GB 5009.150-2016 (GB5009.150-2016) & related versions
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GB 5009.150-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Monascus Pigments in Foods
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the PRC;
State Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 5
5 Analysis steps ... 6
6 Description of the analysis result ... 8
7 Precision ... 9
8 Detection-limit ... 9
Appendix A Standard chromatogram of monascus pigments ... 10
National Food Safety Standard -
Determination of Monascus Pigments in Foods
1 Scope
This Standard specifies the method for determination of monascorubrin, monacolin,
and monascorubramine in foods.
This Standard applies to the determination of three monascus pigments in flavor
fermented milk, jam, fermented bean curd, dried almonds, candy, instant noodle
products, cakes, biscuits, cooked meat products, soy sauce, fruit and vegetable juice
drinks, solid beverages, mixed liquor, jelly and potato chips.
2 Principle
The sample which is extracted by absolute ethanol or 80% ethanol and purified by
solid phase extraction column, shall be detected by liquid chromatography,
characterized by retention time and quantified by external standard method.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, the
water is grade-1 water specified by GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographic pure.
3.1.2 Absolute ethanol (CH3CH2OH): chromatographic pure.
3.1.3 Glacial acetic acid (CH3COOH).
3.1.4 Ammonia acetate (CH3COONH4).
3.2 Preparation of reagents
3.2.1 Acetic acid-ammonium acetate solution (pH=5): accurately weigh 0.77 g of
ammonium acetate in a 1 L volumetric flask; add 900 mL of water to dissolve; use
glacial acetic acid to adjust pH=5.0; add water to fix-volume to 1 L; mix well. Use it
after it is filtered through a 0.45 μm microporous membrane.
4.5 High-speed pulverizer.
4.6 Solid-phase extraction device.
4.7 Organic-phase microporous membrane: pore size of 0.45 μm.
5 Analysis steps
5.1 Sample preparation and storage
5.1.1 Liquid sample
Shake and pack the samples such as fruit juice, fruit juice drink and fruity carbonated
drink; seal at room temperature or refrigerate to store.
5.1.2 Semi-solid sample
For samples such as jelly and flavor fermented milk, take the edible portion to
homogenate; stir evenly; package; seal; refrigerate or freeze to store.
5.1.3 Solid sample
For samples of low-water content, such as biscuits, cakes and cooked meat products,
use the high-speed pulverizer to pulverize; package; seal and store at room
temperature out of the sun. For powdery samples with a uniform shape such as solid
beverage, package directly; seal and store at room temperature out of the sun.
5.2 Sample processing
5.2.1 Liquid sample
Weigh 5 g (accurate to 0.01 g) of uniform sample (if the sample contains carbon
dioxide; first heat to remove it) in a beaker; use an appropriate amount of absolute
ethanol to dissolve it; transfer it to a 50 mL volumetric flask; add absolute ethanol to
fix-volume; shake well. Accurately transfer 20 mL of extract which is mentioned above
to a 100 mL pear-shaped bottle; add 10 mL of absolute ethanol to the pear-shaped
bottle; rotate-concentrate the mixture to near dry at 60°C ± 2°C; use 2 mL of 20%
methanol solution to wash the pear-shaped bottle twice.
Transfer all the above-mentioned extracts to a pre-activated HLB solid-phase
extraction column; control the flow rate at 1 mL/min ~ 2 mL/min; discard the effluent.
Use 5 mL of 40% methanol solution to rinse the purification column; discard the effluent;
then use 5 mL of methanol to elute; control the flow rate at 1 mL/min ~ 2 mL/min;
collect the eluate in a 25 mL graduated centrifuge tube. Use nitrogen to concentrate
the eluent at 45°C; then, use 2 mL of 40% methanol solution to dissolve the residue;
filter it through a 0.45 μm organic filter membrane; test it later.
5.2.2 Semi-solid and solid samples
Weigh 5 g (accurate to 0.01 g) of uniform sample; put it into a 50 mL plastic centrifuge
tube; add 20 mL of 80% ethanol solution; homogenize at 15 000 r/min for 2 min and
at 4 000 r/min for 3 min; transfer the supernatant to a 50 mL volumetric flask; add 20
mL of 80% ethanol solution to the residue and repeat the extraction once; combine the
extracts in the same volumetric flask; use absolute ethanol to fix-volume; shake well.
Accurately transfer 20 mL of extract which is mentioned above to a 100 mL pear-
shaped bottle; add 10 mL of absolute ethanol to the pear-shaped bottle; rotate-
concentrate the mixture to near dry at 60°C ± 2°C; use 2 mL of 20% methanol solution
to wash the pear-shaped bottle twice. Transfer all the above-mentioned purification
liquor to a pre-activated HLB solid-phase extraction column; control and the flow rate
at 1 mL/min ~ 2 mL/min; discard the effluent. Use 5 mL of 40% methanol solution to
rinse the purification column; discard the effluent; then use 5 mL of methanol to elute;
control the flow rate at 1 mL/min ~ 2 mL/min; collect the eluate in a 25 mL graduated
centrifuge tube. Use nitrogen to concentrate the eluent at 45°C; then, use 2 mL of 20%
methanol solution to dissolve the residue; filter it through a 0.45 μm organic filter
membrane; test it later.
5.3 Apparatus reference conditions
Apparatus reference conditions are listed as below:
a) Chromatographic column: high-purity spherical porous silica gel multi-point bonding
C18 amino column, with a column length of 250 mm, an inner diameter of 4.6mm, a
particle size of 5 μm, OR columns of equivalent performance;
b) Mobile phase: A: acetic acid-ammonium acetate solution (pH=5); B: methanol,
gradient elution is shown in Table 1;
c) Column temperature: 40°C;
d) Injection volume: 20 μL;
e) Detection wavelength: that for monascorubramine is 264 nm, and for
monascorubrin and monacolin is 390 nm.
......
| Standard ID | GB 5009.150-2016 (GB5009.150-2016) | | Description (Translated English) | National food safety standard -- Determination of monascas colours in foods | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 7,774 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB/T 5009.150-2003 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | | Standard ID | GB/T 5009.150-2003 (GB/T5009.150-2003) | | Description (Translated English) | Determination of monascas colours in foods | | Sector / Industry | National Standard (Recommended) | | Classification of Chinese Standard | C53 | | Classification of International Standard | 67.040 | | Word Count Estimation | 6,698 | | Date of Issue | 2003-08-11 | | Date of Implementation | 2004-01-01 | | Older Standard (superseded by this standard) | GB/T 17336-1998 | | Drafting Organization | Institute of Industrial Hygiene and Occupational Diseases Chinese Academy of Preventive Medicine | | Administrative Organization | People's Republic of China Ministry of Health | | Proposing organization | Ministry of Health of the People Republic of China | | Issuing agency(ies) | The People Republic of China Ministry of Health, China National Standardization Management Committee | | Summary | This standard specifies: Determination of Monascus pigments by thin layer chromatography. This standard applies to: foods Monascus Pigment determination. |
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