This standard specifies the method for determining vitamin B2 by fluorescence method and high pressure liquid chromatography. Method 1 of this standard is applicable to the determination of vitamin B2 content in milk powder; Method 2 is applicable to the determination of vitamin B2 in infant formula and milk powder. GB/T 5413.12-1997 Determination of vitamin B2 in infant formula and milk powder GB/T5413.12-1997 Standard download decompression password: www.bzxz.net

GB/T 5413. 12-1997
This standard provides two methods. Method 1 is the fluorescence method, which has good reproducibility and high accuracy. Method 2 is the high pressure liquid chromatography method, which has a fast determination speed.
This standard recommends that method 1 is the arbitration method.
This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry Federation.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting units of this standard are the National Dairy Quality Supervision and Inspection Center and the Zhejiang Light Industry Research Institute. The participating drafting units of this standard are the Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Harbin Morinaga Dairy Co., Ltd., and Nestle (China) Investment Services Co., Ltd.
The main drafters of this standard are Li Maosheng, Ren Yiping, Huang Baifen, Lang Zhaobin, Chen Qingjun, and Wang Yun. 268
National Standard of the People's Republic of China
Infant and young children formula foods and milk powder
Determination of vitamin B2
Milk powder and formula foods for infant and young children--Determination of vitamin B2 content1Scope
This standard specifies the method for the determination of vitamin B by fluorescence method and high pressure liquid chromatography. GB/T 5413.12-1997
Replaces GB5413--85
Method 1 of this standard is applicable to the determination of vitamin B content in milk powder; method 2 is applicable to the determination of vitamin B2 in infant and young children formula foods and milk powder.
Method Fluorescence method
2 Method summary
The sample is digested in a dilute hydrochloric acid solution, the pH value is adjusted, filtered, and the protein and other substances are removed. The filtrate is oxidized with potassium permanganate to remove interferences. The vitamin B6 in this solution can produce the maximum fluorescence intensity at an excitation wavelength of 440nm and an emission wavelength of 565nm. The vitamin B6 content can be obtained by comparing it with the standard sample.
3 Reagents
All reagents, if the specifications are not specified, are analytically pure. All experimental water, if no other requirements are specified, refers to grade tertiary water. 3. 1 Hydrochloric acid: c(HCI) is 0. 1 mol/L. 3.2 Sodium hydroxide: c(NaOH) is 400g/L. 3.3 Glacial acetic acid.
3.4 ​​Potassium permanganate: c(KMnO6) is 40g/L. 3.5 Hydrogen peroxide: volume fraction is 3%.
3.6 Sodium dithionite (NaS,O,·2H,O). 3.7 Vitamin B, standard solution
3.7.1 Vitamin B, standard stock solution, concentration is 100μg/mL. Accurately weigh 50.0mg of vitamin Bz dried overnight in the dark under phosphorus pentoxide conditions, dissolve in 0.02mol/L acetic acid, dilute to 500mL, and store in a refrigerator under toluene conditions. 3.7.2 Vitamin Bz standard intermediate solution, concentration is 10μg/mL. Dilute 100mL of standard stock solution to 1000mL with 0.02mol/L acetic acid and store in a refrigerator under toluene conditions. 3.7.3 Standard working solution, vitamin Bz concentration is 1g/mL. Dilute 10mL of standard intermediate solution to 100mL with water. Prepare on the same day. Approved by the State Bureau of Technical Supervision on May 28, 1997 and implemented on September 1, 1998
4 Instruments
Common laboratory instruments and:
4.1 Fluorescence spectrophotometer.
5 Operation steps
5.1 Extraction
GB/T 5413. 12-1997
Accurately weigh about 5g of sample in a 150mL conical flask, add 60ml of hydrochloric acid (3.1), cover the flask mouth with aluminum foil, digest for 30min under 137.2~161.7kPa pressure (125℃), adjust pH to 5.8 with sodium hydroxide (3.2), and then adjust pH to 4.5 with hydrochloric acid (3.1), transfer the extract to a 100mL volumetric flask, make up to volume with distilled water, filter, draw 25mL of filtrate into a 250mL volumetric flask, and dilute to scale with distilled water.
5.2 Oxidation of extract
5.2.1 Draw 4 portions of sample extract (5.1), 10mL each, into 4 test tubes. 5.2.2 Add 1 mL of standard working solution (3.7.3) to each of the two test tubes. 5.2.3 Add 1 mL of water to each of the other two test tubes. 5.2.4 Add 1 mL of glacial acetic acid (3.3) to each test tube and mix well. 5.2.5 Add 0.5 mL of potassium permanganate solution (3.4) to each test tube and mix well. 5.2.6 After 2 minutes, add 0.5 mL of hydrogen peroxide (3.5) to each test tube and mix well to make it fade. 5.3 Fluorescence measurement
Measure the fluorescence intensity of each tube in turn, with an excitation wavelength of 440 nm and an emission wavelength of 565 nm. After the fluorescence measurement of the sample liquid tube (without the standard working solution), add 20 mg of sodium dithionite (3.6) to each tube and mix well. Measure the fluorescence immediately and read the reading within 5 seconds.
6 Description of analysis results
(, -I)×c× D
Vitamin B2 content in sample (mg/100g) = (T. -7, ×10×m 100× 100
Wherein: 1.-
Blank sample fluorescence value;
I.Sample fluorescence value;
1.Sample plus standard fluorescence value;
c—-Standard mass concentration, μg/mL;
DSample dilution multiple;
m—-Sample mass, g.
Note: Ts- s
Is - I
The value must be between 0.66 and 1.5.
7 Allowable difference
The difference between two determinations of the same sample shall not exceed 5% of the average value of the two determinations. Method 2 High pressure liquid chromatography
8 Method summary
....(1)
The sample is hydrolyzed and enzymatically hydrolyzed in a dilute hydrochloric acid environment at high temperature, separated by an ODSC1g reverse phase chromatographic column, detected in a fluorescence detector (E: 462, Em: 522nm), and the content of vitamin B2 is quantified by the external standard method. 270
9 Reagents
GB/T5413.12-1997
All reagents, if the specifications are not specified, are of analytical grade; all experimental water, if no other requirements are specified, All refer to grade tertiary water. 9.1 Concentrated hydrochloric acid.
9.2 Anhydrous sodium acetate.
9.3 Glacial acetic acid.
9.4 Methanol: chromatographic grade.
9.5 Riboflavin (vitamin B,): standard. 9.6 Hydrochloric acid solution: c(HCl) is 0.1mol/L. Take 4.5mL of concentrated hydrochloric acid and dissolve it in 500mL of deionized water. 9.7 Hydrochloric acid solution: c(HCl) is 0.01mol/L. Take 50mL of the above 0.1mol/L hydrochloric acid and prepare 500mL. 9.8 Sodium acetate solution (pH=4.5): c(NaAc) is 0.05mol/L. Weigh 4.10g of sodium acetate solid, prepare 1000mL, and adjust to pH=4 with glacial acetic acid. 5.
9.9 Sodium acetate solution: c(NaAc) is 2.0mol/L. Weigh 16.61g sodium acetate and prepare 100mL. 9.10 Mixed enzyme solution: 30g/L. Weigh 3.6g of amylase and papain, dissolve in 100mL 2mol/L sodium acetate solution. 9.11 Mobile phase preparation: Mix 0.05mol/L sodium acetate solution and methanol in appropriate proportions. 9.12 Standard solution
9.12 .1 Standard stock solution, the concentration of vitamin B2 is 500μg/mL. Accurately weigh 0.0500g of vitamin Bz, dissolve it with 0.01mol/L hydrochloric acid (9.7) and make up to volume in a 100mL volumetric flask, and store it in a refrigerator.
9.12.2 Standard working solution, the concentration of vitamin B2 is 0.5μg/mL. When measuring, dilute the standard stock solution 1000 times with double distilled water, and filter the resulting solution through a 0.3μm filter membrane. 10 Instruments and equipment
Common laboratory instruments and:
10.1 High performance liquid chromatograph or high performance liquid chromatograph with equivalent performance. 10.2 Fluorescence spectrometer (with 9μL flow cell) or fluorescence detector with equivalent performance with wavelength tunable function. 10.3 C1s reverse phase chromatographic column (dp5μm, 25cm×4.6mm) or chromatographic column with equivalent performance. 10.4 Tissue crusher (10000~~12000r/min). 10.5 High pressure sterilizer.
11 Operation steps
11.1 Sample processing
11.1.1 Sample weighing: Put the sample into the crusher and crush it, then accurately weigh 5.0~10.0 g (containing more than 5 μg of vitamin B2) in a conical flask.
11.1.2 Hydrolysis: Add an appropriate amount of hydrochloric acid (9.6) to control the acid concentration in the sample solution to about 0.1 mol/L. After shaking the sample solution in the conical flask, put it into a high-pressure sterilizer and keep it at 121°C for 30 minutes. After cooling, take it out and shake it gently several times. 11.1.3 Enzymolysis: Cool to below 40°C, add 2.5 mL of mixed enzyme solution (9.10) respectively, shake well, and place it in a 37°C incubator overnight.
11.1.4 Volume adjustment: Use 0.1 mol/L sodium hydroxide to adjust the pH value of the sample solution to about 6.0, and then use twice distilled water to adjust the volume to 100 mL. 11.1.5 Filtration: Filter the sample solution through quantitative filter paper, and collect a small amount of filtrate for injection. 11.2 Sample determination
11.2.1 Chromatographic conditions
Mobile phase: 0.05mol/L sodium acetate solution (9.8): methanol (9.4) (65:35, volume ratio). 271
Speed: 1.00mL/min.
GB/T5413.12—1997
Detection wavelength: excitation wavelength 462nm, emission wavelength 522nm. 12 Expression of analysis results
cX F×V× 100
Content of vitamin Bz in sample (mg/100g) = m × 100
Where: c—concentration of injected sample solution, μg/mL; F—dilution factor;
V—fixed volume, mL,
—mass of sample, g.
13 Allowable errorwwW.bzxz.Net
13.1 Repeatability
The difference between the results of two measurements of the same sample shall not exceed 5% of the average value. 13.2 Reproducibility
The difference between the results of two measurements of the same sample in two laboratories shall not exceed 5% of the average value. 272
, (2)
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