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GB 7300.203-2020: Feed Additive - Part 2: Vitamins, provitamins and chemically well-defined substances having similar effect - Betaine
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GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 21515-2008
Feed additive - Part 2: Vitamins, provitamins and chemically
well-defined substances having similar effect - Betaine
ISSUED ON: MAY 29, 2020
IMPLEMENTED ON: DECEMBER 1, 2020
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Chemical name, molecular formula, relative molecular mass, and structural formula
... 4
4 Requirements ... 5
5 Test method ... 5
6 Inspection rules ... 20
7 Labeling, packaging, transportation, and storage ... 21
8 Shelf life ... 22
Appendix A (Informative) Chromatogram of betaine standard solution ... 23
Feed additive - Part 2: Vitamins, provitamins and chemically
well-defined substances having similar effect - Betaine
1 Scope
This part of GB 7300 specifies the requirements, test methods, inspection rules, labeling,
packaging, transportation, storage, and shelf life of the feed additive betaine.
This part applies to natural betaine products that are separated from beet molasses and
processed, and feed additive betaine products that are chemically synthesized from
sodium chloroacetate and trimethylamine.
2 Normative references
The following documents are essential to the application of this document. For the dated
referenced documents, only the versions with the indicated dates are applicable to this
document; for the undated referenced documents, only the latest version (including all
the amendments) is applicable to this document.
GB/T 601 Chemical reagent - Preparations of reference titration solutions
GB/T 602 Chemical reagent - Preparations of standard solutions for impurity
GB/T 603 Chemical reagent - Preparations of reagent solutions for use in test
methods
GB/T 6682 Water for analytical laboratory use - Specification and test methods
GB 10648 Feed label
GB/T 13079-2006 Determination of total arsenic in feeds
GB/T 14699.1 Feeding stuffs - Sampling
3 Chemical name, molecular formula, relative molecular mass,
and structural formula
Chemical name: Trimethylglycine
Molecular formula: C5H11NO2
5.1 Appearance characteristics
Take an appropriate amount of the sample, place it on a white porcelain plate, and
visually inspect its color and shape under natural light.
5.2 Identification test
5.2.1 Reagents
5.2.1.1 Hydrochloric acid solution: hydrochloric acid + water = 1 + 4 (volume ratio).
5.2.1.2 Bismuth potassium iodide solution: Take 0.85 g of bismuth subnitrate, dissolve
it in 10 mL of acetic acid, and add 40 mL of water. Take 40 g of potassium iodide,
dissolve it in water, and dilute the solution to 100 mL. Mix the above two solutions in
equal volumes and store the mixture in a brown glass container.
5.2.1.3 Bismuth potassium iodide hydrochloric acid solution: Take 1 mL of bismuth
potassium iodide solution (5.2.1.2), add 2 mL of hydrochloric acid solution (5.2.1.1),
and add water to 10 mL (prepare the solution newly when it will be used).
5.2.2 Identification method
5.2.2.1 Weigh 0.5 g of the sample, add 1 mL of water to dissolve, and add 2 mL of
bismuth potassium iodide hydrochloric acid solution (5.2.1.3). Shake the mixture to
produce an orange-red precipitate.
5.2.2.2 Take the solution in 5.3.1.4.1 and measure it with the instrument according to
5.3.1.4.3.3. The retention time is consistent with that of the standard solution.
5.3 Determination of betaine content
5.3.1 Ion chromatography (the arbitration method)
5.3.1.1 Method principle
The sample is dissolved in water, the solution is diluted to an appropriate concentration,
and a cation exchange column and a non-suppressed conductivity detector are used for
separation and determination. The quantitative analysis is carried out by the external
standard method.
5.3.1.2 Reagents and materials
5.3.1.2.1 Standard betaine (≥99%).
5.3.1.2.2 Methanesulfonic acid stock solution: Accurately pipette 2 mL of
methanesulfonic acid into a 100 mL volumetric flask, dilute to volume with water, and
shake well. The concentration is 300 mmol/L.
5.3.1.2.3 Mobile phase: Accurately pipette 10 mL of methanesulfonic acid stock
solution (5.3.1.2.2) into a 1000 mL volumetric flask, dilute to volume with water, and
shake well. The concentration is 3.0 mmol/L. Ultrasonic degassing for 5 min~10 min.
Prepare the solution newly when it will be used.
5.3.1.2.4 Betaine standard stock solution: Weigh 0.10 g (accurate to 0.0001 g) of
standard betaine (5.3.1.2.1) that has been dried at 105 °C±2 °C for 4 h; put it in a 100
mL volumetric flask, make up to volume with water and shake well. The solution
concentration is 1000 μg/mL.
5.3.1.2.5 Filter membrane: 0.45 μm, hydrophilic.
5.3.1.3 Instruments and equipment
5.3.1.3.1 Ion chromatograph: It has a cation exchange analytical column or another
analytical column with comparable performance and a conductivity detector.
5.3.1.3.2 Analytical balance: The sense quantity is 0.1 mg.
5.3.1.3.3 Electrically heated drying oven: The temperature can be controlled at
105 °C±2 °C.
5.3.1.4 Analysis steps
5.3.1.4.1 Extraction
Weigh about 0.25 g (accurate to 0.0001 g) of the sample that has been dried in an oven
at 105°C to a constant weight, place it in a 100 mL volumetric flask, add about 70 mL
of water, and make up the volume to the mark after the sample dissolved. Take 2 mL of
the solution to a 50 mL volumetric flask, make up the volume to the mark, shake well,
and pass it through a 0.45 μm filter membrane; let the filtrate stand for later test.
5.3.1.4.2 Preparation of a series of standard solutions
Accurately pipette 1.00 mL, 5.00 mL, 10.00 mL, 20.00 mL, and 50.00 mL of betaine
standard stock solution (5.3.1.2.4) into 100 mL volumetric flasks respectively, dilute to
volume with water, and shake well. The concentrations of the series of standard
solutions are 10.0 μg/mL, 50.0 μg/mL, 100.0 μg/mL, 200.0 μg/mL, and 500.0 μg/mL.
Prepare these solutions newly when they will be used soon.
5.3.1.4.3 Determination
5.3.1.4.3.1 Reference conditions for ion chromatograph
Chromatographic column: a cation exchange analytical column with 4 mm of inner
diameter and 250 mm of length, and 4.5 μm of packing particle size; or other
chromatographic columns with equivalent performance.
5.3.2 Perchloric acid titration method
5.3.2.1 Method principle
The sample that has been dried to constant weight is dissolved in glacial acetic acid,
and the perchloric acid is used as the standard titration solution; the crystal violet is
used as the indicator for titration, and when the color changes from purple to blue-green,
it is the end point of the titration.
5.3.2.2 Reagents or materials
5.3.2.2.1 Glacial acetic acid.
5.3.2.2.2 Acetic anhydride.
5.3.2.2.3 Crystal violet indicator solution: 5 g/L.
5.3.2.2.4 Perchloric acid standard titration solution: c(HClO4) = 0.1 mol/L.
5.3.2.3 Instruments and equipment
5.3.2.3.1 Acid burette: 25 mL.
5.3.2.3.2 Analytical balance: The sense quantity is 0.1 mg.
5.3.2.4 Test procedure
Weigh about 0.2 g (accurate to 0.0001 g) of the sample that has been dried in an oven
at 105 °C to constant weight, add 20 mL of glacial acetic acid (5.3.2.2.1) to dissolve the
sample, add 10 mL of acetic anhydride (5.3.2.2.2) and shake well; then, add 2 drops of
crystal violet indicator solution (5.3.2.2.3), and titrate the mixture with perchloric acid
standard titration solution (5.3.2.2.4) until the solution turns blue-green; do a blank test
at the same time.
5.3.2.5 Test data processing
The content w2 of betaine in the sample is expressed in mass fraction (%) and calculated
according to formula (2):
where:
c2 -- the concentration of perchloric acid standard titration solution, in moles per
liter (mol/L);
V2 -- the volume of perchloric acid standard titration solution consumed when the
sample is titrated, in milliliters (mL);
V20 -- the volume of perchloric acid standard titration solution consumed by the
blank test, in milliliters (mL);
m2 -- the mass of the sample, in grams (g);
M2 -- the molar mass of betaine, in grams per mole (g/mol) (M=117.15).
5.3.2.6 Result presentation
The measurement result is expressed as the arithmetic mean of parallel determinations,
and the result is rounded to three significant figures.
5.3.2.7 Repeatability
The absolute difference between two independent determination results obtained under
repeatability conditions is not more than 0.2%.
5.4 Loss on drying
5.4.1 Instruments and equipment
5.4.1.1 Electrically heated drying oven: The temperature can be controlled at
105 °C±2 °C.
5.4.1.2 Desiccator: The calcium chloride or color-changing silica gel is used as the
desiccant.
5.4.2 Determination steps
Weigh about 1 g of the sample (accurate to 0.0001 g) into a weighing dish with constant
weight, put it in an electrically heated drying oven (5.4.1.1) at 105 °C±2 °C, open the
cover of the weighing dish, and dry the sample for 3 h. Take the weighing dish out and
cover it, put it in a desiccator (5.4.1.2), cool it to room temperature, and weigh it. Repeat
the drying for 1 h, and weigh it until the weight is constant.
5.4.3 Test data processing
The loss on drying w3 of the sample is expressed in mass fraction (%) and calculated
according to formula (3):
where:
m30 -- the mass of the sample before drying, in grams (g);
gram (g).
5.5.4 Result presentation
The measurement result is expressed as the arithmetic mean of parallel determinations,
and the result is rounded to two significant figures.
5.5.5 Repeatability
The absolute difference between two independent determination results obtained under
repeatability conditions is not more than 0.1%.
5.6 Determination of total arsenic content (in As)
The determination shall be carried out according to the provisions of 5.4.1.3 in GB/T
13079-2006.
5.7 Determination of heavy metal content (in Pb)
5.7.1 Principle
Heavy metal ions and sulfide ions that have a charge of minus two form colored sulfide
precipitates in a weak acid medium. When the content of heavy metal elements is low,
a stable tan suspension is formed, which can be used for the visual colorimetric
determination of heavy metals.
5.7.2 Reagents or materials
5.7.2.1 Hydrochloric acid.
5.7.2.2 Nitric acid.
5.7.2.3 Ammonium acetate.
5.7.2.4 Glycerol.
5.7.2.5 Thioacetamide.
5.7.2.6 Ammonia.
5.7.2.7 Lead nitrate.
5.7.2.8 Phenolphthalein indicator solution: 10 g/L.
5.7.2.9 Hydrochloric acid solution: 7 mol/L.
5.7.2.10 Ammonia test solution: Take 400 mL of ammonia water, and add water to
dilute it to 1000 mL.
5.7.2.11 Acetate buffer solution (pH 3.5): Take 25 g of ammonium acetate (5.7.2.3), add
25 mL of water to dissolve it, add 38 mL of 7mol/L hydrochloric acid solution (5.7.2.9),
adjust the pH to 3.5, and add water to volume to 1000 mL.
5.7.2.12 Sodium hydroxide solution: c(NaOH) = 1 mol/L.
5.7.2.13 Thioacetamide test solution: Weigh 4 g of thioacetamide (5.7.2.5), add water
to dissolve it to 100 mL, and store it in a refrigerator. Before use, take 1.0 mL and add
it to 5.0 mL of the mixed solution, which is composed of 15 mL of sodium hydroxide
solution (5.7.2.12), 5.0 mL of water and 20 mL of glycerol (5.7.2.4); heat the mixture
on a water bath for 20 s, cool, and immediately use.
5.7.2.14 Lead standard stock solution: Weigh 0.160 g of lead nitrate (5.7.2.7), put it in
a 1000 mL volumetric flask, add 5 mL of nitric acid (5.7.2.2) and 50 mL of water to
dissolve it, dilute to the mark with water, and shake well. The solution concentration is
100 μg/mL. Store at 2 °C~8 °C after sealing, and the validity period is 3 months. Or
directly purchase lead standard solution with a concentration of 100 μg/mL.
5.7.2.15 Lead standard working solution: Accurately pipette 10 mL of lead standard
stock solution (5.7.2.14) into a 100 mL volumetric flask, dilute with water to the mark,
and shake well. The solution concentration is 10 μg/mL. Prepare the solution newly
when it will be used soon.
5.7.3 Test procedure
5.7.3.1 Preparation of test solution
Weigh 1.00 g±0.01 g of the sample in a crucible, carefully carbonize it in an electric
furnace, and then put it into a high-temperature furnace and burn it at 550 °C for 3 h.
After cooling, add 0.5 mL of nitric acid and evaporate to dryness. After the nitrogen
oxide vapor is completely removed, let it cool, add 2 mL of hydrochloric acid, and place
it on a water bath to evaporate to dryness; add 15 mL of water, add 2 drops of
phenolphthalein indicator solution (5.7.2.8), and then add dropwise the ammonia test
solution (5.7.2.10) until it reacts with the phenolphthalein indicator solution and the
solution turns neutral; add 2 mL of acetate buffer (pH 3.5) (5.7.2.11), and dissolve it
with slight heat.
5.7.3.2 Determination
Filter the test solution and transfer it to a Nessler colorimetric tube, add water to dilute
it to 25 mL, then add 2 mL of thioacetamide test solution (5.7.2.13), and shake well;
place it in the dark for 2 min, and then see through from top to bottom; the color of the
test solution shall not be darker than the standard colorimetric solution.
Standard colorimetric solution: Pipette 1.00 mL lead standard working solution, and at
the same time, treat it in the same way as the processing to the sample.
The measurement result is expressed as the arithmetic mean of parallel determinations,
and the result is rounded to three significant figures.
5.8.7 Repeatability
The absolute difference between the two independent determination results obtained
under repeatability conditions is not more than 30 mg/kg.
5.9 Determination of sodium chloride (NaCl) content
5.9.1 Synthetic betaine
5.9.1.1 Method principle
The chloride ions in the sample are dissolved in the aqueous solution, and the silver
nitrate standard solution is added to react with the chloride to form a silver chloride
precipitate, and the excess silver nitrate solution is titrated with the ammonium
thiocyanate or potassium thiocyanate standard solution.
5.9.1.2 Reagents or materials
5.9.1.2.1 Nitric acid.
5.9.1.2.2 Ferric ammonium sulfate saturated solution: It is prepared with ferric
ammonium sulfate [NH4Fe(SO4)2 • 12H2O].
5.9.1.2.3 Silver nitrate standard titration solution: c(AgNO3) = 0.1 mol/L.
5.9.1.2.4 Ammonium thiocyanate or potassium thiocyanate standard titration solution:
c(NH4SCN) = 0.1 mol/ L, c(KSCN) = 0.1 mol/L.
5.9.1.3 Instruments and equipment
5.9.1.3.1 Analytical balance: The sense quantity is 0.1 mg.
5.9.1.3.2 Acid burette: 25 mL.
5.9.1.4 Test procedure
Weigh about 10 g (accurate to 0.0001 g) of the sample that has been dried at 105 °C in
an oven to constant weight in advance, and dissolve it in 100 mL of water in a conical
flask. Add 5 mL of nitric acid (5.9.1.2.1), 2 mL of ferric ammonium sulfate saturated
solution (5.9.1.2.2), and add 2 drops of ammonium thiocyanate or potassium
thiocyanate standard titration solution dropwise from a burette that has been filled up
to the scale 0 with ammonium thiocyanate or potassium thiocyanate standard titration
solution (5.9.1.2.4).
The absolute difference between two independent determination results obtained under
repeatability conditions is not more than 0.1%.
5.9.2 Natural Betaine
5.9.2.1 Instruments and equipment
5.9.2.1.1 Analytical balance: The sense quantity is 0.1 mg.
5.9.2.2 Reagents
5.9.2.2.1 Chloride stock solution (1 mL of solution contains 0.1 mg of Cl- ): Weigh
0.165 g of sodium chloride that has been burned to constant weight at 500 ℃~600 ℃,
dissolve it in ultrapure water, and dilute to 1000 mL.
5.9.2.2.2 Chloride standard solution (1 mL of solution contains 0.01 mg of Cl-):
Accurately pipette 10 mL of chloride stock solution (5.9.2.2.1) into a 100 mL
volumetric flask, dilute with ultrapure water to volume.
5.9.2.2.3 Nitric acid solution: 25%. Measure 308 mL of nitric acid and dilute it to 1000
mL with ultrapure water.
5.9.2.2.4 Silver nitrate solution: 17 g/L. Weigh 1.7 g of silver nitrate, dissolve it in
ultrapure water, and dilute the solution to 100 mL.
5.9.2.3 Test procedure
Weigh 5 g of the sample (accurate to 0.01 g) in a beaker, dissolve it with ultrapure water,
and dilute the solution to 100 mL. After filtration, take 10 mL of the filtrate into a 25
mL Nessler colorimetric tube.
Accurately pipette 5 mL of chloride standard solution (5.9.2.2.2) into another 25 mL
Nessler colorimetric tube.
Add 1 mL of nitric acid solution (5.9.2.2.3) and 1 mL of silver nitrate solution (5.9.2.2.4)
into the above-mentioned two Nessler colorimetric tubes, and add ultrapure water to
dilute to 25 mL. Shake well and let them stand for 10 min.
The turbidity of the sample solution shall not be greater than that of the standard
solution.
5.10 Determination of sulfate content (in SO2-4 )
5.10.1 Method principle
According to the principle that barium ions and sulfate ions form barium sulfate
precipitates, the content of sulfate can be judged by comparing the turbidity of the
sample solution and the standard solution.
5.10.2 Instruments and equipment
Electronic balance: The sense quantity is 0.01 g.
5.10.3 Reagents
5.10.3.1 Sulfate standard solution: The concentration of SO2-4 is 1000 mg/L, which can
be purchased directly.
5.10.3.2 Hydrochloric acid solution: 10%.
5.10.3.3 Barium chloride solution: 120 g/L.
5.10.4 Test procedure
Weigh about 10 g of the sample, accurate to 0.01 g, put it into a 200 mL volumetric
flask, dissolve it in water, and make up to the mark; shake well and filter it. Accurately
pipette 20 mL of filtrate in a 50 mL beaker.
In addition, pipette 19 mL of water into another 50 mL beaker, and add 1 mL of sulfate
standard solution (5.10.3.1). According to the sulfate content (in SO2-4 ) contained in the
sample, different volumes of sulfate standard solution can be added.
Add 1 mL of hydrochloric acid solution (5.10.3.2) and 3 mL of barium chloride solution
(5.10.3.3) into the above-mentioned two 50 mL beakers respectively, mix well, and let
them stand for 10 min; compare the turbidity between the sample solution and the
standard solution.
The turbidity of the sample solution shall not be greater than that of the standard
solution.
6 Inspection rules
6.1 Batch grouping
Products that are produced continuously or in the same shift with the same raw materials
and the same production process are regarded as a batch.
6.2 Sampling
It shall be carried out according to the provisions of GB/T 14699.1.
6.3 Factory inspection
The factory inspection items include betaine content, appearance and characteristics,
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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