-2005
1 Scope
This standard specifies the method for the determination of chlortetracycline in feed by high performance liquid chromatography (HPLC).
This standard is applicable to the determination of chlortetracycline in compound feed, concentrated feed and premix feed. The detection limit is 1n g and the minimum detectable concentration is 4 mg/kg.
2 Normative references
The terms in the following documents become the terms of this standard by reference in this standard. Any dated reference document, followed by
Some amendments (not including errata content) or revisions do not apply to this standard. However, parties to agreements based on this standard are encouraged to study whether the latest versions of these documents can be used. For undated references, the latest edition applies to this standard.
GB / T 6 682 Analytical laboratory water specifications and test methods
GB / T 14699.1 Feed sampling
3 Principle of the method
The chlortetracycline in the feed was extracted with hydrochloric acid-acetone solution, adjusted to pH 1.0-1.2, shaken, filtered, separated by reverse phase column, detected by ultraviolet detector, and quantitatively analyzed by external standard method.
4 test and materials
The reagents used in this standard are of analytical grade unless otherwise stated. The water is distilled water, and the chromatographic water is deionized water, which meets the requirements of GB/T 6682 water.
4.1 sodium hydroxide solution: 400 g / L.
4.2 oxalic acid (oxalic acid) solution: [c (1/2HZC20,) = 0.01 mol / L].
4.3 Hydrochloric acid solution: [c(HCI)=4 mol/L].
4.4 Extract: Acetone decahydrochloric acid solution (4.3) + water = 13 10 1 6.
4.5 Mobile phase: oxalic acid solution (4.2) + acetonitrile (chromatographically pure) + methanol (chromatographically pure) = 10 10 3 12.
4.6 chlortetracycline standard solution:
4.6.1 The standard solution of chlortetracycline accurately weighs the chlortetracycline standard 0.012 90g (content greater than 96.9%, stored in silica gel dryer), accurate to 0.1 mg, placed in a 50m L volumetric flask, with extract ( 4.4) Dissolve and dilute to the mark, shake well, the concentration is 250g / L,
Stored in a refrigerator at 4 ° C ~ 6 ° C, valid for one week.
4.6.2 chlortetracycline standard working solution accurately transfer 4 mI- chlortetracycline standard stock solution ((4.6.1) in a 10 mL volumetric flask, dilute to the mark with ultrapure water, shake up, the concentration is 100 g /L, now available.
5 instruments, equipment
5.1 Centrifuge: 3 000 r/mine
5.2 pH meter: accuracy 0.0 1m V.
5.3 Constant temperature oscillator: 300 r/min.
5.4 microporous membrane (pore size 0. 45 pm).
5.5 Analytical balance: Sensing 0. 1 mgo
5.6 Analytical balance: Sensitivity 0.0 1m g.
GB/T 19684-2005
5.7 GE-200 high performance liquid chromatography GE-200 high pressure infusion pump, GE-200 UV detector, Zhejiang University Zhida N2010 chromatography workstation, VertexC18 column.
6 Sample selection and preparation
According to GB/T 14699.1, a representative sample is selected from 200g to 500g, which is reduced to 100g by the quarter method, pulverized through a 0.45mm aperture sieve, thoroughly mixed, and stored in a grinding bottle for use.
7 Analysis steps
7.1 Weigh the sample lg-10g (chlortetracycline content is greater than or equal to 40m g / kg), accurate to 0.1 mg, placed in a conical flask, add people
100 mL extract (4.4), shake by hand for 2 min, then adjust the pH to 1. 0. 2 (record the consumption of hydrochloric acid solution, as a dilution factor), cover the plug, and set the constant temperature to oscillate. 30 (3) (oscillation speed 110 r / min) oscillate 30 mine
7.2 Pour the extract (7.1) into a centrifuge tube and centrifuge at 3000 r/min for 15 min.
7.3 The supernatant was taken and filtered through a filter (5.4), and the filtrate was used as a sample solution.
7.4 HPLC measurement parameter setting:
Analytical column C18: column length 150mm, inner diameter 4.6mm, particle size 5um (or similar analytical column);
Column temperature: room temperature;
Detector: UV detector, detection wavelength 375nm;
Mobile phase velocity: 1.0 m L/min;
Injection volume: 10ul-20ul
7.5 Quantitative determination: The multi-point external standard method is used to calculate the content of chlortetracycline based on the linear regression equation of concentration and peak area (g/L).
8 Calculation of measurement results
8.1 The mass X (mg) of chlortetracycline contained in each kilogram of sample is calculated according to formula (1):
X=m1\×n
In the formula:
X—the mass of chlortetracycline contained in each kilogram of sample, in milligrams (mg);
M1--HPLC sample chromatographic peak corresponding to the mass of chlortetracycline, the unit is micrograms (ug);
M—the mass of the sample in grams (g);
n - dilution factor.
8.2 The results of the measurements are expressed as the arithmetic mean of the parallel samples and are retained to one decimal place.
9 allowed difference
The relative deviation of the results of two parallel determinations is not more than 7%.
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