GB 5413.20-2013 English PDF

GB 5413.20-2013: National Food Safety Standard Determination of choline in infant foods and dairy products
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GB 5413-2013
Translated English of Chinese Standard. GB5413.20‐2013 
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Determination of Choline in Infant Food and
Dairy Food
Issued on. November 29, 2013 Issued on. June 1, 2014
Issued by. National Health and Family Planning Commission of the
People’s Republic of China
Table of Contents
Foreword ... 3 
1  Scope ... 4 
The First method  Enzyme colorimetric method ... 4 
2  Principle ... 4 
3  Reagents and materials ... 4 
4  Instruments and equipment ... 5 
5  Analytical steps ... 6 
6  Expression of analysis results ... 7 
7  Precision ... 9 
8  Others ... 9 
The Second Method  Reinecke salt spectrophotometric method ... 9 
9  Principle ... 9 
10  Reagents and materials ... 9 
11  Instruments and equipment ... 10 
12  Analytical steps ... 10 
13  Expression of analysis results ... 11 
14  Precision ... 12 
15  Others ... 12 
Foreword 
This Standard replaces GB/T 5413.20-1997 Milk Powder and Formula Foods for
Infant and Young Children - Determination of Choline. Compared with GB/T
5413.20-1997, main changes of this Standard are as follows.
— The name of this Standard has been modified;
— The composition of the chromogenic agent used for the enzymatic reaction
has been modified;
— The second method of Reinecke salt spectrophotometric method has been
added.
National Food Safety Standard
Determination of Choline in Infant Food and Dairy Food
1  Scope 
This Standard specifies the determination method for the choline in the infant food
and dairy food.
This Standard is applicable to the determination for the choline in the infant food
and dairy food.
The First method  Enzyme colorimetric method 
2  Principle 
The choline in a sample turns to Free State after acid hydrolysis. Then the
coloured matter is created by the reaction with chromogenic agent after the
choline is disposed by enzymatic oxidation. The different shade of the colour is
proportional to the content of choline, within a certain concentration range.
3  Reagents and materials 
Note. Unless otherwise noted, the reagent used in this method is analytically pure; and the
water is grade III water specified in GB/T 6682.
3.1 Reagents
3.1.1 Tris (trihydroxymethyl 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. Placed at -20°C for preservation.
3.1.6 Peroxidase. Placed at 2°C-8°C for preservation.
3.1.7 4-amino antipyrine (C11H13N3O).
3.1.8 Phospholipase D. Placed at -20°C for preservation.
3.2 Reagent preparation
3.2.1 Hydrochloric acid (1mol/L). Take 85mL of concentrated hydrochloric acid
and dilute it with water to 1000mL.
3.2.2 Hydrochloric acid (3mol/L). Take 125mL of concentrated hydrochloric acid
and dilute it with water to 500mL.
3.2.3 Tris buffer solution (0.05mol/L). pH=8.0±0.2.
Dissolve 6.057g of Tris into 500mL of distilled water. Adjust the pH value to
8.0±0.2 with 1mol/L of hydrochloric acid. Adjust to 1000mL of constant volume
with distilled water. This solution can be stored in the 4°C refrigerator for one
month.
3.2.4 The chromogenic agent for enzyme reaction. Take 100-120 activity-unit of
choline oxidase; 250-280 activity-unit of peroxidase; 75-100 activity-unit of
phospholipase D; 15mg of 4-aminoantipyrine; and 50mg of phenol into a 100mL
volumetric flask. Dilute it with 0.05mol/L Tris buffer solution to the scale. Prepare
when using.
3.2.5 Sodium hydroxide solution (500g/L). Weigh 500g of sodium hydroxide
and dissolve in water. Dilute to 1000 mL.
3.3 Standard substance
Standard substance of choline bitartrate (C9H19NO7). Purity≥99%.
3.4 Standard solution preparation
3.4.1 Standard stock solution of choline hydroxide (2.5mg/mL). Weigh 523 mg
of choline bitartrate which has been dried to constant weight at temperature of
102°C±2°C into a 100mL volumetric flask. Dilute to scale with distilled water. It is
stored in the 4°C±2°C refrigerator. Store for less than one week.
3.4.2 Standard working solution of choline hydroxide (250μg/mL). Absorb
10.0mL of standard stock solution to a 100mL volumetric flask. Dilute it with water
to the scale. Prepare when using.
4  Instruments and equipment 
4.1 Balances. The sensitivities are 0.01g and 0.1mg.
4.2 Thermostat water bath. The temperature can be controlled at 70°C±2°C and
37°C±2°C.
liquid sample in conical flask with grinding mouth. Add 50mL of extracting solution
of barium hydroxide-methanol-trichloromethane. Connect to the reflux device after
mixing evenly. Perform the hydrolysis extraction for 4h in the water bath at the
temperature of 79±2°C. Shake once every 1h to avoid sample caking. After the
hydrolysis extraction, remove the conical flask to cool to room temperature. Filter it.
Use the mixed liquor of glacial acetic acid-methanol to wash the filter residue for 3
~ 4 times. Collect the washing liquor with a 100mL volumetric flask. Use methanol
to fix the volume to scale. Mix well.
12.2 Drawing of standard curve
12.2.1 Preparation of chromatographic column
Use latex tube to connect the chromatographic column and the water dropper.
Use a small amount of degreasing cotton to block off the bottom of
chromatographic column. Pour in about 5cm height of florisil. Soak with methanol
for standby application.
12.2.2 Chromatography
Absorb 0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of choline hydroxide
standard solution respectively. Inject the solution into the chromatographic column.
When the solution fully enters into the column bed, wash the chromatographic
column with 5mL of methanol, 10mL of methanol, and 20mL of methyl acetate
successively. Add 5mL of ammonium reineckate solution. Wash off the
superfluous ammonium reineckate with appropriate glacial acetic acid until the
Tripoli appears the original white without ammonium reineckate on the
chromatographic column. Wash off the pink choline ammonium reineckate with
acetone. Collect the eluent into a 10mL volumetric flask. Use the acetone to fix the
volume (if the eluent is muddy, the 0.45μm filter membrane shall be used).
Determine the light absorption value of solution at wavelength 526nm. Take the
choline bitartrate content as the abscissa (mx) and the light absorption value as
the ordinate; draw standard curve.
12.3 Determination of sample
Absorb 10mL of hydrolysed sample (12.1) into the chromatographic column. The
rest of the operations shall be carried out in accordance with the 12.2.2. Find out
the content of choline bitartrate in 10mL of hydrolysed sample from the standard
curve.
13 Expression of analysis results 
The choline in sample shall be counted by choline hydroxide, expressed in
milligram per hectogram (mg/100g). Calculate according to the Formula (3).
GB 5413-2013
Translated English of Chinese Standard. GB5413.20‐2013 
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Determination of Choline in Infant Food and
Dairy Food
Issued on. November 29, 2013 Issued on. June 1, 2014
Issued by. National Health and Family Planning Commission of the
People’s Republic of China
Table of Contents
Foreword ... 3 
1  Scope ... 4 
The First method  Enzyme colorimetric method ... 4 
2  Principle ... 4 
3  Reagents and materials ... 4 
4  Instruments and equipment ... 5 
5  Analytical steps ... 6 
6  Expression of analysis results ... 7 
7  Precision ... 9 
8  Others ... 9 
The Second Method  Reinecke salt spectrophotometric method ... 9 
9  Principle ... 9 
10  Reagents and materials ... 9 
11  Instruments and equipment ... 10 
12  Analytical steps ... 10 
13  Expression of analysis results ... 11 
14  Precision ... 12 
15  Others ... 12 
Foreword 
This Standard replaces GB/T 5413.20-1997 Milk Powder and Formula Foods for
Infant and Young Children - Determination of Choline. Compared with GB/T
5413.20-1997, main changes of this Standard are as follows.
— The name of this Standard has been modified;
— The composition of the chromogenic agent used for the enzymatic reaction
has been modified;
— The second method of Reinecke salt spectrophotometric method has been
added.
National Food Safety Standard
Determination of Choline in Infant Food and Dairy Food
1  Scope 
This Standard specifies the determination method for the choline in the infant food
and dairy food.
This Standard is applicable to the determination for the choline in the infant food
and dairy food.
The First method  Enzyme colorimetric method 
2  Principle 
The choline in a sample turns to Free State after acid hydrolysis. Then the
coloured matter is created by the reaction with chromogenic agent after the
choline is disposed by enzymatic oxidation. The different shade of the colour is
proportional to the content of choline, within a certain concentration range.
3  Reagents and materials 
Note. Unless otherwise noted, the reagent used in this method is analytically pure; and the
water is grade III water specified in GB/T 6682.
3.1 Reagents
3.1.1 Tris (trihydroxymethyl 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. Placed at -20°C for preservation.
3.1.6 Peroxidase. Placed at 2°C-8°C for preservation.
3.1.7 4-amino antipyrine (C11H13N3O).
3.1.8 Phospholipase D. Placed at -20°C for preservation.
3.2 Reagent preparation
3.2.1 Hydrochloric acid (1mol/L). Take 85mL of concentrated hydrochloric acid
and dilute it with water to 1000mL.
3.2.2 Hydrochloric acid (3mol/L). Take 125mL of concentrated hydrochloric acid
and dilute it with water to 500mL.
3.2.3 Tris buffer solution (0.05mol/L). pH=8.0±0.2.
Dissolve 6.057g of Tris into 500mL of distilled water. Adjust the pH value to
8.0±0.2 with 1mol/L of hydrochloric acid. Adjust to 1000mL of constant volume
with distilled water. This solution can be stored in the 4°C refrigerator for one
month.
3.2.4 The chromogenic agent for enzyme reaction. Take 100-120 activity-unit of
choline oxidase; 250-280 activity-unit of peroxidase; 75-100 activity-unit of
phospholipase D; 15mg of 4-aminoantipyrine; and 50mg of phenol into a 100mL
volumetric flask. Dilute it with 0.05mol/L Tris buffer solution to the scale. Prepare
when using.
3.2.5 Sodium hydroxide solution (500g/L). Weigh 500g of sodium hydroxide
and dissolve in water. Dilute to 1000 mL.
3.3 Standard substance
Standard substance of choline bitartrate (C9H19NO7). Purity≥99%.
3.4 Standard solution preparation
3.4.1 Standard stock solution of choline hydroxide (2.5mg/mL). Weigh 523 mg
of choline bitartrate which has been dried to constant weight at temperature of
102°C±2°C into a 100mL volumetric flask. Dilute to scale with distilled water. It is
stored in the 4°C±2°C refrigerator. Store for less than one week.
3.4.2 Standard working solution of choline hydroxide (250μg/mL). Absorb
10.0mL of standard stock solution to a 100mL volumetric flask. Dilute it with water
to the scale. Prepare when using.
4  Instruments and equipment 
4.1 Balances. The sensitivities are 0.01g and 0.1mg.
4.2 Thermostat water bath. The temperature can be controlled at 70°C±2°C and
37°C±2°C.
liquid sample in conical flask with grinding mouth. Add 50mL of extracting solution
of barium hydroxide-methanol-trichloromethane. Connect to the reflux device after
mixing evenly. Perform the hydrolysis extraction for 4h in the water bath at the
temperature of 79±2°C. Shake once every 1h to avoid sample caking. After the
hydrolysis extraction, remove the conical flask to cool to room temperature. Filter it.
Use the mixed liquor of glacial acetic acid-methanol to wash the filter residue for 3
~ 4 times. Collect the washing liquor with a 100mL volumetric flask. Use methanol
to fix the volume to scale. Mix well.
12.2 Drawing of standard curve
12.2.1 Preparation of chromatographic column
Use latex tube to connect the chromatographic column and the water dropper.
Use a small amount of degreasing cotton to block off the bottom of
chromatographic column. Pour in about 5cm height of florisil. Soak with methanol
for standby application.
12.2.2 Chromatography
Absorb 0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of choline hydroxide
standard solution respectively. Inject the solution into the chromatographic column.
When the solution fully enters into the column bed, wash the chromatographic
column with 5mL of methanol, 10mL of methanol, and 20mL of methyl acetate
successively. Add 5mL of ammonium reineckate solution. Wash off the
superfluous ammonium reineckate with appropriate glacial acetic acid until the
Tripoli appears the original white without ammonium reineckate on the
chromatographic column. Wash off the pink choline ammonium reineckate with
acetone. Collect the eluent into a 10mL volumetric flask. Use the acetone to fix the
volume (if the eluent is muddy, the 0.45μm filter membrane shall be used).
Determine the light absorption value of solution at wavelength 526nm. Take the
choline bitartrate content as the abscissa (mx) and the light absorption value as
the ordinate; draw standard curve.
12.3 Determination of sample
Absorb 10mL of hydrolysed sample (12.1) into the chromatographic column. The
rest of the operations shall be carried out in accordance with the 12.2.2. Find out
the content of choline bitartrate in 10mL of hydrolysed sample from the standard
curve.
13 Expression of analysis results 
The choline in sample shall be counted by choline hydroxide, expressed in
milligram per hectogram (mg/100g). Calculate according to the Formula (3).