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GB 5413.20-2022English215 Add to Cart 0-9 seconds. Auto delivery. National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children GB 5413.20-2022 Valid GB 5413.20-2022
GB 5413.20-2013English70 Add to Cart 0-9 seconds. Auto delivery. National Food Safety Standard Determination of choline in infant foods and dairy products GB 5413.20-2013 Obsolete GB 5413.20-2013
GB/T 5413.20-1997English199 Add to Cart 2 days Milk powder and formula foods for infant and young children--Determination of choline GB/T 5413.20-1997 Obsolete GBT 5413.20-1997
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GB 5413.20-2022: PDF in English
GB 5413.20-2022
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard – Determination of Choline in
Foods, Milk and Milk Products for Infants and Young Children
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
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Apparatus ... 6
5 Analysis Procedures ... 6
6 Presentation of the Analysis Results ... 7
7 Precision ... 8
8 Others ... 8
9 Principle ... 9
10 Reagents and Materials ... 9
11 Apparatus ... 10
12 Analytical Procedures ... 10
13 Presentation of the Analysis Results ... 12
14 Precision ... 12
15 Others ... 13
16 Principle ... 13
17 Reagents and Materials ... 13
18 Apparatus ... 15
19 Analytical Procedures ... 15
20 Presentation of Analysis Results ... 18
21 Precision ... 18
22 Others ... 18
Appendix A Ion Chromatogram of Choline Standard Solution ... 19
Appendix B Choline Standard Solution Mass Spectrum Scanning Diagram, Standard
Solution and Internal Standard Multi-Reaction Monitoring (MRM) Diagram ... 21
National Food Safety Standard – Determination of Choline in
Foods, Milk and Milk Products for Infants and Young Children
1 Scope
This Standard specifies the method for the determination of choline in food, milk and milk
products for infants and young children.
This Standard applies to the determination of choline in food, milk and milk products for infants
and young children.
The First Method – Enzymic Colorimetric Method
2 Principle
The specimen is hydrolyzed by acid; and then reacts with the color developer to form colored
substances after enzymatic action. The alkali content is proportional to the external standard
method. Within a certain concentration range, the color depth is proportional to the choline
content, and the external standard method is used for quantification.
3 Reagents and Materials
Unless otherwise specified, the reagents used in this method are all analytically pure; and the
water is Class-3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Tris(hydroxymethyl)aminomethane [(CH2OH)3CNH2].
3.1.2 Phenol (C6H5OH).
3.1.3 Concentrated hydrochloric acid (HCl).
3.1.4 Sodium hydroxide (NaOH).
3.1.5 Choline oxidase: ≥10U/mg; store at -20 ℃.
3.1.6 Peroxidase: ≥250U/mg; store at 2℃~8℃.
3.1.7 4-Aminoantipyrine (C11H13N3O).
3.1.8 Phospholipase D: ≥60U/mg, store at -20°C.
3.2 Preparation of reagents
3.2.1 Hydrochloric acid solution (1 mol/L): Pipette 85 mL of concentrated hydrochloric acid
and inject it into about 900 mL of water; and dilute to 1000mL.
3.2.2 Hydrochloric acid solution (3 mol/L): pipette 250 mL of concentrated hydrochloric acid
and inject it into about 600 mL of water; and dilute to 1000mL.
3.2.3 Tris(hydroxymethyl)aminomethane buffer solution (Tris) (0.05 mol/L): Accurately TAKE
6.057g of Tris(hydroxymethyl)aminomethane and dissolve it in 500 mL of water; use
hydrochloric acid solution (1 mol/L) to adjust the pH to 8.0±0.2; and then make a constant
volume of 1000mL with water. This solution can be stored in a refrigerator at 4°C for 1 month.
3.2.4 Color developer: take 120U of choline oxidase, 280U of peroxidase, 100U of
phospholipase D, 15 mg of 4-aminoantipyrine and 50 mg of phenol into a 100 mL volumetric
flask; and use 0.05mol/L Tris buffer solution to dissolve and made the constant volume. It shall
be prepared for immediate use.
3.2.5 Sodium hydroxide solution (500g/L): Take 500g of sodium hydroxide; dissolve in water
and dilute to 1000mL; and store in a plastic container.
3.3 Standard product
Choline bitartrate standard product (C9H19NO7, relative molecular mass: 253.25, CAS number:
87-67-2): purity ≥ 99%, or the reference material certified by the state and awarded the
reference material certificate.
3.4 Preparation of standard solution
3.4.1 Choline (calculated as choline hydroxide, relative molecular mass 121.18) standard stock
solution (2500 mg/L): Accurately take 522.5mg of choline bitartrate that is baked at 102℃± 2℃
to constant weight; dissolve in water and transfer to a 100mL volumetric flask to make a
constant volume; and mix well. Store at 4°C below in the dark; the validity period is 3 months.
3.4.2 Choline standard working solution (250 mg/L): Pipette 10.0 mL of choline standard stock
solution into a 100 mL volumetric flask; make a constant volume with water; mix well; store at
4°C below in the dark; the validity period is 1 month.
3.5 Materials
3.5.1 0.45μm aqueous filter membrane syringe filter.
3.5.2 Syringe: 5mL or equivalent.
3.00 mL of color developer, respectively; mix well; the colorimetric tube is placed in a water
bath at 37°C±2°C for 15 min for thermal-insulation reaction.
5.2.2 Specimen preparation
Prepare 2 colorimetric tubes (B, C); both add 100μL of the solution to be analyzed; add 3.00
mL of water to colorimetric tube B, add 3.00 mL of color developer to colorimetric tube C; mix
well; place the colorimetric tubes in a water bath at 37°C±2°C for 15 min for thermal-insulation
reaction.
5.2.3 Colorimetric determination
Take out the standard series working solutions and specimens from the water bath and cool to
room temperature. At a wavelength of 505nm, use water as a blank; and use 1cm microcuvette
to measure the absorbance. Draw the standard curve by taking the concentration of choline
standard solution as the abscissa, and taking the absorbance value of the standard solution
minus the absorbance value of the reagent blank (colorimetric tube A) as the ordinate.
6 Presentation of the Analysis Results
6.1 Calculation of net absorbance value
Usually formulated reagents shall produce a slight color; and the filtrate is not colorless due to
hydrolysis. In order to remove these interference factors, The respective blank values
(colorimetric tubes A and B) must be subtracted from the total absorbance value.
The net absorbance value of the specimen is calculated according to Formula (1):
Where;
A - the net absorbance value of the specimen;
At - total absorbance value (colorimetric tube C));
Ab - reagent absorbance value (colorimetric tube A);
Ae - absorbance value of filtrate (colorimetric tube B).
Ab and Ae shall be no greater than 20% of the total absorbance value.
6.2 Calculation of choline content
The choline content (by choline hydroxide) in the specimen shall be calculated according to
Formula (2):
The Second Method – Ion Chromatography
9 Principle
The specimen is hydrolyzed by acid, purified by solid phase extraction column, then separated
by ion chromatography; detected by conductivity detector, and quantified by external standard
method.
10 Reagents and Materials
Unless otherwise specified, the reagents used in this method are all analytically pure; and the
water is the Class-1 water specified in GB/T 6682.
10.1 Reagents
10.1.1 Concentrated hydrochloric acid (HCl).
10.1.2 Methanesulfonic acid (CH4O3S): chromatographically pure.
10.2 Preparation of reagents
10.2.1 Hydrochloric acid solution (1 mol/L): Pipette 85 mL of concentrated hydrochloric acid
and inject it into about 900 mL of water; and dilute to 1000mL.
10.2.2 Hydrochloric acid solution (1.7 mol/L): Pipette 145 mL of concentrated hydrochloric
acid and inject it into about 800 mL of water; and dilute to 1000mL.
10.2.3 Methanesulfonic acid solution (6 mmol/L): Pipette 0.390mL of methanesulfonic acid
and dilute to 1000mL.
10.2.4 Methanesulfonic acid solution (15 mmol/L): Pipette 0.974mL of methanesulfonic acid
and dilute to 1000mL.
10.2.5 Methanesulfonic acid solution (25 mmol/L): Pipette 1.62mL of methanesulfonic acid
and dilute to 1000mL.
10.3 Standard products
Choline bitartrate standard product (C9H19NO7, relative molecular mass: 253.25, CAS number:
87-67-2): purity ≥ 99%; or a standard substance certified by the state and granted a standard
substance certificate.
10.4 Preparation of standard solutions
10.4.1 Choline (by choline hydroxide, relative molecular mass 121.18) standard stock solution
water bath after mixing; take hydrolysis for 3h (shaking every 30min). Cool the hydrolyzate to
room temperature; transfer it to a 50mL volumetric flask; and make constant volume to the
mark with water; mix well for later use.
12.1.1.2 Semi-solid or solid specimens
Accurately take 2.5g (accurate to 0.001g) of semi-solid or solid specimen into a 50mL
colorimetric tube; add 25mL of 1 mol/L hydrochloric acid solution; cover it; and vortex until
there is no agglomeration in the specimen solution. After mixing, put it into a water bath at
70°C±2°C for hydrolysis for 3 h (shaking every 30 min). Cool the hydrolyzate to room
temperature; transfer it to a 50mL volumetric flask; and make constant volume to the mark with
water; mix well for later use.
12.1.2 Specimen purification
C18 solid phase extraction cartridge (1.0mL) is passed through successively with 10mL of
methanol and 15mL of water before use; and stand for activation for 30min. Dilute the
extraction solution 50 times (the dilution ratio can be adjusted appropriately according to the
concentration of choline in the specimen, and it shall be no less than 10 times) with water; take
about 15 mL of the diluted solution; pass through a 0.45μm aqueous filter membrane and a C18
solid phase extraction cartridge (1.0mL); discard the first 3mL; and collect the later eluate for
testing.
12.2 Instrument reference conditions
a) Parameters of ion chromatographic column: High-capacity cationic exchange columns
equipped with carboxyl groups, such as IonPac CS12A 4 mm × 250 mm (with IonPac
CG12A protective column of 4mm × 50mm) or IonPac CS19 4mm × 250mm (with
IonPac CG19 protective column of 4mm × 50mm), or equivalent chromatographic
column.
b) IonPac CS12A equivalent eluting: 15 mmol/L methane sulfonic acid solution equivalent
eluting; and the collection time is 25 min. IonPac CS19 equivalent eluting: 6 mmol/L
methane sulfonic acid solution equivalent eluting, the collection time is 25 min.
c) Flow rate: 1.0mL/min.
d) Conductive detector: Equipped with inhibitors or equivalent inhibitors.
e) Sample-injection quantity: 100μL.
12.3 Drawing of the standard curve
The standard series working liquid is injected into the ion color spectrometer to determine the
corresponding conductivity peak area or peak height. Draw the standard curve by taking the
concentration of the standard series working solution as the abscissa, and taking the
repeatability conditions shall not exceed 10 % of the arithmetic mean.
15 Others
When the sampling quantity of solid or semi -solid specimens is 2.5g, the detection limit of the
method is 2 mg/100g; and the limit of quantification is 6 mg/100g. When the sampling quantity
of liquid specimen is 10g, the detection limit of the method is 0.5mg/100g; and the limit of
quantification is 2 mg/100g.
The Third Method - Liquid Chromatography - Series Mass Spectrometry
16 Principle
After the specimen is hydrolyzed by acid, it adjusts the pH and is filtered; then it is measured
and confirmed through liquid chromatography – series mass spectrometry; and it is quantified
by the Isotope internal standard method.
17 Reagents and Materials
Unless otherwise instructed, the reagents used in this method are analytically pure; and water
is Class-1 water specified by GB/T 6682.
17.1 Reagents
17.1.1 Formic acid (HCOOH): Chromatographically pure.
17.1.2 Acetylene (CH3CN): Chromatographically pure.
17.1.3 Ammonium formate (HCOONH4): Purity ≥ 99.9 %.
17.1.4 Concentrated hydrochloric acid (HCL).
17.1.5 Sodium hydroxide (NaOH): Purity ≥ 99.9 %.
17.2 Preparation of reagents
17.2.1 Ammonium formate aqueous solution (10 mmol/L): Take 0.63g (accurate to 0.01g) of
ammonium formate. After dissolving with water, adjust pH to 5.0±0.1 by formic acid; and
transfer to 1000 mL volumetric flask; and make constant volume by water to the mark; and mix
well.
17.2.2 Hydrochloric acid solution (1 mol/L): Pipette 85 mL of concentrated hydrochloric acid,
and inject it into about 900 mL of water; and dilute to 1000mL.
17.2.3 Sodium hydroxide solution (1mol/L): Take 2.0g (accurate to 0.01g) of sodium hydroxide;
dissolve with water and dilute to 50mL.
17.2.4 Acetylene aqueous solution (80%): Take 80mL of acetylene; and dilute to 100mL with
water.
17.3 Standard products
17.3.1 Standard products (C9H19NO7, relative molecular mass: 253.25, CAS No.: 87-67-2):
Purity ≥99 %; standard substances that are certified by the state and awarded standard substance
certificates.
17.3.2 Stable isotope internal standard: chlorine chloride-d4 (C5H10NOD4Cl, relative molecular
mass: 143.65, CAS number: 285979-70-6): Purity ≥99.8 %, or equivalent.
17.4 Preparation of standard solutions
17.4.1 Choline (by choline hydroxide, relative molecular mass 121.18) standard stock solution
(100 mg/L): Accurately take 20.90mg of choline bitartrate that are baked to constant weight at
102℃ ± 2°C; dissolve with 80 % acetonitrile aqueous solution and make constant volume to
100mL. After shaking well, transfer the solution to the brown glass bottle; store it at 4°C below
in the dark; and the validity period is 1 month.
17.4.2 Choline standard work solution (1.0 mg/L): Pipette 1.00 mL of the above choline
standard stock solution into a 100 mL volumetric flask; and use 10 mmol/L ammonium formate
aqueous solution to make constant volume; mix well; and it shall be prepared for immediate
use.
17.4.3 Choline-d4 internal standard stock solution (1000 mg/L): Accurately take 13.3 mg of
chlorine chloride-d4; use the 80% acetonitrile aqueous solution to dissolve and transfer it to the
10mL brown volumetric flask; and make constant volume; mix well; store at 4°C in the dark;
and the validity period is 3 months.
17.4.4 Choline-d4 internal standard working solution (1.0 mg/L): Pipette 0.10mL of the above
choline-d4 internal standard stock solution into the 100mL volumetric flask; and use 10mmol/L
ammonium formate aqueous solution to make the constant volume; mix well; store at 4°C
below in the dark; and the validity period is 1 month.
17.4.5 Standardized series working solution: Accurately pipette the above choline standard
working solution of 0.100 mL, 0.200 mL, 0.500 mL, 1.00 mL, 1.50 mL, 2.00mL to 10 mL
volumetric flasks, respectively. Among them, each add 500μL of choline-d4 internal standard
working solution; use 10 mmol/L ammonium formate aqueous solution to make constant
volume; mix well. The concentrations of the choline in such standard series are 0. 010mg/L,
0.020mg/L, 0.050mg/L, 0.100mg/L, 0.150mg/L, 0.200mg/L. It shall be prepared for immediate
use.
......

BASIC DATA
Standard ID GB 5413.20-2022 (GB5413.20-2022)
Description (Translated English) National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children
Sector / Industry National Standard
Classification of Chinese Standard C53
Word Count Estimation 15,182
Date of Issue 2022-06-30
Date of Implementation 2022-12-30
Older Standard (superseded by this standard) GB 5413.20-2013
Administrative Organization National Health Commission
Issuing agency(ies) State Administration for Market Regulation