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GB 5009.287-2022 (GB5009.287-2022)

GB 5009.287-2022_English: PDF (GB5009.287-2022)
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BASIC DATA
Standard ID GB 5009.287-2022 (GB5009.287-2022)
Description (Translated English) National food safety standard - Determination of bixinin foods
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 8,826
Date of Issue 2022-06-30
Date of Implementation 2022-12-30
Administrative Organization National Health Commission
Issuing agency(ies) State Administration for Market Regulation

Standards related to: GB 5009.287-2022

GB 5009.287-2022
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Bixin in Foods
ISSUED ON. JUNE 30, 2022
IMPLEMENTED ON. DECEMBER 30, 2022
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope... 3 
2 Principle... 3 
3 Reagents and Materials... 3 
4 Instruments and Equipment... 5 
5 Analytical Procedures... 5 
6 Expression of Analytical Results... 7 
7 Precision... 7 
8 Others... 8 
Appendix A Liquid Chromatograms of Norbixin and Bixin... 9 
National Food Safety Standard -
Determination of Bixin in Foods
1 Scope
This Standard specifies the method of liquid chromatography for the determination of bixin
(bixin and norbixin) in foods.
This Standard is applicable to the determination of bixin and norbixin in cheese, processed
cheese and similar products, margarine and similar products, non-dairy creamers, frozen drinks,
jams, chocolate and chocolate products, candies, grains and grain products, baked goods,
western-style ham, meat sausages, compound seasonings, beverages, jelly and puffed food.
2 Principle
Use ammonia ethanol solution to extract the specimen. After degreasing with petroleum ether
and acidification with acetic acid, use dichloromethane to extract it. Then, adopt liquid
chromatography to separate it; use diode array detector or ultraviolet detector to detect it. Use
the external standard method to quantify it.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are analytically pure; the water
is Grade-1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH4O). chromatographically pure.
3.1.2 Anhydrous ethanol (C2H6O). chromatographically pure.
3.1.3 Acetonitrile (C2H3N). chromatographically pure.
3.1.4 petroleum ether. with a boiling range of 30 C ~ 60 C.
3.1.5 Dichloromethane (CH2Cl2). chromatographically pure.
3.1.6 2,6-di-tert-butyl-4-methylphenol (C15H24O, BHT for short).
3.1.7 Concentrated ammonia water (NH3  H2O), with a concentration of 25% ~ 28%.
3.1.8 Glacial acetic acid (C2H4O2).
3.2 Preparation of Reagents
3.2.1 BHT-methanol solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of methanol to
dissolve it.
3.2.2 BHT-ethanol solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of anhydrous ethanol
to dissolve it.
3.2.3 70% aqueous acetonitrile. measure-take 350 mL of acetonitrile and 150 mL of water, mix
them up; add 0.5 g of BHT (3.1.6); shake to dissolve it.
3.2.4 5% ammonia ethanol solution. measure-take 50 mL of ammonia water and 950 mL of
anhydrous ethanol, mix them up; add 1 g of BHT (3.1.6); shake to dissolve it.
3.2.5 BHT-petroleum ether solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of petroleum
ether to dissolve it.
3.2.6 BHT-dichloromethane solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of
dichloromethane to dissolve it.
3.2.7 2% acetic acid aqueous solution. measure-take 20 mL of glacial acetic acid; add it to 980
mL of water; mix it up.
3.3 Reference Substances
3.3.1 Norbixin (C24H28O4, CAS. 542-40-5). purity  98.0%, or reference substances certified
by the state and awarded with a reference substance certificate.
3.3.2 Bixin (C25H30O4, CAS. 6983-79-5). purity  98.0%, or reference substances certified by
the state and awarded with a reference substance certificate.
3.4 Preparation of Standard Solutions
3.4.1 Standard stock solution (100 mg/L). accurately weigh-take 10.0 mg of norbixin reference
substance (3.3.1) with the converted content into a 100 mL brown volumetric flask; add BHT-
methanol solution (3.2.1) to dissolve and reach a constant volume, then, mix it up. Accurately
weigh-take 10.0 mg of bixin reference substance (3.3.2) with the converted content into a 100
mL brown volumetric flask; add BHT-ethanol solution (3.2.2) to dissolve and reach a constant
volume, then, mix it up. Store the standard stock solution at 18 C in the dark. The shelf life
is 6 months.
3.4.2 Mixed standard working solution. respectively absorb-take 0.05 mL, 0.1 mL, 0.2 mL, 0.5
mL, 1.0 mL, 2.0 mL and 5.0 mL of the norbixin and bixin standard stock solution (3.4.1) into a
10 mL brown volumetric flask. Add 70% aqueous acetonitrile (3.2.3) and reach a constant
volume; mix it up. The concentration of the norbixin and bixin mixed standard series working
solutions is respectively. 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 5.0 mg/L, 10 mg/L, 20 mg/L and 50
mg/L. Prepare them right before use.
Weigh-take 0.5 g ~ 5 g (accurate to 0.01 g) of specimen into a 50 mL plastic centrifuge tube
with a stopper. Add 2 g ~ 5 g of diatomaceous earth; use a glass rod to thoroughly mix the
sample and diatomaceous earth; stir, until it becomes loose and granular. Add 20 mL of 5%
ammonia ethanol solution (3.2.4). The following steps are the same as 5.1.2.1.
5.1.2.3 Liquid samples
Weigh-take 0.5 g ~ 5 g (accurate to 0.01 g) of specimen into a 50 mL plastic centrifuge tube
with a stopper. Add 5 mL of 5% ammonia ethanol solution (3.2.4). Reserve it for purification.
5.1.3 Sample purification
Add 2 mL of ammonia water to the specimen extract; use water to dilute to about 20 mL. Add
20 mL of BHT-petroleum ether solution (3.2.5); perform vortex oscillation for 1 min; centrifuge
it and discard the upper layer of solution. Repeatedly add 20 mL of BHT-petroleum ether
solution (3.2.5); perform vortex oscillation for 1 min; centrifuge it and discard the upper layer
of solution. Add 2 mL of glacial acetic acid and 25 mL of BHT-dichloromethane solution (3.2.6)
to the lower layer of solution; perform vortex oscillation for 1 min; centrifuge it and discard the
upper layer of solution. Add 1 mL of BHT-ethanol solution (3.2.2) to the lower layer of
dichloromethane solution. At 35 c, adopt the nitrogen concentration device to concentrate to
the volume of about 1 mL. Use BHT-ethanol solution (3.2.2) to assist the transferring of the
residual solution to a 5 mL brown volumetric flask; reach a constant volume and shake it well.
Use 0.45 m filter membrane (3.5.2) to filter it into an injection vial. Reserve it for liquid
chromatographic determination.
NOTE. avoid light during the pre-processing.
5.2 Instrument Reference Conditions
5.2.1 Chromatographic column. C18 chromatographic column, column length. 150 mm, inner
diameter. 4.6 mm, particle size. 5 m, or equivalent chromatographic column.
5.2.2 Mobile phase. acetonitrile + 2% aqueous acetic acid = 70 + 30.
5.2.3 Flow rate. 1.0 mL/min.
5.2.4 Column temperature. 30 C.
5.2.5 Injection volume. 10 L.
5.2.6 Detection wavelength. 458 nm.
5.3 Drawing of Standard Curve
Respectively inject the mixed standard working solution prepared in 3.4.2 into the liquid
chromatograph (see Figure A.1 in Appendix A for the liquid chromatogram of the cis-norbixin
and cis-bixin standard solutions). Use the retention time for qualitative determination to
determine the corresponding peak area; take the mass concentration of the mixed standard
...