GB/T 5413.11-1997 Determination of vitamin B1 in infant formula and milk powder
Some standard content:
GB/T5413.11~1997
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.
Method 1 of this standard 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: National Dairy Quality Supervision and Inspection Center, Jianjiang Light Industry Research Institute. The participating drafting units of this standard are: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Harbin Morinaga Dairy Co., Ltd., and Nestle (China) Investment Service Co., Ltd.
The main drafters of this standard are: Li Maosheng, Ren Yiping, Huang Baifen, Lang Zhaobin, Chen Qingjun, and Wang Yun. 263.
National Standard of the People's Republic of China
Infant and young children formula foods and milk powder
Determination of vitamin B
Milk powder and formula foods for infant and young children-Determination of vitamin B, content1Scope
This standard specifies the method for the determination of vitamin B, by fluorescence method and reversed phase high pressure liquid chromatography. GB/T 5413. 11--1997
Replaces GB5413-85
Method 1 of this standard is applicable to the determination of vitamin B in milk powder, and method 2 is applicable to the determination of vitamin B in infant and young children formula foods and milk powder.
Method 1 Fluorescence method
2 Principle of the method
The sample is digested in a dilute hydrochloric acid solution, filtered, and substances such as protein are removed. If the vitamin B in the sample is in a free state, it can be directly oxidized to thiamethoxam in alkaline potassium ferrocyanide and emit fluorescence under ultraviolet light. The intensity of the fluorescence is proportional to the thiamethoxam. If the vitamin B is in a bound state, it must be enzymatically converted to a free state, and then the fluorescence detection is performed after the impurities are removed by chromatography. The excitation wavelength E used in the detection is 365nm, and the emission wavelength Em is 435nm.
3 Reagents
All reagents, if the specifications are not specified, refer to analytical grade; all experimental water, if no other requirements are specified, refers to grade tertiary water. 3.1 Hydrochloric acid: c(HCI) is 0. 1mol/L. 3.2 Sodium hydroxide: c(NaOH) is 150g/L. 3.3 Isobutanol: redistilled.
3.4 Sodium acetate: c(NaAc) is 2mol/L
3.5 Acidic ethanol: volume fraction is 20%. Adjust pH to 3.5-4.3 with hydrochloric acid. 3.6 Amylase (biochemical reagent) solution: 100g/L 3.7 Neutral potassium fluoride: cKCl) is 250g/L. Dissolve 250g potassium chloride in 1000mL water. 3.8 Acidic potassium fluoride: c(KCI) is 250g/L. Add 8.5mL hydrochloric acid to 1L neutral potassium chloride solution. 3.9 Potassium ferrocyanide: c[K.Fe(CN). J is 10g/L, prepare on the day of use. 3.10 Oxidant.
3.11 Standard solution
3.11.1 Vitamin B, standard stock solution, concentration is 100μg/mL. Accurately weigh 50.0 mg (in a phosphorus pentoxide desiccator to constant weight) of analytically pure vitamin B, hydrochloride, dissolve in about 400 mL of 20% volume fraction acidic ethanol solution (3.5), and dilute to volume in a 500 mL brown volumetric flask, and store at about 10°C. Take 4 mL of 10 g/L potassium ferrocyanide (3.9), dilute to 100 mL with 150 g/L sodium hydroxide solution (3.2), and place in a dark place for use within 4 hours.
GB/T 5413.11-1997
3. 11. 2 Vitamin B, standard working solution, concentration is 3 μg/mL. Take 3mL of the above standard stock solution and dilute it to 100mL with 0.1mol/L hydrochloric acid (3.1). It must be freshly prepared for each experiment. 3.12 Acetic acid, volume fraction is 3%~5%.
Dilute 30mL of glacial acetic acid with water to 1000mL. 3.13 Activated artificial zeolite, 40~60 mesh, chemically pure. Activation of zeolite: Weigh 100g of artificial zeolite in a large beaker, add ten times the volume of hot acetic acid (3.12), boil for 15min under stirring, discard the acetic acid solution after it is clear, repeat this three times, add five times the volume of hot acidic potassium chloride solution (3.8), boil for 15min under stirring, discard the potassium chloride solution after it is clear, repeat this three times, then wash with hot water until there is no chloride ion, filter with a Buchner funnel, dry in an oven at 100℃, and store in an airtight wide-mouth bottle. 4 Instruments
Common laboratory instruments and:
4.1 Fluorescence spectrophotometer.
5 Operating steps
5.1 Extraction
Accurately weigh about 3g of sample in a 150mL conical flask, add 50mL of hydrochloric acid (3.1), cover the flask mouth with aluminum foil, digest for half an hour at a pressure of 137.2-161.7kPa (125℃), and at the same time, draw 5mL of standard working solution (3.11.2) in a 150mL conical flask, add about 50mL of hydrochloric acid (3.1), digest for half an hour at a pressure of 137.2-161.7kPa (125℃), cool, adjust the pH to 4.5 with sodium acetate (3.4), then add 5mL of enzyme solution (3.6), keep warm overnight at 37℃, adjust the pH to 3.5 after cooling, add water to a 100mL volumetric flask to make up the volume, and filter.
5.2 Chromatography
5.2.1 Weigh about 2g of activated artificial zeolite (3.13) and wet-load it into the column. 5.2.2 Take 10mL of the filtrate and pass it through the zeolite column for chromatography. The flow rate is controlled at ≤0.5mL/min and the filtrate is discarded. 5.2.3 Wash the zeolite three times with nearly boiling distilled water, 5mL each time, and then wash the column five times with 70-80℃ acidic potassium chloride (4.8), 4~~4.5mL each time, discard 1mL of the initial filtrate, collect the filtrate in a 25mL volumetric flask, cool it, and dilute it to the mark with acidic potassium chloride solution (4.8). 5.3 Oxidation
Add 1.5g sodium chloride or potassium chloride solid and 5mL standard sample (5.2.3) to four 50mL test tubes respectively. Immediately add 3mL oxidant (3.9) to two of the test tubes, mix, and immediately add 13mL isobutyl alcohol (3.3). Shake vigorously for more than 15s, let stand and separate the layers, and wait for fluorescence measurement. Do not add oxidant to the other two tubes, but replace them with 3mL sodium hydroxide (3.2) as blanks. At the same time, draw 5mL of sample solution (5.2.3) into the other four test tubes, and do the same as above. 5.4 Fluorescence measurement
Aspirate the supernatant from each test tube for fluorescence measurement. 6 Expression of analysis results
Vitamin B in sample (mg/100g) = (=1.)×m×1000(I, -)×c×5
Wherein: 1.---—sample fluorescence value;
I——sample blank fluorescence value;
Iad~—standard sample fluorescence value;
I.—standard sample blank fluorescence value;
c—--standard sample concentration, μg/mL;
7 Allowable difference
Sample mass, g.
GB/T 5413.11-1997
The difference between two measured values of the same sample shall not exceed 5% of the average value of the two measurements. Method 2 Reversed-phase high-pressure liquid chromatography
8 Method summary
The sample is hydrolyzed and enzymatically hydrolyzed at high temperature in a dilute hydrochloric acid environment, and the sample solution is derivatized with alkaline potassium ferrocyanide. After extraction with n-butane, it is separated by an ODSCts reverse-phase column and detected under the conditions of a fluorescence detector (E: 375nm, Em: 435nm). The content of vitamin B is quantified by the external standard method. 9 Reagents
9. 1 n-Butanol.
9.2 Potassium ferrocyanide.
9.3 Concentrated hydrochloric acid.
9.4 Anhydrous sodium acetate. bZxz.net
9.5 Glacial acetic acid.
9.6 Methanol: chromatographically pure.
9.7 Thiamine (vitamin B): standard substance. 9.8 Potassium ferrocyanide solution: c[K, Fe(CN). J is 10g/L. Weigh 2g potassium ferricyanide, dissolve and dilute to 200mL. 9.9 Sodium hydroxide solution: c (NaOH) is 100g/L. Weigh 20g sodium hydroxide solid, dissolve and dilute to 200mL9.10 Alkaline potassium ferrocyanide solution: 5mL of 10g/L potassium ferrocyanide solution (9.8) and 200mL of 100g/L sodium hydroxide solution (9.9) are mixed. 9.11 Hydrochloric acid solution: c (HCl) is 0.1mol/L. Absorb 4.5mL concentrated hydrochloric acid, dissolved in 500mL deionized water. 9.12 Hydrochloric acid solution: c(HCl) is 0.01mol/L. Take 50mL of the above 0.1mol/L hydrochloric acid solution and prepare 500mL. 9.13 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= 4.5 with glacial acetic acid.
9.14 Sodium acetate solution: c(NaAc) is 2.0mol/L. Weigh 16.61g of sodium acetate and prepare 100mL. 9.15 Mixed enzyme solution, 30g/L. Weigh 3.6g of amylase and papain each, and dissolve in 100mL 2mol/L sodium acetate solution. 9.16 Mobile phase preparation: Mix 0.05mol/L sodium acetate solution with methanol in appropriate proportions. 9.17 Standard solution
9.17.1 Vitamin B, standard stock solution, concentration is 500μg/mL. Accurately weigh 0.0500g vitamin B, (9.7), dissolve it with 0.01mol/L hydrochloric acid (9.11) and make up to volume in a 100mL volumetric flask, and store it in a refrigerator.
9.17.2 Vitamin B, standard working solution, concentration is 0.5μg/mL. Dilute the standard stock solution 1000 times with deionized water, and filter the resulting solution through a 0.3um filter membrane. 10 Instruments
10.1 High performance liquid chromatograph or high performance liquid chromatograph with equivalent performance. 10.2 Fluorescence spectrometer (with 9L flow cell) or equivalent performance fluorescence detector with wavelength tunable function. 10.3 C18 reverse phase chromatographic column (dp5um, 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 treatment
GB/T 5413.11—1997
11.1.1 Weighing: Put the sample into the crusher and crush it, then accurately weigh 5.0~10.0g (containing vitamin B, 5μg or more) into a conical flask.
11.1.2 Hydrolysis: Add an appropriate amount of hydrochloric acid solution (9.11) to control the acid concentration in the sample solution to about 0.1mol/L, shake the sample solution in the conical flask, put it into the high pressure sterilizer, keep it at 121℃ for 30min, take it out after cooling, and shake it gently several times. 11.1.3 Enzymatic hydrolysis: Cool to below 40℃, add 2.5mL of mixed enzyme solution (9.15) respectively, shake well, and place in an incubator at 37℃ overnight.
11.1.4 Volume adjustment: Adjust the pH value to about 6.0 with 0.1mol/L sodium hydroxide solution, and then adjust the volume to 100mL with double distilled water. 11.1.5 Filtration: Filter the sample solution through quantitative filter paper. 11.1.6 Derivatization: Take 10.00mL of the filtrate (11.1.5) in a 25mL stoppered measuring cylinder, add 5mL of alkaline potassium ferrocyanide (9.10) for oxidation, mix well, and then add 10.00mL of n-butanol (9.1) for extraction. After strong shaking, let the n-butanol phase (upper layer) stand for stratification and inject. 11.2 Sample determination
11.2.1 Chromatographic conditions
Mobile phase: 0.05mol/L sodium acetate solution (9.13): methanol (9.6) (65:35, volume ratio). Flow rate: 1.00 mL/min.
Detection wavelength: excitation wavelength 375nm, emission wavelength 435nm. Injection volume: 20μL.
12 Expression of analysis results
Content of vitamin B12 in sample (mg/100g) = C×FXVX100m X 1000
Where: c——concentration of injected sample solution, μg/mL; F-—dilution multiple;
V——fixed volume, mL;
m—mass of sample, g.
13 Allowable error
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 different laboratories shall not exceed 5% of the average value. 267
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