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GB 5413.20-2022 English PDF (GB 5413.20-2013, GB/T 5413.20-1997)

GB 5413.20-2022_English: PDF (GB5413.20-2022)
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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 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 Obsolete GB 5413.20-2013
GB/T 5413.20-1997English199 Add to Cart 2 days [Need to translate] Milk powder and formula foods for infant and young children--Determination of choline Obsolete GB/T 5413.20-1997


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Older Standard (superseded by this standard) GB 5413.20-2013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

BASIC DATA
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Older Standard (superseded by this standard) GB 5413.20-1997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.

BASIC DATA
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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