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
Determination of vitamin B2 in feeds
ICS 65.120
B46
National Standards of People's Republic of China
Replace GB/T 14701-2002
Determination of vitamin B2 in feed
Published on.2019-06-04
2020-01-01 implementation
State market supervision and administration
China National Standardization Administration issued
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard replaces GB/T 14701-2002 "Determination of Vitamin B2 in Feed".
Compared with GB/T 14701-2002, the main technical contents of this standard are revised as follows.
--- Revised the scope of application 1, method 2 and the limits of detection and quantitation (see Chapter 1, Chapter 1 of the.2002 edition);
---Arbitration law for different feed types (see Chapter 1, Chapter 1 of the.2002 edition);
--- Revised the pulverization size of the sample (see Chapter 3, Chapter 3 of the.2002 edition);
--- Supplemented the standard chromatogram of vitamin B2 under UV detection and fluorescence detection chromatography (see Appendix A, Figure A.1)
And Figure A.2).
This standard is proposed and managed by the National Feed Industry Standardization Technical Committee (SAC/TC76).
This standard was drafted. Institute of Agricultural Quality Standards and Testing Technology, Chinese Academy of Agricultural Sciences [National Feed Quality Supervision and Inspection Center
(Beijing)].
The main drafters of this standard. Li Lan, Suo Decheng, Wei Shulin.
The previous versions of the standards replaced by this standard are.
---GB/T 14701-1993, GB/T 14701-2002.
Determination of vitamin B2 in feed
1 Scope
This standard specifies the fluorescence spectrophotometry and high performance liquid chromatography for the determination of vitamin B2 (ie riboflavin C17H20N10) in feed.
The method 1 of this standard is applicable to the determination of vitamin B2 in animal and plant feed ingredients, compound feed and concentrated feed. The limit of quantification is
0.25 mg/kg.
This standard method 2 is applicable to the determination of vitamin B2 in vitamin premix feed, compound premix feed, and concentrated feed.
When the detector is tested, the limit of quantification is 5 mg/kg; when it is detected by UV detector, the limit of quantification is 10 mg/kg.
Determination of vitamin B2 in concentrated feed, method 1 is arbitration method; vitamin premix feed, additive premix feed vitamin
Determination of B2, the method of high performance liquid chromatography fluorescence detector in Method 2 is an arbitration method.
2 Normative references
The following documents are indispensable for the application of this document. For dated references, only dated versions apply to this article.
Pieces. For undated references, the latest edition (including all amendments) applies to this document.
GB/T 6682 Analytical laboratory water specifications and test methods
GB/T 14699.1 Feed sampling
Preparation of GB/T 20195 animal feed samples
3 Method 1 Fluorescence spectrophotometry
3.1 Principle
Vitamin B2 (riboflavin) produces green fluorescence under 440 nm UV excitation, and its fluorescence intensity and nuclear yellow in a certain concentration range
The content of the prime is proportional. Reduction of riboflavin to non-fluorescent substance by sodium dithionite, the ratio of fluorescence intensity difference to fluorescence intensity before and after reduction
The value is used to calculate the amount of vitamin B2 in the sample.
3.2 Reagents or solutions
Unless otherwise stated, the reagents used are analytically pure reagents, and the water is distilled water, which meets the third grade water or equivalent purity in GB/T 6682.
Water.
3.2.1 Sodium hydroxide solution. 0.05 mol/L.
3.2.2 Sodium hydroxide solution. 1.0 mol/L.
3.2.3 Hydrochloric acid solution. 0.1 mol/L.
3.2.4 Hydrochloric acid solution. 1.0 mol/L.
3.2.5 Sodium dithionite (Na2S2O4).
3.2.6 Potassium permanganate solution. 40g/L.
3.2.7 Glacial acetic acid.
3.2.8 Glacial acetic acid solution, 0.02 mol/L. 1.8 mL of glacial acetic acid was diluted with water to 1000 mL.
3.2.9 Hydrogen peroxide solution, 100 mL/L, prepared on the day of analysis.
3.2.10 Vitamin B2 standard solution
3.2.10.1 Vitamin B2 stock solution I. Weigh the vitamin B2 standard dried in a phosphorus pentoxide dryer for 24 hours (purity is greater than
95%) 25mg (accurate to 0.0001g) in a 250mL brown Erlenmeyer flask, add about.200mL glacial acetic acid solution (3.2.8), in a boiling water bath
Boil until dissolved, cool and transfer to a 250 mL brown volumetric flask and dilute to the mark with glacial acetic acid solution (3.2.8). Adding toluene
Cover, 2 ° C ~ 8 ° C refrigerator, storage period of 6 months. This solution contains 0.1 mg/mL of vitamin B2.
3.2.10.2 Vitamin B2 stock solution II. Take vitamin B2 stock solution I (3.2.10.1) 10mL diluted with glacial acetic acid solution (3.2.8) to
100mL, placed in a brown volumetric flask, added with toluene, stored in a refrigerator at 2 ° C ~ 8 ° C, the shelf life of 3 months. This solution contains 10μg/mL
Vitamin B2.
3.2.10.3 Vitamin B2 standard working solution. Take 10 mL of vitamin B2 stock solution II (3.2.10.2), dilute with water to 100 mL, before analysis
preparation. This solution contained 1 μg/mL of vitamin B2.
3.2.11 fluorescein standard solution
3.2.11.1 Fluorescein stock solution. Weigh 0.050g of fluorescein, dilute to 1000mL with water, and place it in a brown bottle at 2°C~8°C.
Save. This solution contained 50 μg/mL of fluorescein.
3.2.11.2 Fluorescein standard working solution. Take 1mL fluorescein stock solution (3.2.11.1), dilute to 1000mL with water, and place in brown bottle
Store in a refrigerator at 2°C~8°C. This solution contained 0.05 μg/mL of fluorescein.
3.2.12 bromocresol green pH indicator
Take 0.1 g of bromocresol green, dissolve it with 2.8 mL of sodium hydroxide solution (3.2.1), and dilute to.200 mL with water. Discoloration range
pH 3.6~5.2.
3.3 Instrumentation
3.3.1 Analytical balance. Sensitivity 0.0001g.
3.3.2 Electric heated water bath.
3.3.3 Test tube with stoppered glass. 15 mL.
3.3.4 Fluorescence spectrophotometer.
3.4 Sample preparation
According to the provisions of GB/T 14699.1, select a representative sample of at least 500g, use the quarter method to reduce to 100g;
Samples were prepared according to GB/T 20195, ground, passed through a 0.425 mm sieve, mixed, placed in a closed container, and stored at low temperature in the dark.
3.5 Test procedure
The following operations should avoid strong light exposure.
3.5.1 Preparation of sample solution
Weigh 1g~2g (accurate to 0.001g) of feed ingredients, compound feed and concentrated feed in 100mL brown conical flask. plus
Into a 65 mL hydrochloric acid solution (3.2.3), boil in a boiling water bath for 30 min. Shake the Erlenmeyer Bottle every 5min~10min at the beginning of heating
Once, in case the sample is agglomerated. After cooling to room temperature, adjust the pH to 6.0~6.5 with sodium hydroxide solution (3.2.2), immediately add hydrochloric acid solution.
(3.2.4) Adjust the pH to 4.5 (bromocresol green indicator becomes grass green). Transfer to a 100mL brown volumetric flask and dilute with water until engraved
degree. Filtration through medium speed ashless filter paper, discarding the first 5 mL~10 mL solution, and collecting the filtrate in a 100 mL brown Erlenmeyer flask. Rounding
The supernatant is diluted with dilute hydrochloric acid to check the protein. If a precipitate is formed, continue to add sodium hydroxide solution, and shake it vigorously to precipitate it completely. Again
Filtered as the liquid to be tested.
3.5.2 Impurity oxidation
Into each of the three 15mL graduated tubes (3.3.3) of a, b, c, inhale the sample solution (3.5.1) 10mL, and do parallel, add to the test tube a
1 mL of water was added, and 1 mL of vitamin B2 standard working solution (3.2.10.3) was added to the test tube b. Then add 1 mL of glacial acetic acid (3.2.7),
After shaking and mixing, add 0.5 mL of potassium permanganate solution (3.2.6) one by one, mix by shaking, let stand for 2 min, then add hydrogen peroxide solution one by one.
(3.2.9) 0.5 mL of shaking, so that the potassium permanganate color subsided within 10 s. The cover is shaken to allow the bubbles to escape.
3.5.3 Determination
The fluorimeter was adjusted with a fluorescein standard working solution (3.2.11.2) to stabilize it at a certain value as a fixed condition for the operation of the instrument. Tune
The whole excitation wavelength is 440 nm, the emission wavelength is 525 nm, and the fluorescence intensity of the test tube a and the test tube b is measured, and the sample solution is excited and irradiated in the instrument.
Not more than 10s; add 20mg of sodium dithionite (3.2.5) in test tube c, shake to dissolve, and let the gas in the test tube escape, quickly determine
Its fluorescence intensity acts as a fluorescent blank. If the solution is cloudy, it cannot be read.
3.6 Test data processing
The content of vitamin B2 in the sample is expressed in mass fraction in milligrams per kilogram (mg/kg), calculated according to formula (1).
Wi=
T1-T3
T2-T1×
M0
m ×
V1×
n (1)
In the formula.
T1---the fluorescence intensity of test tube a (test solution plus water);
T2---the fluorescence intensity of test tube b (test solution plus standard);
T3---the fluorescence intensity of test tube c (test solution plus sodium dithionite);
M0---the amount of vitamin B2 standard added, in micrograms (μg);
V --- the initial volume of the test solution, in milliliters (mL);
V1---The volume of the test solution is taken in test time, the unit is milliliter (mL);
m --- sample mass in grams (g);
n --- dilution factor;
T1-T3
T2-T1
The value should be 0.66~1.5, otherwise the concentration of the sample solution should be adjusted, and the sample volume or dilution factor should be adjusted.
The results are expressed as the arithmetic mean of the parallel measurements, with three significant figures retained.
3.7 precision
For feeds with a vitamin B2 content of less than 5 mg/kg, two independent determinations obtained under repeated conditions are arithmetically flat
The difference between the means is not more than 15% of the arithmetic mean of the two measured values;
For feeds with a vitamin B2 content greater than 5 mg/kg and less than 50 mg/kg, two independent tests were obtained under repetitive conditions.
The difference between the determined result and its arithmetic mean is not more than 10% of the arithmetic mean of the two measured values;
For feeds with a vitamin B2 content greater than 50 mg/kg, two independent determinations obtained under repeated conditions and their arithmetic
The difference between the means is not more than 5% of the arithmetic mean of the two measured values.
4 Method 2. High Performance Liquid Chromatography
4.1 Principle
After the riboflavin in the sample is ultrasonically extracted by an acidic solution, the centrifuged and filtered sample solution is injected into a high performance liquid chromatography reverse phase chromatography system.
Separation was performed in the system, and the content of vitamin B2 was calculated by an external standard method using a UV detector (diode matrix detector) or a fluorescence detector.
4.2 Reagents or solutions
Unless otherwise specified, the reagents used are of analytical grade, water is distilled water, and chromatographic water is deionized water, which meets the first level of GB/T 6682.
Water regulations.
4.2.1 Disodium edetate dihydrate (EDTA). excellent grade pure.
4.2.2 Sodium heptane sulfonate (PICB7). excellent grade pure.
4.2.3 Sodium dihydrogen phosphate. excellent grade pure.
4.2.4 Glacial acetic acid. excellent grade pure.
4.2.5 Triethylamine. chromatographically pure.
4.2.6 Methanol. Chromatographically pure.
4.2.7 Extract. In a 1000 mL volumetric flask, weigh 50 mg (accurate to 0.001 g) of EDTA (4.2.1) and add about 700 mL of deionized
Water, ultrasound completely dissolves EDTA. Add 25 mL of glacial acetic acid (4.2.4), 5 mL of triethylamine (4.2.5), and dilute to the mark with deionized water.
Shake well.
4.2.8 Sodium dihydrogen phosphate solution. Weigh 3.9 g of sodium dihydrogen phosphate, dissolve in 1000 mL of deionized water, and pass through the membrane for use.
4.2.9 Mobile phase I. In a 1000 mL volumetric flask, weigh 50 mg (accurate to 0.001 g) EDTA (4.2.1), 1.1 g (accurate to
0.001 g of sodium heptane sulfonate (4.2.2), about 700 mL of deionized water was added, and the solid was completely dissolved by sonication. Add 25mL glacial acetic acid
(4.2.4), 5mL of triethylamine (4.2.5), dilute to the mark with deionized water and shake. Adjust the pH of the solution to glacial acetic acid and triethylamine to
3.70 ± 0.10, over 0.45 μm filter. 800 mL of this solution was mixed with.200 mL of methanol (4.2.6), degassed by ultrasonication, and set aside.
4.2.10 Vitamin B2 standard solution
4.2.10.1 Vitamin B2 Standard stock solution. Weigh vitamin B2 (purity greater than 95%) dried in a phosphorus pentoxide dryer for 24 hours.
10mg (accurate to 0.0001g) in a 250mL Erlenmeyer flask, add 1mL glacial acetic acid (4.2.4) and boil for 30min in boiling water bath until solid
The pellet was completely dissolved, taken out and cooled to room temperature, transferred to a 250 mL brown volumetric flask, and made up to the mark with deionized water. Living in this solution
The concentration of B2 is 50μg/mL, and it can be stored in the refrigerator at 2°C~8°C for 6 months.
4.2.10.2 Vitamin B2 Standard working fluid. Determination of vitamin premixed feed samples, direct use of vitamin B2 standard stock solution
The standard solution in (4.2.10.1) is used as the standard solution on the machine; the composite premixed feed and concentrated feed sample should be accurately taken in 5mL dimension.
The standard stock solution of B2 (4.2.10.1) was placed in a 50 mL brown volumetric flask and the volume was adjusted to the mark with the extract. Vitamins in the standard working fluid
The concentration of B2 was 5 μg/mL, which was diluted before analysis.
4.3 Instrumentation
4.3.1 High Performance Liquid Chromatograph. with UV de...