GB/T 14701-2019Determination of vitamin B2 in feeds (English PDF)

3.2.11.1 Fluorescein stock solution: weigh

0.050 g of fluorescein; use water to dilute to 1 000 mL; place it in a brown bottle in the refrigerator at 2°C ~ 8°C to store. The solution contains 50 µg/mL of fluorescein.

3.2.11.2 Fluorescein standard working solution: take 1 mL of fluorescein stock solution (3.2.11.1); use water to dilute to 1 000 mL; place in a brown bottle in a refrigerator at 2°C ~ 8°C to store. The solution contains 0.05 µg/mL of fluorescein.

3.2.12 Bromocresol green pH indicator Take

0.1 g of bromocresol green; add

2.8 mL of sodium hydroxide solution (3.2.1) to dissolve; then, add water to dilute to 200 mL. The color-changing range is pH 3.6~5.2.

3.3 Instruments and apparatuses

3.3.1 Analytical balance: sensitivity of 0.000 1 g.

3.3.2 Electric constant-temperature water bath.

3.3.3 Glass scale test tube with stopper: 15 mL. 3.3.4, Fluorescence spectrophotometer.

3.4 Sample preparation According to the provisions of GB/T 14699.1, select at least 500 g of representative sample; use quartering to reduce it to 100 g; prepare samples according to GB/T 20195; grind; pass

0.425 mm hole sieve; mix well; place in a sealed container; keep out of the sun and store at low temperature for use.

3.5 Test steps The following operations shall be taken without strong light exposure.

3.5.1 Preparation of sample solution Weigh 1 g ~ 2 g (accurate to

0.001

g) of feed materials, compound feed and concentrated feed in a 100 mL brown conical flask. Add 65 mL of hydrochloric acid solution (3.2.3); boil for 30 min in a boiling water bath. At the beginning of the heating, shake the conical flask once every 5 min ~ 10 min, so as to prevent the sample from agglomerating. After it is cooled to room temperature, use sodium hydroxide solution (3.2.2) to adjust the pH to 6.0 ~ 6.5; immediately add hydrochloric acid solution (3.2.4) to adjust the pH to 4.5 (bromocresol green indicator becomes grass green). Transfer to a 100 mL brown volumetric flask; m0 -- the amount of the added vitamin B2 standard, in micrograms (µg); V -- the initial volume of the test solution, in milliliters (mL); V1 -- the volume of the taken test solution during the test, in milliliters (mL); m -- sample mass, in grams (g); n -- dilution ratio; The value of shall be 0.66 ~ 1.5; otherwise, the concentration of the sample solution shall be adjusted, and the sample volume or the dilution ratio shall be adjusted. The determination result is expressed in the arithmetic mean of the parallel determinations, with three significant figures retained.

3.7 Precision For feeds whose vitamin B2 content is less than 5 mg/kg, the difference BETWEEN the two independent determination results that are obtained under repeated conditions AND their arithmetic mean is not more than 15% of the arithmetic mean of the two determination values; For feeds whose vitamin B2 content is more than 5 mg/kg but less than 50 mg/kg, the difference BETWEEN the two independent determination results that are obtained under repeated conditions AND their arithmetic mean is not more than 10% of the arithmetic mean of the two determination values; For feeds whose vitamin B2 content is more than 50 mg/kg, the difference BETWEEN the two independent determination results that are obtained under repeated conditions AND their arithmetic mean is not more than 5% of the arithmetic mean of the two determination values;

4.2 Reagents or solutions

4.2.10.2 Vitamin B2 standard working solution: for the determination of vitamin premix feed sample, the standard solution in the standard stock solution of vitamin B2 (4.2.10.1) can be directly used as the standard solution of the upper machine; for the determination of compound premix feed, concentrated feed samples, accurately absorb 5 mL of vitamin B2 standard stock solution (4.2.10.1) in a 50 mL brown volumetric flask; use extract to dilute to the mark. The concentration of vitamin B2 in the standard working solution is 5 µg/mL; dilute before analysis.

4.3 Instruments and apparatuses

4.3.1 High-performance liquid chromatograph: with UV detector (diode matrix detector) or fluorescence detector.

4.3.2 pH meter: with temperature control; accuracy of 0.01.

4.3.3 Constant-temperature water bath: 0°C ~ 100°C.

4.3.4 Needle filter: with a 0.45 µm water filter.

4.4 Preparation of samples Sample according to the provisions of GB/T 14699.1; select at least 500 g of representative sample; use quartering to reduce it to 100 g; prepare samples according to GB/T 20195; grind; pass through

0.425 mm hole sieves; mix well; place in a sealed container; keep out of the sun and store at low temperature for use.

4.5 Test steps The following operations shall be taken without strong light exposure.

4.5.1 Preparation of sample solution Weigh

0.25 g ~

0.50 g (accurate to 0.000 1

g) of vitamin premix feed or 2 g ~ 3 g (accurate to

0.001

g) of compound premix feed, concentrated feed; add about 70 mL of extract (4.2.7) in a 100 mL brown conical flask; boil in a 100°C water bath for 30 min to 40 min; in the first few minutes, shake the conical flask to prevent solid agglomeration. After cooling, transfer to a 100 mL brown volumetric flask; use the extract to fix-volume to the mark; mix and filter. For vitamin premix feeds, it's necessary to use the extract to further dilute for 5 ~ 10 times. All test solutions need to be filtered through a 0.45 µm filter (4.3.4) before filtration by the liquid chromatography.