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GB/T 14701-2019 English PDF (GBT14701-2019)

GB/T 14701-2019 English PDF (GBT14701-2019)

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GB/T 14701-2019: Determination of vitamin B2 in feeds
GB/T 14701-2019
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 14701-2002
Determination of vitamin B2 in feeds
ISSUED ON: JUNE 04, 2019
IMPLEMENTED ON: JANUARY 01, 2020
Issued by: State Administration for Market Regulation;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Method 1: Fluorescence spectrophotometry ... 4 
4 Method 2: High-performance liquid chromatography ... 8 
Appendix A (Informative) Vitamin B2 standard chromatogram ... 13 
Determination of Vitamin B2 in feeds
1 Scope
This Standard specifies the fluorescence spectrophotometry and high-
performance liquid chromatography for the determination of vitamin B2 (namely
riboflavin C17H20N10) in feeds.
Method 1 of this Standard is applicable to the determination of vitamin B2 in
animal and plant feed materials, compound feed and concentrated feed; the
quantitation-limit is 0.25 mg/kg.
Method 2 of this Standard is applicable to the determination of vitamin B2 in
vitamin premix feed, compound premix feed, and concentrated feed. When it is
detected by a fluorescence detector, the quantitation-limit is 5 mg/kg; when it is
detected by an ultraviolet detector, the quantitation-limit is 10 mg/kg.
For the determination of vitamin B2 in concentrated feed, method 1 is the
arbitration method; for the determination of vitamin B2 in vitamin premix feed
and additive premix feed, the high-performance liquid chromatography
fluorescence detector method of method 2 is the arbitration method.
2 Normative references
The following documents are indispensable for the application of this document.
For dated references, only the dated version applies to this document. For
undated references, the latest edition (including all amendments) applies to this
document.
GB/T 6682, Water for analytical laboratory use. Specification and test methods
GB/T 14699.1, Feeding stuffs. Sampling
GB/T 20195, Animal feeding stuffs. Preparation of test samples
3 Method 1: Fluorescence spectrophotometry
3.1 Principle
Vitamin B2 (riboflavin) produces green fluorescence under excitation of 440 nm
ultraviolet light; its fluorescence intensity is proportional to the riboflavin content
in a certain concentration range. Use sodium dithionite to reduce riboflavin to a
non-fluorescent substance; use the ratio OF the difference between the
3.2.11 Fluorescein standard solution
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 Method 2: High-performance liquid chromatography
4.1 Principle
After the riboflavin in the sample is ultrasonically extracted by an acidic solution,
inject the centrifuged and filtered sample solution into a high-performance liquid
chromatography reverse-phase chromatography system for separation; use a
UV detector (diode matrix detector) or a fluorescence detector to detect; use
the external standard method to calculate the content of vitamin B2.
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...
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