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GB/T 13087-2020 English PDF (GBT13087-2020)
GB/T 13087-2020 English PDF (GBT13087-2020)
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GB/T 13087-2020: Determination of isothiocyanates in feeds
GB/T 13087-2020
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 13087-1991
Determination of Isothiocyanates in Feeds
ISSUED ON: JULY 21, 2020
IMPLEMENTED ON: FEBRUARY 01, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Principle ... 4
4 Reagents and Materials ... 4
5 Apparatus ... 6
6 Sample ... 6
7 Test Procedures ... 6
8 Test Data Processing ... 7
9 Precision ... 9
Appendix A (Informative) Gas Chromatogram of Isothiocyanate Standard
Solution ... 10
Determination of Isothiocyanates in Feeds
1 Scope
This Standard specifies the gas chromatography method for the determination of
isothiocyanate in feeds.
This Standard is applicable to the determination of isothiocyanate in compound feeds,
concentrated feeds, concentrate supplements and feed ingredients.
The detection limit of this Standard is 2mg/kg; and the limit of quantification is 5mg/kg.
2 Normative References
The following documents are essential to the application of this document. For the
dated documents, only the versions with the dates indicated are applicable to this
document; for the undated documents, only the latest version (including all the
amendments) is applicable to this document.
GB/T 5520 Inspection of Grain and Oils - Germination Test of Seeds
GB/T 6682 Water for Analytical Laboratory Use – Specification and Test Methods
GB/T 20195 Animal Feeding Stuffs－Preparation of Test Samples
3 Principle
The glucosinolate in the specimen is in the buffer solution with pH 7.0; under the action
of white myrosinase, generate allyl isothiocyanate, butenyl isothiocyanate and
pentenyl isothiocyanate. Use the methyl chloride to extract, dehydrate, filter; and then
is determined by gas chromatograph; and quantified by internal standard method. The
result is calculated as per allyl isothiocyanate.
4 Reagents and Materials
Unless otherwise specified, only use analytically-pure reagents.
4.1 Water: Class-I water specified in GB/T 6682.
4.2 Dichloromethane.
4.3 Petroleum ether (boiling range 30°C ~60°C).
4.4 Anhydrous sodium sulfate.
4.5 Citric acid solution (0.1mol/L): accurately weigh 4.20g of citric acid (C6H8O7·H2O)
and dissolve in water; dilute by water and make constant volume to 200mL; and mix
evenly. Prepare for current use.
4.6 Disodium hydrogen phosphate solution (0.2mol/L): accurately weigh 28.39g of
anhydrous disodium hydrogen phosphate (Na2HPO4) and dissolve in water; dilute by
water and make constant volume to 1L; and mix evenly. Prepare for current use.
4.7 Hydrochloric acid solution (0.01mol/L): accurately pipette 0.9mL of concentrated
hydrochloric acid; dilute by water and make constant volume of 1L; and mix evenly.
4.8 Sodium hydroxide solution (0.01mol/L): accurately take 0.40g of sodium hydroxide
and dissolve in water, dilute with water and dilute to 1L, and mix.
4.9 Buffer solution with pH 7.0: take 176.5 mL of citric acid solution (4.5) in a 1000 mL
volumetric flask; dilute by disodium hydrogen phosphate solution (4.6) and make the
constant volume to 1000 mL; mix evenly; and use hydrochloric acid solution (4.7) or
sodium hydroxide solution (4.8) to adjust the pH to 7.0. Prepare for current use.
4.10 White myrosinase: take 50g of white mustard (Sinapis alba L.) seeds (make the
germination test in accordance with GB/T 5520; the germination rate shall be greater
than 85% within 72h; and the storage period shall not exceed two years); after crushing,
place them in a 500mL beaker. Add 100mL of petroleum ether (4.3); stir for 2min; let
stand; discard the supernatant liquid; repeat degreasing for 10 times; make the fat
content less than 2%. Put the solvent in a fume hood to evaporate dry; and then
pulverize again; 80% passed through the 0.28mm test sieve; pack in an enclosed
container; store at -18°C below; valid for 6 months. In order to ensure the activity of
white myrosinase, the environment temperature shall be kept below 30°C during the
preparation process. Pay attention to a small amount and multiple times during the two
crushing to prevent the crusher from overheating.
4.11 Butyl isothiocyanate internal standard solution (0.1mg/mL): accurately take 50mg
(accurate to 0.00001g) of butyl isothiocyanate [CH3(CH2)3NCS, CAS number: 592-82-
5, purity ≥99%] in a 500mL brown volumetric flask; dissolve and make constant volume
by dichloromethane; mix evenly. Store at -18°C below; valid for 3 months.
4.12 Isothiocyanate mixed standard solution (1.0mg/mL): accurately take 10mg
(accurate to 0.00001g) of allyl isothiocyanate (CH2 = CHCH2NCS, CAS number: 57-
06-7, purity ≥ 99.7%], butenyl isothiocyanate [CH2 =CHCH2CH2NCS, CAS number:
3386-97-8, purity ≥95.0%], pentenyl isothiocyanate [CH2 = CHCH2CH2CH2NCS, CAS
number: 18060-79-2, purity ≥95.0%] in a 10mL brown volumetric flask; dissolve and
make constant volume by butyl isothiocyanate internal standard solution (4.11); mix
7.2 Reference conditions for gas chromatography
Chromatographic column: polyethylene glycol (FFAP) capillary column, length of 30m,
inner diameter of 0.32mm, film thickness of 0.25μm, or equivalent.
Chromatographic column temperature: starting temperature 100°C, keep for 10min;
increase to 200°C at 10°C/min; keep for 2min.
Inlet temperature: 220°C.
Detector temperature: 230°C.
Carrier gas: high purity nitrogen.
Carrier gas flow rate: 1.0mL/min.
Hydrogen flow rate: 35mL/min.
Air flow rate: 400mL/min.
Makeup flow rate: 20mL/min.
Injection volume: 1μL.
Split ratio: 5:1.
7.3 Determination
7.3.1 Determination of standard solution and specimen solution
Under the best conditions of the instrument, take the isothiocyanate mixed standard
series working solution (4.13) and specimen solution (7.1) on the machine to determine.
Refer to Appendix A for the gas chromatogram of each isothiocyanate standard
solution.
7.3.2 Qualitative and quantitative
Be qualitative by retention time, the retention time of each isothiocyanate in the
specimen solution shall be consistent with the retention time of each isothiocyanate in
the standard solution; and the relative deviation is within ±2.5%. The internal standard
method is used for quantification.
8 Test Data Processing
The content of isothiocyanate (calculated by allyl isothiocyanate) in the specimen is
calculated in mass fraction w; and the value is expressed in mg/kg, and calculated
according to Formula (1):
Get QUOTATION in 1-minute: Click GB/T 13087-2020
Historical versions: GB/T 13087-2020
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GB/T 13087-2020: Determination of isothiocyanates in feeds
GB/T 13087-2020
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 13087-1991
Determination of Isothiocyanates in Feeds
ISSUED ON: JULY 21, 2020
IMPLEMENTED ON: FEBRUARY 01, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Principle ... 4
4 Reagents and Materials ... 4
5 Apparatus ... 6
6 Sample ... 6
7 Test Procedures ... 6
8 Test Data Processing ... 7
9 Precision ... 9
Appendix A (Informative) Gas Chromatogram of Isothiocyanate Standard
Solution ... 10
Determination of Isothiocyanates in Feeds
1 Scope
This Standard specifies the gas chromatography method for the determination of
isothiocyanate in feeds.
This Standard is applicable to the determination of isothiocyanate in compound feeds,
concentrated feeds, concentrate supplements and feed ingredients.
The detection limit of this Standard is 2mg/kg; and the limit of quantification is 5mg/kg.
2 Normative References
The following documents are essential to the application of this document. For the
dated documents, only the versions with the dates indicated are applicable to this
document; for the undated documents, only the latest version (including all the
amendments) is applicable to this document.
GB/T 5520 Inspection of Grain and Oils - Germination Test of Seeds
GB/T 6682 Water for Analytical Laboratory Use – Specification and Test Methods
GB/T 20195 Animal Feeding Stuffs－Preparation of Test Samples
3 Principle
The glucosinolate in the specimen is in the buffer solution with pH 7.0; under the action
of white myrosinase, generate allyl isothiocyanate, butenyl isothiocyanate and
pentenyl isothiocyanate. Use the methyl chloride to extract, dehydrate, filter; and then
is determined by gas chromatograph; and quantified by internal standard method. The
result is calculated as per allyl isothiocyanate.
4 Reagents and Materials
Unless otherwise specified, only use analytically-pure reagents.
4.1 Water: Class-I water specified in GB/T 6682.
4.2 Dichloromethane.
4.3 Petroleum ether (boiling range 30°C ~60°C).
4.4 Anhydrous sodium sulfate.
4.5 Citric acid solution (0.1mol/L): accurately weigh 4.20g of citric acid (C6H8O7·H2O)
and dissolve in water; dilute by water and make constant volume to 200mL; and mix
evenly. Prepare for current use.
4.6 Disodium hydrogen phosphate solution (0.2mol/L): accurately weigh 28.39g of
anhydrous disodium hydrogen phosphate (Na2HPO4) and dissolve in water; dilute by
water and make constant volume to 1L; and mix evenly. Prepare for current use.
4.7 Hydrochloric acid solution (0.01mol/L): accurately pipette 0.9mL of concentrated
hydrochloric acid; dilute by water and make constant volume of 1L; and mix evenly.
4.8 Sodium hydroxide solution (0.01mol/L): accurately take 0.40g of sodium hydroxide
and dissolve in water, dilute with water and dilute to 1L, and mix.
4.9 Buffer solution with pH 7.0: take 176.5 mL of citric acid solution (4.5) in a 1000 mL
volumetric flask; dilute by disodium hydrogen phosphate solution (4.6) and make the
constant volume to 1000 mL; mix evenly; and use hydrochloric acid solution (4.7) or
sodium hydroxide solution (4.8) to adjust the pH to 7.0. Prepare for current use.
4.10 White myrosinase: take 50g of white mustard (Sinapis alba L.) seeds (make the
germination test in accordance with GB/T 5520; the germination rate shall be greater
than 85% within 72h; and the storage period shall not exceed two years); after crushing,
place them in a 500mL beaker. Add 100mL of petroleum ether (4.3); stir for 2min; let
stand; discard the supernatant liquid; repeat degreasing for 10 times; make the fat
content less than 2%. Put the solvent in a fume hood to evaporate dry; and then
pulverize again; 80% passed through the 0.28mm test sieve; pack in an enclosed
container; store at -18°C below; valid for 6 months. In order to ensure the activity of
white myrosinase, the environment temperature shall be kept below 30°C during the
preparation process. Pay attention to a small amount and multiple times during the two
crushing to prevent the crusher from overheating.
4.11 Butyl isothiocyanate internal standard solution (0.1mg/mL): accurately take 50mg
(accurate to 0.00001g) of butyl isothiocyanate [CH3(CH2)3NCS, CAS number: 592-82-
5, purity ≥99%] in a 500mL brown volumetric flask; dissolve and make constant volume
by dichloromethane; mix evenly. Store at -18°C below; valid for 3 months.
4.12 Isothiocyanate mixed standard solution (1.0mg/mL): accurately take 10mg
(accurate to 0.00001g) of allyl isothiocyanate (CH2 = CHCH2NCS, CAS number: 57-
06-7, purity ≥ 99.7%], butenyl isothiocyanate [CH2 =CHCH2CH2NCS, CAS number:
3386-97-8, purity ≥95.0%], pentenyl isothiocyanate [CH2 = CHCH2CH2CH2NCS, CAS
number: 18060-79-2, purity ≥95.0%] in a 10mL brown volumetric flask; dissolve and
make constant volume by butyl isothiocyanate internal standard solution (4.11); mix
7.2 Reference conditions for gas chromatography
Chromatographic column: polyethylene glycol (FFAP) capillary column, length of 30m,
inner diameter of 0.32mm, film thickness of 0.25μm, or equivalent.
Chromatographic column temperature: starting temperature 100°C, keep for 10min;
increase to 200°C at 10°C/min; keep for 2min.
Inlet temperature: 220°C.
Detector temperature: 230°C.
Carrier gas: high purity nitrogen.
Carrier gas flow rate: 1.0mL/min.
Hydrogen flow rate: 35mL/min.
Air flow rate: 400mL/min.
Makeup flow rate: 20mL/min.
Injection volume: 1μL.
Split ratio: 5:1.
7.3 Determination
7.3.1 Determination of standard solution and specimen solution
Under the best conditions of the instrument, take the isothiocyanate mixed standard
series working solution (4.13) and specimen solution (7.1) on the machine to determine.
Refer to Appendix A for the gas chromatogram of each isothiocyanate standard
solution.
7.3.2 Qualitative and quantitative
Be qualitative by retention time, the retention time of each isothiocyanate in the
specimen solution shall be consistent with the retention time of each isothiocyanate in
the standard solution; and the relative deviation is within ±2.5%. The internal standard
method is used for quantification.
8 Test Data Processing
The content of isothiocyanate (calculated by allyl isothiocyanate) in the specimen is
calculated in mass fraction w; and the value is expressed in mg/kg, and calculated
according to Formula (1):
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