GB 5413.20-2022_English: PDF (GB5413.20-2022)
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National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children
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National Food Safety Standard Determination of choline in infant foods and dairy products
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Milk powder and formula foods for infant and young children--Determination of choline
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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| Word Count Estimation | 15,182 | Date of Issue | 2022-06-30 | Date of Implementation | 2022-12-30 | Older Standard (superseded by this standard) | GB 5413.20-2013 | Administrative Organization | National Health Commission | Issuing agency(ies) | State Administration for Market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 | Standard ID | GB 5413.20-2013 (GB5413.20-2013) | Description (Translated English) | National Food Safety Standard Determination of choline in infant foods and dairy products | Sector / Industry | National Standard | Classification of Chinese Standard | X82 | Classification of International Standard | 67.100.10 | Word Count Estimation | 11,169 | Older Standard (superseded by this standard) | GB 5413.20-1997 | Drafting Organization | National Food Industry Standardization Technical Committee | Administrative Organization | National Health & Family Planning Commission of PRC | Regulation (derived from) | China Food & Drug Administration [2013] No. 234, November, 1, 2013 | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China | Summary | This Standard, from June 1, 2014, substitutes GB/T 5413. 20-1997 Determination of choline in infant foods and dairy products. This standard specifies the baby food, dairy products Determination of choline. | Standard ID | GB/T 5413.20-1997 (GB/T5413.20-1997) | Description (Translated English) | Milk powder and formula foods for infant and young children--Determination of choline | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | X82 | Classification of International Standard | 67.100.10 | Word Count Estimation | 4,463 | Date of Issue | 1997/5/28 | Date of Implementation | 1998/9/1 | Older Standard (superseded by this standard) | GB 5413-1985 | Drafting Organization | Nestle (China) Investment Services Co., Ltd. | Administrative Organization | National Dairy Standardization Center | Regulation (derived from) | National Health Planning Commission Bulletin 2013 No. 7 | Proposing organization | China Light Industry Association | Issuing agency(ies) | State Bureau of Technical Supervision | Summary | This Standard specifies the determination of choline. This Standard is applicable to the determination of food and infant formula milk powder choline. The standard minimum detection limit of about 2mg/100g. |
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.
......
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).
......
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