GB 5009.208-2016 English PDF (GB5009.208-2016)

Delivery: 3 seconds. Download true-PDF + Invoice.
Get QUOTATION in 1-minute: Click GB 5009.208-2016
Historical versions: GB 5009.208-2016
Preview True-PDF (Reload/Scroll if blank)

GB 5009.208-2016: Food safety national standard -- Determination of biogenic amines in food
GB 5009.208-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of
Biogenic Amine in Food
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the
People’s Republic of China;
China Food and Drug Administration.
Table of Contents
Foreword ... 3 
Method I Liquid Chromatography ... 4 
1 Scope ... 4 
2 Principle ... 4 
3 Reagents and Materials ... 4 
4 Instruments and Equipment ... 8 
5 Analytical Procedures ... 8 
6 Expression of Analytical Result ... 11 
7 Precision ... 11 
8 Others ... 11 
Method II Spectrophotometry ... 13 
9 Scope ... 13 
10 Principle ... 13 
11 Reagents and Materials ... 13 
12 Analytical Procedures ... 14 
13 Result Calculation ... 15 
14 Precision ... 16 
15 Others ... 16 
Appendix A Liquid Chromatogram of 9 Biogenic Amine Standard Solutions and
Internal Standard Derivatives ... 17 
National Food Safety Standard - Determination of
Biogenic Amine in Food
Method I Liquid Chromatography
1 Scope
This Standard specifies the determination method for tryptamine, -phenylethylamine,
putrescine, cadaverine, histamine, octopamine, tyramine, spermidine and spermine
content in food.
This Standard is applicable to the determination of alcohol (wine, beer and yellow rice
wine, etc.), condiments (vinegar and soy sauce), aquatic products (fish and its products;
shrimp and its products), biogenic amine in meat.
2 Principle
Aquatic products (fish and its products; shrimp and its products) and meat: use 5%
trichloroacetic acid to extract sample; use n-hexane to remove fat. After extraction and
purification through trichloromethane-n-butanol (1 + 1) solution, use Dansyl chloride
for derivation. Use C18 chromatographic column for separation. Use high-performance
liquid chromatography-UV detector for detection. Use internal standard method for
quantification.
Alcohol (wine, beer and yellow rice wine, etc.) and sediments (vinegar and soy sauce):
use Dansyl chloride for derivation. Use C18 chromatographic column for separation.
Use high-performance liquid chromatography-UV detector for detection. Use internal
standard method for quantification.
3 Reagents and Materials
Unless it is otherwise stipulated, all reagents used in this Method are analytically pure;
water is Grade-1 water stipulated in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN): chromatographically pure.
3.1.2 Acetone (C3H6O): chromatographically pure.
3.1.3 Ether(C4H10O): re-evaporated.
3.2.7 Saturated sodium bicarbonate solution: weigh-take 15 g of sodium bicarbonate;
add 100 mL of water to dissolve it; take the supernatant as the saturated solution.
3.2.8 50 mg/mL sodium glutamate solution: accurately weigh-take 5.0 g of sodium
glutamate; use saturated sodium bicarbonate solution to dissolve it and reach a
constant volume of 100 mL.
3.2.9 0.01 mol/L ammonium acetate solution that contains 1% acetic acid: weigh-take
0.77 g of ammonium acetate; dissolve it in water; then, transfer it into a 1,000 mL
volumetric flask. Add 10 mL of formic acid; use water to reach a constant volume to
the scale.
3.2.10 Mobile phase A: measure-take 100 mL of 0.01 mol/L ammonium acetate
solution that contains 1% acetic acid; add it to 900 mL of acetonitrile.
3.2.11 Mobile phase B: measure-take 900 mL of 0.01 mol/L ammonium acetate
solution that contains 1% acetic acid; add it to 100 mL of acetonitrile.
3.3 Standard Substances
3.3.1 Histamine dihydrochloride (C5H9N32HCl, CAS No.: 56-92-8) standard
substance (purity > 99%).
3.3.2 -phenylethylamine hydrochloride (C8H11NHCl, CAS No.: 64-04-0) standard
substance (purity > 98%).
3.3.3 Tyramine hydrochloride (C8H11NOHCl, CAS No.: 60-19-5) standard substance
(purity > 98%).
3.3.4 Putrescine dihydrochloride (C4H12N22HCl, CAS No.: 333-93-7) standard
substance (purity > 98%).
3.3.5 Cadaverine dihydrochloride (C5H14N22HCl, CAS No.: 1476-39-7) standard
substance (purity > 98%).
3.3.6 Tryptamine hydrochloride (C10H12N2HCl, CAS No.: 61-54-1) standard substance
(purity > 99%).
3.3.7 Spermine tetrahydrochloride (C10H26N44HCl, CAS No.: 306-67-2) standard
substance (purity > 97%).
3.3.8 Spermidine trihydrochloride (C7H19N33HCl, CAS No.: 334-50-9) standard
substance (purity > 97%).
3.3.9 Octopamine hydrochloride (C8H11NO2HCl, CAS No.: 770-05-9) standard
substance (purity > 97%).
3.3.10 1,7-Diaminoheptane (C7H18N2, CAS No.: 646-19-5) internal standard substance
5.1.3.1 Fat removal: transfer-take 10 mL of the above-mentioned sample extract into
a 25 mL test tube with a plug. Add 0.5 g of sodium chloride; conduct vortex oscillation,
till sodium chloride completely dissolves. Then, add 10 mL of n-hexane; conduct vortex
oscillation for 5 min. Place it still for stratification; then, discard the upper layer of
organic phase. Add 10 mL of n-hexane to the lower layer of sample solution to conduct
fat removal one more time.
5.1.3.2 Extraction: transfer-take 5 mL of the above-mentioned post-fat-removal sample
solution into a 10 mL centrifuge tube with a plug. Use 5 mol/L sodium hydroxide
solution (several drops) to adjust pH to around 12.0. Add 5 mL of N-
butanol/trichloromethane (1 + 1) mixing solution. Conduct vortex oscillation for 5 min.
At 5,000 r/min, conduct centrifugation for 5 min. Then, transfer the upper layer of
organic phase into another 10 mL centrifuge tube with a plug. Re-extract the lower
layer of the sample solution; combine the extract. Use N-butanol/trichloromethane (1
+ 1) to dilute to the scale. Take 5 mL of the extract; add 200 L of hydrochloric acid (1
mol/L); thoroughly mix it up. In 40 °C water bath, use nitrogen to blow it to dryness.
Add 1 mL of hydrochloric acid (0.1 mol/L); conduct vortex oscillation. Thoroughly
dissolve the residue. Reserve it for derivation.
5.1.4 Derivation
5.1.4.1 Derivation of sample: successively add 1 mL of saturated sodium bicarbonate
solution, 100 L of sodium hydroxide solution (1 mol/L) and 1 mL of derivatization
reagent to the above-mentioned sample solution, which is reserved for derivation.
Conduct vortex and mixing for 1 min, then, place it in 60 °C constant-temperature water
bath to conduct derivation for 15 min. Then, take it out; add 100 L of sodium glutamate
solution; conduct oscillation mixing. At 60 °C, conduct constant-temperature reaction
for 15 min. Take it out, then, cool it down to room temperature. Add 1 mL of water to
each centrifuge tube; conduct vortex mixing for 1 min. In 40 °C water bath, use nitrogen
to blow it, so as to remove acetone (around 1 mL). Add 0.5 g of sodium chloride;
conduct vortex oscillation, till sodium chloride completely dissolves. Then, add 5 mL of
ether; then, conduct vortex oscillation for 2 min; place it still for stratification. Then,
suck out the upper layer of organic phase (the ether layer); re-extract once. Combine
the ether extract; in 40 °C water bath, use nitrogen to blow it to dryness. Add 1 mL of
acetonitrile to conduct vortex oscillation, so that the residue can completely dissolve.
Use 0.22 m membrane needle filter to filter it into a small sample injection bottle.
Reserve it for determination.
5.1.4.2 Derivation of the standards: respectively transfer-take 1 mL of biogenic amine
standard series solution; place it in a 10 mL test tube with a plug. Successively add
250 L of internal standard working solution (100 mg/...