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This standard replaces GB 5413.20-2013 National food safety standard—Determination of choline in infant food and dairy products.
The following main changes have been made with respect to GB 5413.20-2013:
——Method II—Reinecke salt spectrophotometry is deleted and Ion chromatography is added as Method II;
——Liquid chromatography tandem mass spectrometry was added as Method III.
National food safety standard—
Determination of choline in infant food and dairy products
1 Scope
This standard specifies the methods for determination of choline in infant food and dairy products.
This standard is applicable to the determination of choline in infant food and dairy products.
Method I—Enzymatic colorimetry
2 Principle
After acid hydrolysis and enzyme action, the specimen reacts with chromogenic reagent to produce colored substances. In a certain concentration range, the color depth is proportional to choline content, which is quantified by external standard method.
3 Reagents and materials
Unless otherwise specified, analytically-pure reagents and Class-III water specified in GB/T 6682 are used for the purpose of this method.
3.1 Reagents
3.1.1 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: ≥ 10 U/mg, stored at -20°C.
3.1.6 Peroxidase: ≥ 250 U/mg, stored at 2°C to 8°C.
3.1.7 4-aminoantipyrine(C11H13N3O).
3.1.8 Phospholipase D: ≥ 60 U/mg, stored at -20°C.
3.2 Preparation of reagents
3.2.1 Hydrochloric acid solution (1 mol/L): inject 85 mL concentrated hydrochloric acid into about 900 mL water and dilute it to 1,000 mL.
3.2.2 Hydrochloric acid solution (3 mol/L): inject 250 mL concentrated hydrochloric acid into about 600 mL water and dilute it to 1,000 ml.
3.2.3 Trimethylol aminomethane buffer solution (Tris) (0.05 mol/L): weigh 6.057 g trimethylol aminomethane accurately and dissolve it in 500 ml water, and adjust the pH to 8.0±0.2 with hydrochloric acid solution (1 mol/L), set the volume to 1,000 ml with water. This solution can be stored in refrigerator at 4°C for 1 month.
3.2.4 The chromogenic reagent: place 120 U of choline oxidase, 280 U of peroxidase, 100 U of phospholipase D, 15 mg of 4-aminoantipyrine and 50mg of phenol in a 100 ml volumetric flask and dissolve it in 0.05 mol/L Tris buffer solution and set its volume. Freshly prepare before use.
3.2.5 Sodium hydroxide solution (500g/L): weigh 500 g sodium hydroxide, dissolve it in water and dilute it to 1,000mL, and store it in a plastic container.
3.3 Standard sample
Standard sample of choline tartrate hydrogen salt (C9H19NO7, relative molecular weight: 253.25, CAS No.: 87-67-2): purity ≥ 99%, or certified by China and granted with reference material certificate.
3.4 Preparation of standard solutions
3.4.1 Standard stock choline (in term of choline hydroxide, with relative molecular weight of 121.18) solution (2500 mg/L): accurately weigh 522.5 mg of choline hydrogen tartrate dried to constant weight at 102°C± 2°C, dissolve it with water, transfer it to a 100 mL volumetric flask for constant volume, and mix it well. Store it in dark below 4°C, with a shelf life of 3 months.
3.4.2 Standard working choline solution (250 mg/L): pipette 10.0 mL standard stock choline solution of in a 100mL volumetric flask, set the volume with water, mix it well, and store it in dark below 4°C, with a shelf life of 1 month.
3.5 Materials
3.5.1 0.45 μm waterborne membrane needle filter.
3.5.2 Syringe: 5 mL or equivalent.
4 Apparatus
4.1 Analytical balance: with a sensitivity of 0.1 mg and 0.001 g.
4.2 Constant temperature water bath device: the temperature can be controlled at 70°C± 2°C and 37°C± 2°C.
4.3 pH meter: with accuracy of 0.01.
4.4 Spectrophotometer.
5 Analytical procedures
5.1 Sample preconditioning
5.1.1 Sample pretreatment
Accurately weigh 20 g (accurate to 0.001 g) of uniformly mixed liquid specimen in a 100 mL conical flask, add 10 mL of 3 mol/L hydrochloric acid solution, cover it with stopper and mix it well.
Accurately weigh 5 g (accurate to 0.001 g) of uniformly mixed semi-solid or solid specimens in a 100 mL conical flask, add 30mL of 1mol/L hydrochloric acid solution, add a stopper and mix it well.
5.1.2 Hydrolysis
Place the container containing the specimen in a water bath at 70°C ± 2°C keep it for 3 h (shake every 30min) to cool it to room temperature. Adjust pH to 3.5 to 4.0 with sodium hydroxide solution (500 g/L), transfer it to a 50 mL volumetric flask, and set its volume with water to scale.
5.1.3 Filtering
Filter the hydrolysate with filter paper. If the filtrate is not clear, filter it again with a 0.45 μm water-based membrane needle filter. Collect filtrate for testing.
5.2 Determination
5.2.1 Plotting of standard curve
Pipette 2.00 mL, 4.00 mL, 6.00 mL, 8.00 mL and 10.00 mL standard working solutions of choline (250mg/L) into a volumetric flask of 10 mL, set their volume to scale, mix them well, and then prepare them into the standard series working solutions with concentrations of 50.0 mg/L, 100 mg/L, 150 mg/L, 200 mg/L and 250 mg/L. Prepare six colorimetric tubes, one of which is used as reagent blank (A), add 100 μL water, the other five of which are correspondingly added with 100 μL standard series working solution, and then add 3.00 mL chromogenic reagent respectively, mix them well, and put the colorimetric tubes in a water bath at 37°C ± 2°C for heat preservation and reaction for 15 min.
5.2.2 Specimen preparation
Prepare two colorimetric tubes (B, C), add 100 μL of solution to be analyzed, add 3.00 ml of water into colorimetric tube B, add 3.00 ml of chromogenic agent into colorimetric tube C, mix well, and keep the colorimetric tubes in 37°C ± 2°C water bath for 15 min.
5.2.3 Colorimetric determination
Take out the standard series working solutions and samplespecimens from the water bath and cool them to room temperature. At the wavelength of 505nm, take water as blank, and measure the absorbance value with 1 cm microcuvette. Plot the standard curve with the concentration of standard choline solution as the abscissa and the absorbance value of standard solution minus the absorbance value of reagent blank (colorimetric tube A) as the ordinate.
6 Expression of analysis results
6.1 Calculation of net absorbance value
Generally, the reagent prepared will produce 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 using Formula (1):
A=At-Ab-Ae (1)
where,
A——the net absorbance value of the specimen;
At——the total absorbance value (colorimetric tube C);
Ab——the absorbance value of reagent (colorimetric tube A);
Ae——the absorbance value of filtrate (colorimetric tube B).
Ab and Ae shall not be greater than 20% of the total absorbance value.
6.2 Calculation of choline content
The choline content (in term of choline hydroxide) in the specimen is calculated using Formula (2):
(2)
where,
X——the choline content (in term of choline hydroxide) in the specimen, mg/100g;
c——the concentration of choline corresponding to the net absorbance value found from the standard curve, mg/L;
V——-the diluted volume of the hydrolysate (constant volume of the hydrolysate, usually 50 mL), mL;
f——the dilution factor.
100——the conversion coefficient;
m——the mass of the specimen, g;
1,000——the conversion factor.
The calculation results shall be rounded off to three significant figures.
7 Precision
The absolute difference in the two results measured independently acquired under repeated conditions shall not exceed 10% of the arithmetic mean value.
8 Others
When the weight of solid or semi-solid specimen is 5 g, the detection limit and the quantitative limit of the method are 1 mg/100g, and 3 mg/100g respectively; when the weight of the liquid specimen is 20 g, the detection limit and quantitative limit are 0.3 mg/100g and 0.8 mg/100g respectively.
Method II—Ion chromatography
9 Principle
Specimens are 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 specified, analytically-pure reagents and Class-I water specified in GB/T 6682 are used for the purpose of this method.
10.1 Reagents
10.1.1 Concentrated hydrochloric acid (HCl).
10.1.2 Methane sulfonic acid (CH4O3S): chromatographically pure.
10.2 Preparation of reagents
10.2.1 Hydrochloric acid solution (1 mol/L): inject 85 ml concentrated hydrochloric acid into about 900ml water and dilute it to 1,000 ml.
10.2.2 Hydrochloric acid solution (1.7 mol/L): measure 145 mL of concentrated hydrochloric acid and inject it into about 800 mL of water, and dilute it to 1,000 mL.
10.2.3 Hydrochloric acid solution (6 mol/L): pipette 0.390 mL concentrated hydrochloric acid and dilute it to 1,000 ml.
10.2.4 Methanesulfonic acid solution (15mmol/L): pipette 0.974 mL of methanesulfonic acid and dilute it to 1,000ml.
10.2.5 Methanesulfonic acid solution (25mmol/L): pipette 1.62 mL of methanesulfonic acid and dilute it to 1,000 ml.
10.3 Standard sample
Standard sample of choline tartrate hydrogen salt (C9H19NO7, relative molecular weight: 253.25, CAS No.: 87-67-2): purity ≥ 99%, or certified by China and granted with reference material certificate.
10.4 Preparation of standard solutions
10.4.1 Standard stock choline (in term of choline hydroxide, with relative molecular weight of 121.18) solution (2500mg/L): accurately weigh 522.5 mg of choline hydrogen tartrate dried to constant weight at 102°C± 2°C, dissolve it with water, transfer it to a 100 mL volumetric flask for constant volume, and mix it well. Store it in dark below 4°C, with a shelf life of 3 months.
10.4.2 Standard working choline solution (100mg/L): pipette 2.0 ml of the above-mentioned standard stock choline solution into a 50ml volumetric flask, set the volume with 15 mmol/L methanesulfonic acid solution, mix it well, and store it in dark below 4°C, with a shelf life of 1 month.
10.4.3 Standard series working Choline solution: pipette 0.200 mL, 0.500 mL, 1.00 mL, 2.50 mL, 5.00 mL of the above standard choline working fluid were respectively into a set of 100 mL volumetric flasks, and then mix it with 15 mmol/L methanesulfonic acid solution at constant volume to prepare standard series working fluid with choline concentration of 0.200 mg/L, 0.500 mg/L, 1.00 mg/L, 2.50 mg/L and 5.00 mg/L. Freshly prepare it before use.
10.5 Materials
10.5.1 0.45 μm waterborne membrane needle filter.
10.5.2 Purification column: C18 solid phase extraction column in 1.0 mL or equivalent.
10.5.3 Syringe: 5 mL or equivalent.
11 Apparatus
11.1 Ion chromatograph (IC): conductance detector.
11.2 Analytical balance: with a sensitivity of 0.1mg and 0.001g.
11.3 Electrothermal constant temperature water bath device: the temperature can be controlled at 70°C ± 2°C.
11.4 Turbine mixer.
12 Analytical procedures
12.1 Sample preconditioning
12.1.1 Specimen extraction
12.1.1.1 Liquid specimen
Accurately weigh 10 g (accurate to 0.001 g) of uniformly mixed liquid specimens in a 50mL colorimetric tube, add 15 mL of 1.7 mol/L hydrochloric acid solution, cover them, mix them well, and hydrolyze them in a water bath at 70°C ± 2°C for 3 h (shake once every 30 min). Cool the hydrolysate to room temperature, transfer it to a 50 mL volumetric flask, set the volume with water to the scale, and mix it well for later use.
12.1.1.2 Semi-solid or solid specimen
Accurately weigh 2.5 g (accurate to 0.001 g) semi-solid or solid specimen in a 50ml colorimetric tube, add 25 ml of 1 mol/L hydrochloric acid solution cover it, whirl it until there is no caking in the specimen solution, mix it well, and put it into a water bath at 70°C ± 2°C for hydrolysis for 3 h (shake once every 30 min). Cool the hydrolysate to room temperature, transfer it to a 50 mL volumetric flask, set the volume with water to the scale, and mix it well for later use.
12.1.2 Specimen purification
Let 10 ml methanol and 15ml water pass C18 solid phase extraction column (1.0 ml) in turn before use, and then have it stand and activated for 30 min. Dilute the extraction solution 50 times with water (the dilution factor can be adjusted according to the concentration of choline in the specimen, not less than 10 times), take about 15 mL of diluted solution and pass it through 0.45 μm aqueous filter membrane and C18 solid phase extraction column (1.0 ml), discard the first 3ml, and collect the eluent to be tested.
12.2 Instrument reference conditions
a) Column parameters for ion chromatography: high capacity cation exchange columns containing carboxyl groups, such as IonPac CS 12A of 4mm × 250mm (with IonPac CG 12A protection column of 4mm × 50mm) or LonPac CS 19 of 4mm × 250mm (with LonPac CG 19 protection column of 4mm × 50mm), or equivalent chromatographic columns.
b) IonPac CS 12A iso-elution: 15 mmol/L methanesulfonic acid solution iso-elution, collection time: 25 min.
IonPac CS 19 iso-elution: 6 mmol/L methanesulfonic acid solution iso-elution, collection time: 25 min.
c) Flow velocity: 1.0 mL/min.
d) Conductivity detector: equipped with suppressor or equivalent suppressor.
e) Injection volume: 100 μL.
12.3 Plotting of standard curve
Inject the standard series working fluids into ion chromatograph respectively, and measure the corresponding conductivity peak area or peak height. Plot the standard curve with the concentration of the standard series working solution as the abscissa and the conductivity peak area or peak height as the ordinate.
See Annex A for ion chromatogram of standard choline solution.
12.4 Determination of specimen solution
Inject the specimen solution into the ion chromatograph to obtain the corresponding conductivity peak area or peak height, and obtain the concentration of choline in the solution to be tested according to the standard curve.
12.5 Blank test
Carry out blank test according to procedures in 12.1 without weighing the specimen. It shall be confirmed that there are no such substances that interfere with the components to be tested.
Foreword i
1 Scope
Method I—Enzymatic colorimetry
2 Principle
3 Reagents and materials
4 Apparatus
5 Analytical procedures
6 Expression of analysis results
7 Precision
8 Others
Method II—Ion chromatography
9 Principle
10 Reagents and materials
11 Apparatus
12 Analytical procedures
13 Expression of analysis results
14 Precision
15 Others
Method III—Liquid chromatography tandem mass spectrometry
