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GB 5413.20-2022: National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children
GB 5413.20-2022
National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children
Book the National Standard of the People's Republic of China
national food safety standards
Determination of choline in infant food and dairy products
Published on 2022-06-30
Implementation on 2022-12-30
National Health Commission of the People's Republic of China
Released by the State Administration for Market Regulation
foreword
This standard replaces GB 5413.20-2013 "National Food Safety Standard for Determination of Choline in Infant Food and Milk Products".
Compared with GB 5413.20-2013, the main changes of this standard are as follows.
--- Deleted the second Farrell's salt spectrophotometry, and added ion chromatography as the second method;
--- Added liquid chromatography-tandem mass spectrometry as the third method.
national food safety standards
Determination of choline in infant food and dairy products
1 range
This standard specifies the method for the determination of choline in food and dairy products for infants and young children.
This standard applies to the determination of choline in infant food and dairy products.
The first method of enzymatic colorimetry
2 Principle
The sample is hydrolyzed by acid, and then reacted with the chromogenic agent to form colored substances after enzymatic action.
The alkali content is proportional to the external standard method.
3 Reagents and materials
Unless otherwise specified, the reagents used in this method are all analytically pure, and the water is the tertiary water specified in GB/T 6682.
3.1 Reagents
3.1.1 Trihydroxymethylaminomethane [(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, stored at -20℃.
3.1.6 Peroxidase. ≥250U/mg, stored at 2℃～8℃.
3.1.7 4-aminoantipyrine (C11H13N3O).
3.1.8 Phospholipase D. ≥60U/mg, store at -20℃.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (1mol/L). measure 85mL of concentrated hydrochloric acid and pour it into about 900mL of water, and dilute to 1000mL.
3.2.2 Hydrochloric acid solution (3mol/L). measure 250mL of concentrated hydrochloric acid and pour it into about 600mL of water, and dilute to 1000mL.
3.2.3 Tris buffer solution (Tris) (0.05 mol/L). Accurately weigh 6.057 g of tris and dissolve in it.
500mL of water, adjust the pH to 8.0±0.2 with hydrochloric acid solution (1mol/L), and then dilute to 1000mL with water. This solution was kept in a refrigerator at 4°C
Can be stored for 1 month.
3.2.4 Color developer. take 120U of choline oxidase, 280U of peroxidase, 100U of phospholipase D, and 15mg of 4-aminoantipyridine
Lin and 50mg phenol were placed in a 100mL volumetric flask, dissolved with 0.05mol/LTris buffer solution and made up to volume. Ready to use.
3.2.5 Sodium hydroxide solution (500g/L). Weigh 500g of sodium hydroxide, dissolve in water and dilute to 1000mL, and store in a plastic container.
3.3 Standard product
Choline bitartrate standard (C9H19NO7, relative molecular mass. 253.25, CAS number. 87-67-2). purity ≥ 99%, or certified by
Home certified reference materials and awarded a 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 (2500mg/L). accurately weighed in
Bake at 102℃±2℃ to constant weight of choline bitartrate 522.5mg, dissolve in water and transfer to a 100mL volumetric flask to dilute to volume, and mix well.
Store below 4°C away from light, valid for 3 months.
3.4.2 Choline standard working solution (250 mg/L). draw 10.0 mL of choline standard stock solution into a 100 mL volumetric flask, and determine with water.
Mix well, store below 4°C away from light, valid for 1 month.
3.5 Materials
3.5.1 0.45μm aqueous filter membrane syringe filter. 3.5.2 Syringe. 5mL or equivalent.
4 Instruments and equipment
4.1 Analytical balance. the sensed quantities are 0.1 mg and 0.001 g, respectively.
4.2 Constant temperature water bath device. the temperature can be controlled at 70℃±2℃ and 37℃±2℃.
4.3 pH meter. accuracy 0.01.
4.4 Spectrophotometer.
5 Analysis steps
5.1 Sample pretreatment
5.1.1 Sample pretreatment
Accurately weigh 20g (accurate to 0.001g) of the mixed liquid sample into a 100mL conical flask, add 10mL of 3mol/L hydrochloric acid solution, stopper and mix.
Accurately weigh 5g (accurate to 0.001g) of the evenly mixed semi-solid or solid sample into a 100mL conical flask, add 30mL of 1mol/L hydrochloric acid solution, stopper and mix.
5.1.2 Hydrolysis
Place the container containing the sample in a water bath at 70°C ± 2°C, place it for 3h (shaking every 30min), and cool it to room temperature. with hydrogen
Sodium oxide solution (500g/L) was adjusted to pH 3.5-4.0, transferred to a 50mL volumetric flask, and the volume was adjusted to the mark with water.
5.1.3 Filtering
Filter the hydrolyzate with filter paper. If the filtrate is not clear, filter it again with a 0.45 μm aqueous filter membrane syringe filter. The filtrate was collected for testing.
5.2 Determination
5.2.1 Preparation of standard curve
Pipette 2.00mL, 4.00mL, 6.00mL, 8.00mL, 10.00mL of choline standard working solution (250mg/L) into 10mL
In the volumetric flask, make up to the mark with water, mix well, and prepare the concentration of 50.0mg/L, 100mg/L, 150mg/L,.200mg/L,
250mg/L standard series working solution. Prepare 6 colorimetric tubes, one colorimetric tube is used as reagent blank (A), add 100 μL of water, add 100 μL of standard series working solution to the other 5 colorimetric tubes, and then add 3.00 mL of chromogenic reagent respectively, mix well, The colorimetric tube was placed in a water bath at 37°C ± 2°C and incubated for 15min.
5.2.2 Sample preparation
Prepare 2 colorimetric tubes (B, C), add 100 μL of the solution to be analyzed, add 3.00 mL of water to colorimetric tube B, add 3.00 mL of chromogenic reagent to colorimetric tube C, mix well, and place the colorimetric tube in the Incubate the reaction in a water bath at 37 °C ± 2 °C for 15 min.
5.2.3 Colorimetric determination
Remove the standard series of working solutions and samples from the water bath and cool to room temperature. At the wavelength of 505nm, use water as blank, use 1cm
Absorbance values were measured in microcuvettes. Take the concentration of the choline standard solution as the abscissa, subtract the reagent blank (colorimetric tube) from the absorbance value of the standard solution
The absorbance value of A) was plotted on the ordinate, and a standard curve was prepared.
6 Presentation of the analysis results
6.1 Calculation of net absorbance value
Usually formulated reagents will produce a slight color, and the filtrate is not colorless due to hydrolysis. In order to remove these interfering factors, it is necessary to
The respective blank values (Cuvette A and Cuvette B) must be subtracted from the total absorbance value.
The net absorbance value of the sample is calculated according to formula (1).
A=At-Ab-Ae (1)
where.
A --- the net absorbance value of the sample;
At---total absorbance value (colorimetric tube C));
Ab---reagent absorbance value (colorimetric tube A);
Ae---the absorbance value of the filtrate (colorimetric tube B).
Ab and Ae should not be greater than 20% of the total absorbance value.
6.2 Calculation of choline content
The content of choline (calculated as choline hydroxide) in the sample is calculated according to formula (2).
The calculation result retains three significant figures.
7 Precision
The absolute difference between the results of two independent determinations obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.
8 Others
When the solid or semi-solid sample weighs 5g, the detection limit of the method is 1mg/100g, and the quantification limit is 3mg/100g;
When the sample weight is 20g, the detection limit of the method is 0.3mg/100g, and the quantification limit is 0.8mg/100g.
The second method of ion chromatography
9 Principles
The samples were hydrolyzed by acid, purified by solid phase extraction column, separated by ion chromatography, detected by conductivity detector, and quantified by external standard method.
10 Reagents and materials
Unless otherwise stated, the reagents used in this method are all analytically pure, and the water is the first-grade 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 Reagent preparation
10.2.1 Hydrochloric acid solution (1mol/L). measure 85mL of concentrated hydrochloric acid and pour it into about 900mL of water, and dilute to 1000mL.
10.2.2 Hydrochloric acid solution (1.7mol/L). measure 145mL of concentrated hydrochloric acid and inject it into about 800mL of water, and dilute to 1000mL.
10.2.3 Methanesulfonic acid solution (6mmol/L). absorb 0.390mL of methanesulfonic acid and dilute to 1000mL.
10.2.4 Methanesulfonic acid solution (15mmol/L). absorb 0.974mL of methanesulfonic acid and dilute to 1000mL.
10.2.5 Methanesulfonic acid solution (25mmol/L). absorb 1.62mL of methanesulfonic acid and dilute to 1000mL.
10.3 Standard products
Choline bitartrate standard (C9H19NO7, relative molecular mass. 253.25, CAS number. 87-67-2). purity ≥ 99%, or certified by
Home certified reference materials and awarded a reference material certificate.
10.4 Preparation of standard solutions
10.4.1 Choline (calculated as choline hydroxide, relative molecular mass 121.18) standard stock solution (2500mg/L). accurately weighed at 102℃±
Bake at 2°C to a constant weight of 522.5 mg of choline bitartrate, dissolve in water, transfer to a 100 mL volumetric flask, and mix. Below 4°C
Store in the dark, valid for 3 months.
10.4.2 Choline standard working solution (100 mg/L). draw 2.0 mL of the above choline standard stock solution into a 50 mL volumetric flask, and use
Dilute to a volume of 15 mmol/L methanesulfonic acid solution, mix well, and store below 4°C in the dark, with a validity period of 1 month.
10.4.3 Choline standard working solution. draw 0.200mL, 0.500mL, 1.00mL, 2.50mL, 0.200mL, 0.500mL, 1.00mL, 2.50mL,
5.00mL was placed in a set of 100mL volumetric flasks, and the 15mmol/L methanesulfonic acid solution was used to dilute to volume and mix to obtain a choline concentration of
Standard series working solution of 0.200mg/L, 0.500mg/L, 1.00mg/L, 2.50mg/L and 5.00mg/L. Ready to use.
10.5 Materials
10.5.1 0.45μm aqueous filter membrane syringe filter.
10.5.2 Purification column. C18 solid phase extraction cartridge 1.0mL or equivalent.
10.5.3 Syringe. 5mL or equivalent.
11 Instruments and equipment
11.1 Ion chromatograph (IC). equipped with a conductivity detector.
11.2 Analytical balance. the inductive quantities are 0.1 mg and 0.001 g, respectively.
11.3 Electrothermal constant temperature water bath device. the temperature can be controlled at 70℃±2℃.
11.4 Vortex mixer.
12 Analysis steps
12.1 Sample preparation
12.1.1 Sample extraction
12.1.1.1 Liquid sample
Accurately weigh 10g (accurate to 0.001g) of the mixed liquid sample into a 50mL colorimetric tube, add 1.7mol/L hydrochloric acid to dissolve it.
Add 15mL of liquid, cover, mix well, put it into a 70℃±2℃ water bath to hydrolyze for 3h (shake every 30min). Cool the hydrolyzate to room temperature, turn
Transfer to a 50mL volumetric flask and dilute to the mark with water, mix well and set aside.
12.1.1.2 Semi-solid or solid sample
Accurately weigh 2.5g (accurate to 0.001g) semi-solid or solid sample into a 50mL colorimetric tube, add 1mol/L hydrochloric acid solution
25mL, cover, vortex until there is no agglomeration in the sample solution, after mixing, put it into a 70℃±2℃ water bath for hydrolysis for 3h (shake every 30min).
Cool the hydrolyzate to room temperature, transfer it to a 50mL volumetric flask and make up to the mark with water, mix well and set aside.
12.1.2 Sample cleanup
The C18 solid phase extraction cartridge (1.0 mL) was passed through successively with 10 mL methanol and 15 mL water before use, and was allowed to stand for activation for 30 min. diluted with water
Extract the solution 50 times (the dilution ratio can be adjusted appropriately according to the concentration of choline in the sample, and it should not be less than 10 times), take the diluted solution about
15mL was passed through a 0.45μm aqueous filter membrane and a C18 solid phase extraction cartridge (1.0mL), the first 3mL was discarded, and the latter eluate was collected for testing.
12.2 Instrument reference conditions
a) Ion chromatography column parameters. a high-capacity cation exchange column with carboxyl groups, such as IonPaccS12A4mm×250mm (with
IonPacCG12A type guard column 4mm×50mm) or IonPacCS194mm×250mm (with IonPacCG19 type
Guard column 4mm × 50mm), or equivalent chromatographic column.
b) IonPacCS12A isocratic leaching. 15mmol/L methanesulfonic acid solution isocratic leaching, collection time 25min.
IonPacCS19 isocratic leaching. 6mmol/L methanesulfonic acid solution isocratic leaching, acquisition time 25min.
c) Flow rate. 1.0mL/min.
d) Conductivity detector. equipped with suppressor or equivalent suppression device.
e) Injection volume. 100 μL.
12.3 Preparation of standard curve
The standard series of working solutions were injected into the ion chromatograph to measure the corresponding conductivity peak area or peak height. with standard series of working fluids
The concentration is the abscissa, and the conductivity peak area or peak height is the ordinate, and a standard curve is drawn.
The ion chromatogram of choline standard solution is shown in Appendix A.
12.4 Determination of sample solution
Inject the sample solution into the ion chromatograph to obtain the corresponding conductivity peak area or peak height, and obtain the concentration of the solution to be tested according to the standard curve.
Alkali concentration.
12.5 Blank test
Without weighing the sample, do a blank test according to the procedure in 12.1.It should be confirmed that it does not contain substances that i...