GB/T 17481-2025Determination of choline chloride in feed additive premix (English PDF)

4.2.5 Methanesulfonic acid solution (24 mmol/L). Weigh

2.304 g of methanesulfonic acid into a 1000 mL beaker, add 800 mL of water, dissolve, and transfer to Dilute to volume with water in a 1000mL volumetric flask and mix well.

4.2.6 Standard stock solution (2 mg/mL). Weigh.200 mg (accurate to

0.1 mg) of choline chloride standard (CAS No.. 67-48-1, pure) Dissolve (with a purity of not less than 99.5%) in a 100 mL volumetric flask, dilute to volume with water, and mix well. Store at 2°C~8°C, shelf life 3 months.

4.2.7 Standard Intermediate Solution (100 µg/mL). Accurately transfer 5 mL of the standard stock solution (4.2.6) into a 100 mL volumetric flask, and dilute with water. Prepare and mix thoroughly. Prepare immediately before use.

4.2.8 Standard series solutions. Accurately transfer appropriate amounts of the standard intermediate solution (4.2.7), dilute and bring to volume with water, mix well, and prepare solutions of the desired mass concentration. The standard series solutions were 1 µg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL, respectively. Prepare fresh before use.

4.2.9 Purification column. Polyvinylbenzene polymer reversed-phase packing, 1 mL.

4.2.10 Microporous filter membrane. 0.45µm, aqueous system.

4.2.11 Filter paper. Qualitative, rapid.

4.3 Instruments and Equipment

4.3.1 Ion chromatograph. equipped with a conductivity detector.

4.3.2 Analytical balance. accuracy

0.1 mg.

4.3.3 Water bath.

4.3.4 Ultrasonic cleaner.

4.3.5 Vortex mixer.

4.3.6 Nitrogen blowing device.

4.4 Samples Prepare samples according to GB/T 20195, at least.200g, crush them so that they all pass through a test sieve with a 0.425mm aperture, mix thoroughly, and load into... Store in a sealed container, away from light.

4.5.1 Sample Solution Preparation

4.5.1.1 Vitamin premixed feed Perform two parallel tests. Weigh 1-2g of the sample (containing 0.01-0.2g of choline chloride, accurate to 0.1mg) and place it in a 100mL container. Add approximately 60 mL of water to a volumetric flask, shake well, place in a 70°C water bath for 20 minutes, sonicate for 10 minutes, remove, cool to room temperature, and dilute to volume with water. (V) Shake well and filter. Take 8 mL of the filtrate and pass it through a purification column (4.2.9) pre-activated with 5 mL methanol (4.2.3) and 15 mL water. Discard the unfiltered solution. Collect 3 mL of filtrate, filter the purified solution through a microporous membrane (4.2.10), and test.

4.5.1.2 Compound premixed feed Perform two parallel tests. Weigh 1-2 g (accurate to

0.1 mg) of the sample and place it in a 100 mL volumetric flask. Add 1-2 g of ED- Disodium TA, 60 mL chloroform-methanol solution (4.2.4), shake well, sonicate for 30 min, shaking once every 10 min, remove and cool to... At room temperature, dilute to volume (V) with chloroform-methanol solution (4.2.4), shake well, and filter. Accurately transfer 8 mL of the filtrate into a centrifuge tube and incubate at 40°C under nitrogen. Blow until nearly dry, accurately add 8 mL of water, vortex to mix, and pass through a purification column pre-activated with 5 mL of methanol (4.2.3) and 15 mL of water. (4.2.9) Discard the first 3 mL of filtrate, collect the purified liquid, filter it through a microporous membrane (4.2.10), and then test it.

4.5.2 Reference conditions for ion chromatography The reference conditions for ion chromatography are as follows:

a) Chromatographic column. Weak acid cation exchange column, 250 mm in length, 4 mm in inner diameter, 8.5 µm in particle size, or equivalent;

b) Guard column. Weak acid cation exchange column, 50 mm in length, 4 mm in inner diameter, 8.5 µm in particle size, or equivalent;

c) Column temperature. 30°C;

d) Eluent. Methanesulfonic acid solution (4.2.5), or an eluent compatible with the ion chromatograph;

f) Injection volume. 25 µL.

4.5.3 Measurement

4.5.3.1 Determination of Standard Solutions and Sample Solutions Under optimal instrument conditions, standard series solutions (4.2.8) and sample solutions (4.5.1) were tested using the instrument. Choline chloride standard solution... The ion chromatogram of the liquid is shown in Appendix A.

4.5.3.2 Qualitative Qualitative analysis is based on retention time. The retention time of choline chloride in the sample solution should be the same as that of choline chloride in the standard series solutions (with equivalent mass concentration). The retention time is consistent.

4.5.3.3 Quantitative analysis A standard curve was plotted with the mass concentration of choline chloride on the x-axis and the peak area (response value) on the y-axis. The correlation coefficient should not exceed [a certain value]. The concentration of choline chloride in the sample solution should be below 0.99.The concentration should be within the linear range of the standard curve. If it exceeds this range, the sample solution should be... Dilute with water and re-measure. During single-point calibration quantification, the mass concentration of the analyte in the sample solution should not differ significantly from the mass concentration of the standard solution. More than 30%. The separation between the target peak and adjacent peaks should be greater than 1.5.

4.6 Experimental Data Processing The choline chloride content w in the sample is calculated using formula (1) for multi-point calibration and formula (2) for single-point calibration. The values are expressed in milligrams per kilogram. It is expressed as (mg/kg). w=rho× V×1000 m×1000 ×f (1) In the formula. rho

--- The mass concentration of choline chloride in the sample solution obtained from the standard curve, expressed in micrograms per milliliter (µg/mL); V

--- Volume of the sample solution, in milliliters (mL); 1000

--- Conversion factor; m

--- Sample mass, in grams (g); f

--- The dilution factor of the sample solution that exceeds the linear range of the standard curve. w= A×rhoS×V×1000 AS×m×1000 ×f (2) In the formula. A

--- The chromatographic peak area of choline chloride in the sample solution; rhoS

--- the mass concentration of choline chloride in the standard solution, expressed in micrograms per milliliter (µg/mL); V

--- Volume of the sample solution, in milliliters (mL); 1000

--- Conversion factor; AS

--- Peak area of choline chloride in standard solution; m