GB/T 18246-2019 PDF English (GB/T 18246-2000: Older version)
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Determination of amino acids in feeds
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GB/T 18246-2019: PDF in English (GBT 18246-2019) GB/T 18246-2019
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 18246-2000
Determination of amino acids in feeds
ISSUED ON: DECEMBER 10, 2019
IMPLEMENTED ON: JULY 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 ... 5
2 Normative references ... 5
3 Conventional acid hydrolysis method ... 6
4 Oxidative acid hydrolysis method ... 9
5 Alkaline hydrolysis method ... 10
6 Acid extraction method ... 10
Appendix A (Normative) Molar mass and quantitation limit of each amino acid ... 13
Appendix B (Informative) Chromatogram of amino acid standard solution ... 14
Determination of amino acids in feeds
1 Scope
This Standard specifies the determination methods for total amino acids (peptide
bonded and free) and free amino acids (natural and added) in feeds, including
conventional acid hydrolysis method, oxidative acid hydrolysis method, alkaline
hydrolysis method and acid extraction method.
The conventional acid hydrolysis method applies to the determination of the content of
sulfur-containing amino acids (cystine, cysteine, methionine) and other amino acids
other than tryptophan (aspartic acid, threonine, serine, glutamic acid, proline, glycine,
alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine)
in feed raw materials, compound feeds, concentrated feeds, concentrate supplements
and additive premixes.
The oxidative acid hydrolysis method applies to the determination of sulfur-containing
amino acids (cystine, cysteine and methionine) in feed raw materials, compound feeds,
concentrated feeds and concentrate supplements. When sodium metabisulfite is used as
the oxidation terminator, other proteolytic amino acids except tyrosine and tryptophan
can be determined; when hydrobromic acid is used as the terminator, other proteolytic
amino acids except tyrosine, phenylalanine, histidine and tryptophan can be determined.
The alkaline hydrolysis method applies to the determination of tryptophan in feed raw
materials, compound feeds, concentrated feeds and concentrate supplements.
The acid extraction method applies to the determination of free amino acids in
compound feeds, concentrated feeds, concentrate supplements and additive premixes.
The quantitative limits of each amino acid are shown in Table A.1 in Appendix A.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
GB/T 6682, Water for analytical laboratory use - Specification and test methods
GB/T 15399, Determination of sulfur amino acids in feeds - Ion exchange
chromatography
GB/T 15400, Determination of tryptophan in feeds
GB/T 20195, Animal feeding stuffs. Preparation of test samples
JJG 1064, Automatic amino acid analyzer
3 Conventional acid hydrolysis method
3.1 Principle
The protein in the feed is hydrolyzed into amino acids at 110 ℃ and 6 mol/L
hydrochloric acid solution, separated by ion exchange chromatography and derivatized
on a ninhydrin column. Proline is determined at a wavelength of 440 nm, and other
amino acids are determined at a wavelength of 570 nm.
3.2 Reagents or materials
Unless otherwise specified, all reagents used in this Standard are analytical reagents.
3.2.1 Water: Shall comply with the requirements of Grade 1 water in GB/T 6682.
3.2.2 Hydrochloric acid: guaranteed reagent.
3.2.3 Hydrochloric acid hydrating solution (6 mol/L): Mix 500 mL of hydrochloric acid
(3.2.2) with 500 mL of water; add 1 g of phenol; mix well.
3.2.4 Sodium citrate buffer solution [pH = 2.2, c(Na+) = 0.2 mol/L]: Weigh 19.6 g of
trisodium citrate into a 1 000 mL volumetric flask; add water to dissolve; then, add 16.5
mL of hydrochloric acid (3.2.2), 5.0 mL of thiodiglycol, and 1 g of phenol; use water
to fix the volume and filter.
3.2.5 Amino acid standard stock solution: Containing 17 kinds of amino acids for
routine protein hydrolysate analysis (aspartic acid, threonine, serine, glutamic acid,
proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine,
phenylalanine, lysine, histidine and arginine). The concentration of each component is
2.50 μmol/mL. Certified standard solutions shall be used.
3.2.6 Amino acid standard working solution: Accurately transfer 2 mL of amino acid
standard stock solution (3.2.5) into a 50 mL volumetric flask; use sodium citrate buffer
solution (3.2.4) to fix the volume, with a concentration of 100 nmol/mL for each amino
acid component; package and seal in ampoules, approximately 1 mL each; store at 2 °C
~ 8 °C, valid for 3 months.
3.2.7 Sodium citrate buffer solutions and ninhydrin solutions of different pH values and
ionic strengths: Prepare or purchase according to the instrument manual.
3.2.8 Liquid nitrogen.
3.3 Instruments and equipment
3.3.1 Automatic amino acid analyzer: equipped with cation exchange column,
ninhydrin post-column derivatization device and 570 nm and 440 nm photometric
detectors.
3.3.2 Balance: sensitivity 0.1 mg and 0.01 mg.
3.3.3 Vacuum pump.
3.3.4 Blowtorch.
3.3.5 Constant temperature drying oven: The temperature can reach 110 ℃ ± 2 ℃.
3.3.6 Centrifuge: speed not less than 4 000 r/min.
3.3.7 Rotary evaporator or concentrator: The temperature can be adjusted between room
temperature and 65 °C, with a temperature control accuracy of ±1 °C and a vacuum
degree as low as 3.3×103 Pa (25 mmHg).
3.4 Test sample
Prepare the sample according to GB/T 20195; crush it through a 0.25 mm sieve; mix it
evenly; store it in a sealed container for later use.
For samples with crude fat content greater than or equal to 5%, the defatted samples
need to be air-dried, mixed, and placed in a sealed container for later use. For samples
with crude fat content less than 5%, they can be weighed directly.
3.5 Test procedure
3.5.1 Sample pretreatment
Perform two tests in parallel. Weigh 50 mg ~ 100 mg of the sample (containing about
7.5 mg ~ 25 mg of protein, accurate to 0.1 mg) into a 20 mL ampoule or hydrolysis tube;
accurately add 10 mL of hydrochloric acid hydrolysis solution (3.2.3); freeze in liquid
nitrogen (3.2.8); use a vacuum pump to evacuate to 7 Pa (≤5×10-2 mmHg); then use a
blowtorch to seal the mouth or fill with nitrogen for 1 min and tighten the tube cap.
Place the ampoule or hydrolysis tube in a constant temperature drying oven at 110 ℃
± 2 ℃ and hydrolyze for 22 h ~ 24 h. Cool; mix well; open the tube; filter; use a pipette
to accurately draw an appropriate amount of filtrate; place it in a rotary evaporator or
concentrator; evaporate to dryness under vacuum at 60 °C. If necessary, add a little
water and repeat the evaporation 1 ~ 2 times. Add 3 mL ~ 5 mL of sodium citrate buffer
solution (3.2.4) to re-dissolve, so that the amino acid concentration in the sample
solution reaches 50 nmol/mL ~ 250 nmol/mL; shake well; filter or centrifuge; take the
supernatant for determination.
3.5.2 Determination
Vi – injection volume of sample solution, in microliters (μL);
F – crude fat content, %.
Report the result as the arithmetic mean of the results of two parallel samples, retaining
two decimal places.
3.7 Precision
Under repeatability conditions, the absolute difference between two independent
measurement results and their arithmetic mean shall not exceed 5% of the arithmetic
mean of the two measurement values when the content is not greater than 0.5%; and
shall not exceed 4% when the content is greater than 0.5%.
4 Oxidative acid hydrolysis method
4.1 Determination of sulfur-containing amino acids
Carry out according to the provisions of GB/T 15399.
4.2 Determination of other amino acids
4.2.1 Principle
When sodium metabisulfite is used as the oxidation terminator, the content of other
protein hydrolyzed amino acids except tyrosine and tryptophan can be determined;
when hydrobromic acid is used as the terminator, the content of other amino acids
including sulfur-containing amino acids except tyrosine, phenylalanine, histidine and
tryptophan can be determined.
4.2.2 Sample pretreatment
Carry out sample pretreatment in accordance with the provisions of GB/T 15399.
4.2.3 Determination
Inject the standard solution containing cysteine, methionine sulfone and amino acids
into the automatic amino acid analyzer; use sodium citrate buffer solution and ninhydrin
solution (3.2.7) of different pH and ionic strength as mobile phase and derivatization
agent; adjust the instrument operation procedures and parameters, and the ratio of buffer
solution reagents for elution, which shall comply with the requirements for the
verification procedures of JJG 1064 Automatic amino acid analyzer; ensure that the
separation degrees of threonine-serine, glycine-alanine, leucine-isoleucine and
methionine sulfone-aspartic acid are not less than 85%, 90%, 80% and 85% respectively.
The chromatogram of the standard solution is shown in Figure B.3 of Appendix B. In
the determination, every 5 samples (i.e., 10 single samples) form a group, and cysteine,
methionine sulfone and amino acid standard solutions are inserted between the groups
6.2.6 Sodium citrate buffer solution [pH = 2.2, c(Na+) = 0.2 mol/L]: same as 3.2.4.
6.2.7 Amino acid standard stock solution: same as 3.2.5.
6.2.8 Amino acid standard working solution: same as 3.2.6.
6.2.9 Sodium citrate buffer solutions and ninhydrin solutions of different pH and ionic
strength: same as 3.2.7.
6.3 Instruments and equipment
6.3.1 Automatic amino acid analyzer: same as 3.3.1.
6.3.2 Balance: same as 3.3.2.
6.3.3 Centrifuge: same as 3.3.6.
6.3.4 Ultrasonic oscillator.
6.4 Test sample
Prepare the sample according to GB/T 20195; crush it through a 0.25 mm sieve; mix it
evenly; store it in a sealed container for later use.
6.5 Procedure
6.5.1 Sample pretreatment
6.5.1.1 Additive premix feed
Perform two tests in parallel. Weigh 1 g ~ 2 g of sample (accurate to 0.2 mg); add 30
mL of hydrochloric acid extraction solution (6.2.3); use ultrasonic oscillator to extract
for 15 min; let it stand for a while; filter the supernatant into a 100 mL volumetric flask;
repeat the extraction twice with 15 mL ~ 20 mL of water; filter the supernatant into the
above volumetric flask; use water to rinse the residue on the extraction flask and filter
paper; fix the volume. If the filtration is too slow during the sample extraction process,
centrifuge for 10 minutes (4 000 r/min), combine the supernatant and add water to fix
the volume. Shake well and use the filtrate for determination.
6.5.1.2 Compound feeds, concentrated feeds and concentrate supplements
Perform two tests in parallel. Weigh 2 g ~ 5 g (accurate to 0.5 mg) of the sample into a
100 mL centrifuge tube; add 50 mL of hydrochloric acid extraction solution (6.2.3); use
an ultrasonic oscillator to perform ultrasonic extraction for 30 min; centrifuge at 4 000
r/min for 10 min; transfer the supernatant to a 100 mL volumetric flask; use 45 mL of
hydrochloric acid extraction solution (6.2.3) to extract the residue once again. After
centrifugation, combine the supernatants and add water to fix the volume; accurately
transfer 10 mL of the filtrate into a 50 mL beaker; add 5 mL of 5-sulfosalicylic acid
solution (6.2.4); stir for 5 min; filter or centrifuge to remove the precipitate; use sodium
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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