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GB/T 5009.217-2008 (GB/T5009.217-2008, GBT 5009.217-2008, GBT5009.217-2008) & related versions
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GB/T 5009.217-2008: PDF in English (GBT 5009.217-2008)
GB/T 5009.217-2008
ICS 67.040
C 53
Determination of Vitamin B12 in Health Foods
Issued by: The Ministry of Health of PRC;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents ... 4
4 Apparatus ... 5
5 Analytical Procedures ... 6
6 Result Calculation ... 8
7 Precision ... 8
8 Chromatogram ... 9
Determination of Vitamin B12 in Health Foods
1 Scope
This Standard specifies the determination of vitamin B12 in heath foods.
This Standard is applicable to the determination of vitamin B12 in heath foods, such as
tablets, capsules, powders and functional beverages.
When this method adopts solid-phase extraction to treat the sample, the sampling
amount is 2.0g, and the detection limit of the method is 1.0µg/g. When taking the
immunoaffinity method to treat the sample, the sampling amount for tablets, capsules
and powders is 2.0g, the detection limit of the method is 0.05µg/g; while the sampling
amount for functionable beverage is 20mL, and the detection limit of the method is
2 Principle
Adopt the solid-phase extraction or immunoaffinity chromatography to enrich the
vitamin B12 in the sample extracting solution and remove the impurities; then analyze
by high performance liquid chromatography.
3 Reagents
Unless otherwise specified, all reagents used in this Standard are of analytical reagent.
The experimental water is Class-1 laboratory water; the conductivity (25°C) is
3.1 Acetonitrile (C2H3N): chromatographic pure.
3.2 Methanol (CH4O): guaranteed reagent.
3.3 Ethanol (C2H6O).
3.4 Tetrabutylammonium chloride (C16H36NCl).
3.5 5% tetrabutylammonium chloride (C16H36NCl) solution: take 5.0g of
tetrabutylammonium chloride; add water to dissolve and dilute to 100mL.
3.6 Chloroform (CHCl3).
5 Analytical Procedures
5.1 Solid-phase extraction method (pretreatment method I) operation
5.1.1 Specimen treatment
Evenly mix 20 pieces of tablet and capsule samples; take 5~10 packets of powder
samples to mix thoroughly.
5.1.2 Extraction
Take 4g~10g (equivalent to about 4µg of vitamin B12, accurate to 0.001g) of specimens
into 50mL centrifuge tube; add 10mL~15mL water; mix evenly; place it into the
ultrasonic cleaner. Ultrasonic extraction is performed for about 10min and then
centrifuge at 4000r/min for 5min. Pipette the supernatant into another 50mL centrifuge
tube. Add about 10mL of water to the residue each time according to the above
procedure; repeat the extraction twice; combine the extract solutions into a 50mL
centrifuge tube.
5.1.3 Purification
Add 1mL of 5% tetrabutylammonium chloride and about 20mL of chloroform into the
extracting solution; use vortex mixer to mix them evenly; centrifuge at 1000r/min for
3min in the centrifuge machine. Transfer the water layer into the evaporating dish;
place it onto the water bath to heat and evaporate to dryness. Dissolve the residue
with ethanol; transfer into the centrifuge tube; ultrasound-dissolve, centrifuge, and
pipette the supernatant into the evaporating dish. Repeat the extraction for twice,
combine the extracting solutions into the evaporating dish. Evaporate the ethanol;
quantitatively transfer the specimen by 5mL of 5% acetonitrile solution to the test tube
for later-use on the solid-phase extraction column.
5.1.4 Solid-phase extraction
Firstly, use 3mL of methanol to activate the solid-phase extraction column; then use
3mL of water to balance the solid-phase extraction column; the speed is 1 drop/s. Add
the appropriate amount of the above-treated specimen onto the solid-phase extraction
column. After loading the specimen, use 5ml of 5% acetonitrile solution as the elution
solvent to rinse the interference materials off the solid-phase extraction column. finally,
use 25% acetonitrile solution to elute off the vitamin B12; collect 0.5mL of elate.
1) This information is given for the convenience of the users of this Standard and does not imply
endorsement of the product. If other equivalent products have the same effect, then these equivalent
products can be used.
5.2 Immunoaffinity method (pretreatment method II) operation procedures
5.2.1 Specimen treatment
Carbonated functional beverage: take 150mL of sample solution and degas for 10min
in ultrasonic wave for later-use.
Fruit or juice-type functional beverage: adjust the pH to 7.0 with 1mol/L sodium
hydroxide; centrifuge in a centrifuge machine at 4000r/min for 10min; filter; and take
filtrate for later-use.
Tablets, capsules, powders: take 20 pieces of tablet and capsule specimens to crush
or mix evenly. Mix evenly the 5 ~ 10 packets of powder specimens.
Take 10g~50g of specimen (equivalent to 25µg~2500µg of vitamin B12; accurate to
0.001g) into 250mL volumetric flask; add 100mL of water; shake and mix evenly. Place
it into the ultrasonic cleaner; after 15-minute ultrasonic extraction, make constant
volume by water; dilute the concentration of the sample solution till the vitamin B12
content in the sample is 25µg/mL. If the pH of the sample solution is 7.0 above; use
citric acid to adjust the pH to be 4.5~7.0; if the pH of the sample solution is 4.5 below;
use phosphate buffer instead of water as the extracting solution to repeat the above
5.2.2 Enrichment and purification
Firstly, use 10mL of water to wash the unbound compound in the small column of
vitamin B12 immunoaffinity purification column; pipette 20mL (5.2.1) of extracting
solution to the purification column; then use 3mL of methanol to elute the vitamin B12
to the evaporating dish; the speed during the whole process is 1 drop/s. Evaporate the
solvent in the 60°C~70°C water bath; use the 1mL of 0.025% trifluoroacetic acid
solution to dissolve; pass the solution through 0.45µm aqueous filter membrane for the
later-use on the high performance liquid chromatograph.
5.3 Reference and analysis conditions of the liquid chromatography
5.3.1 Chromatographic column: C18 (4.6mm×250mm, 5µm) reversed phase column.
5.3.2 Mobile phase: use the gradient elution method, see Table 1.

Standard ID GB/T 5009.217-2008 (GB/T5009.217-2008)
Description (Translated English) Determination of vitamin B12 in health foods
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard C53
Classification of International Standard 67.040
Word Count Estimation 6,679
Date of Issue 2008-12-03
Date of Implementation 2009-03-01
Drafting Organization China Disease Prevention and Control Center of Nutrition and Food Safety
Administrative Organization Ministry of Health
Summary This standard specifies the method for the determination of vitamin B12 in health foods. This standard applies to tablets, determination of the type of functional drinks in health food vitamin B12, capsule, powder. When this method of treatment using solid phase extraction sample, the sample size was 2. 0g, the detection limit was 1. 0��g/g. When the application immunoaffinity treatment samples, tablets, capsule, powder sample volume is 2. 0g, the detection limit was 0. 05��g/g, functional beverage sampling volume of 20mL, the detection limit was 3. 0��g/L.