GB/T 5413.15-1997 Determination of niacin and niacinamide in infant formula and milk powder
Some standard content:
GB/T5413.15--1997
This standard provides two methods. Method 1 is the microbiological method, which is equivalent to the American Association of Analytical Chemists (AOAC) method. Although the operation steps are complicated, the determination results are highly accurate. Method 2 is the high pressure liquid chromatography method, which is a fast and accurate method determined by experiments.
Method 1 of this standard is the arbitration method.
From the date of implementation, this series of standards will replace GB5413-85. 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 unit of this standard: National Dairy Quality Supervision and Inspection Center. Participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Yang Jinbao, E Laiming, Wang Yun, Liu Bo. 281
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of niacin and niacinamide
Milk powder and formula foods for infant and young children-Determination of vitamin PP content1Scope
GB/T 5413.15-1997
Replaces GB5413-85
This standard specifies the method for the determination of niacin and niacinamide by microbiological method and reversed phase high pressure liquid chromatography. This standard method is applicable to the determination of niacin and niacinamide in infant formula foods and milk powder, and method 2 is applicable to the determination of niacin and niacinamide in infant formula milk powder.
Method Microbiological method
2Method Summary
The content of niacin and niacinamide is evaluated by measuring the acidity of the growth of Lactobacillus plantarum. Reagents, strains and culture media
All reagents, if not specified, are of analytical grade. All experimental water, if not specified otherwise, is grade III water. 3.1 Sulfuric acid solution, c(H,SO,) is 10 mol/L. Slowly add 280 mL of concentrated sulfuric acid with a mass fraction of 95% to 98% to 600 mL of water, stirring while adding, cooling, and constant volume to 1000 mL.
3.2 Sulfuric acid solution, c(H,SO,) is 1 mol/L. Dilute 100 mL of 10 mol/L sulfuric acid solution (3.1) with water to 1000 mL. 3.3 Sodium hydroxide solution, c(NaOH) is 150 g/L. Dissolve 150 g of sodium hydroxide in a beaker containing 400 mL of water, place the beaker in a cold water bath, cool, dilute with water to 1000 mL, stir, and transfer to a reagent bottle. 3.4 Hydrochloric acid solution, c(HCI) is 0.1 mol/L. Take 10 mL of 10 mol/L hydrochloric acid solution and dilute it to 1000 mL with water. 3.5 Sodium hydroxide solution, c(NaOH) is 0.1 mol/L. Dilute 100 mL of 1 mol/L sodium hydroxide solution to 1 L with water and calibrate with potassium hydrogen phthalate. 3.6 Ethanol solution, volume fraction is 25%. Dilute 250 mL of 95% ethanol solution with water to 950 mL. 3.7 Bacterial species: Lactobacillus plantarum. 3.8 Culture medium
3.8.1 Lactobacillus agar culture medium: 15 g of photolysis Chen, 10 g of glucose, 100 mL of tomato juice, 2 g of potassium dihydrogen phosphate, 1 g of polysorbate monooleate, 10 g of agar, add distilled water to 1000 mL, pH 6.8±0.2 (25°C). Approved by the State Bureau of Technical Supervision on May 28, 1997, 282. Implementation on September 1, 1998. GB/T 5413.15---1997. 3.8.2 Lactobacillus broth culture medium: 15 g of photolyzed Chen, 10 g of glucose, 100 mL of tomato juice, 2 g of potassium dihydrogen phosphate, 1 g of polysorbate monooleate, add distilled water to 1000 mL, pH 6.8±0.2 (25°C). 3.8.3 Culture medium for niacin determination: Casamino Acids 12g, glucose 40g, sodium acetate 20g, L-cystine 0.4g, DL-tryptophan 0.2g, adenine hydrochloride 20mg, guanine hydrochloride 20mg, uracil 20mg, thiamine hydrochloride 200μg, calcium pantothenate 200μg, pyridoxine hydrochloride 400μg, riboflavin 400μg, β-aminobenzoic acid 100μg, biotin 0.8μg, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.4g, sodium fluoride 20mg, ferrous sulfate 20mg, manganese sulfate 20mg. Add distilled water to 1000mL, pH 6.7±0.2 (25℃). 4 Instruments
Common laboratory instruments and:
pH meter.
5 Preparation
5.1 Preparation of inoculation
Pipette part of the bacteria from the stock strain of Lactobacillus planlarum into a sterilized 10mL liquid culture medium. Choose a constant temperature between 30℃ (±0.5℃) and 35℃ (±0.5℃) for 18 to 24h. 5.2 Preparation of standard solution
5.2.1 Nicotinic acid standard stock solution, with a concentration of 100μg/mL. Take out the standard sample from the phosphorus pentoxide desiccator, accurately weigh 50~~60mg of niacin, dissolve it in the ethanol solution (3.6), continue to add ethanol solution to make the accurate content of niacin 100μg/mL, and put it in the refrigerator. 5.2.2 Nicotinic acid standard intermediate solution, with a concentration of 10μg/ml. Pipette 100mL from the standard stock solution (5.2.1), add 25% ethanol solution to 1L, and store in a refrigerator. 5.2.3 Standard working solution, nicotinic acid concentration 100mg/mL. Pipette 5.0mL from the standard intermediate solution (5.2.2), dilute with water to 500.0mL. Prepare the standard solution afresh before each use. 6 Operation steps
6.1 Preparation of the assay solution
Accurately weigh a certain amount of sample, containing about 0.1mg nicotinic acid. Transfer to a 250mL beaker, add 2mol/L sulfuric acid solution 10 times the mass of the dry powder.
Mix the sample thoroughly, wash the sample on the edge of the bottle mouth with 1mol/L sulfuric acid solution (3.2), sterilize at 121℃ for 30min, and cool. Stir thoroughly until the particles are dispersed.
Stir well, adjust pH to 6.0-6.5 with sodium hydroxide solution (3.3), quickly add hydrochloric acid solution (3.4) until no more protein is produced (pH 4.5 is the isoelectric point of many proteins), dilute the mixture to 100mL with water, and filter. If it is difficult to filter, centrifuge or filter through a filter plate. Pipette 25mL of supernatant into a 100mL flask, stir well, adjust pH to 6.8 (with 3.5), transfer to a 250ml. graduated volumetric flask, dilute to the scale with water, and filter again if floccules are produced. 6.2 Preparation of standard curve
Add distilled water, standard solution and culture medium to the test tube in the order of Table 1, in triplicate. Table 1
Test tube No.
Distilled water, mL
Standard solution, mL
Culture medium, mL
6.3Sample
GB/T5413.15—1997
Add distilled water, sample and culture medium to the test tube in the order of Table 2, in triplicate. Table 2
Test tube No.
Distilled water, mL
Sample, mL
Culture medium, mL
6.4Sterilization
Directly sterilize all test tubes, cool to the culture temperature or quickly put them into a circulating water bath to make the color form the lightest. Ensure uniform heating and cooling conditions (too many sterilization tubes or too close distances can have adverse effects in the autoclave). Sterilize at 121℃ for 10 minutes. 6.5 Inoculation
Aseptically inoculate each tube by adding a drop of appropriate inoculum. Except for test tube No.1 in the standard curve. Cover the lid and shake all tubes thoroughly.
Choose a constant temperature between 28 and 40℃ (±0.5℃) and incubate for 60 to 72 hours. 6.7 Determination (acidity method)
The determination is invalid if the test tube is contaminated by any foreign microorganisms. Predict the reaction by visual inspection of each test tube. The uninoculated tube is clear and no other bacteria grow in the standard solution and sample solution. Using bromovanillin blue as an indicator, titrate the solution in each tube with sodium hydroxide solution (3.5), or use pH 6.8 as the potentiometric titration endpoint. If the titration reaction of the inoculated blank is equal to or higher than 1.5mL, the determination result should be ignored. Usually the reaction of the standard solution in 5.0mL is equal to the titer of sodium hydroxide solution 8~~~~12mL of c(NaOH)=0.1mol/L. 7 Expression of analytical results
Niacin content in sample (mg/100g or 100mL)=Where: X——Average content of niacin in sample obtained from the curve, ug, F dilution factor,
8 Allowable difference
The mass or volume of the sample, g or mL.
The difference between the two determinations of the same sample shall not exceed 10% of the average value of the two determinations. Method 2 Reversed-phase high-pressure liquid chromatography
9 Summary of the method
After the sample is extracted with hot water and the protein is precipitated with acid, it is separated with a reversed-phase C1: chromatographic column and quantified with a UV detector. 10 Reagents
10. 1 Hydrochloric acid solution: c(HCI) is 5. 0mol/L. 10.2 Sodium hydroxide solution: c(NaOH) is 5.0 mol/L. 10.3 Perfluoric acid: volume fraction is 60%.
10.4 Anhydrous methanol: chromatographic grade.
10.5 Isopropanol: chromatographic grade.
10.6 Sodium octane sulfonate: high grade.
10.7 Standard solution
GB/T5413.151997
10.7.1 Nicotinic acid and nicotinamide standard intermediate solution, concentration is 100μg/mL. Weigh 10.0 mg each of nicotinic acid and nicotinamide standard products, dissolve in water, and then dilute to 100mL volumetric flasks respectively. 10.7.2 Nicotinic acid and nicotinamide mixed standard working solution, concentration is 5μg/mL. Take 5.0mL of the above standard intermediate solution in a 100mL volumetric flask and make up to volume with water (the concentration of niacin and niacinamide is 5μg/mL). 11 Instruments
Common laboratory instruments and:
11.1 High performance liquid chromatograph: with UV detector. 11.2 Chromatographic column: 15cm×4.6mm, or reverse phase Ct column with equivalent performance. 11.3 Acidity meter.
11.4 Ultrasonic oscillator.
12 Operation stepsbZxz.net
12.1 Sample pretreatment
12.1.1 Sample weighing: Accurately weigh 5.000g of sample and put it into a 100mL conical flask. 12.1.2 Extraction: Add 25mL of distilled water at about 60℃ to the above conical flask, shake it, and then shake it in an ultrasonic oscillator for 10min.
12.1.3 Precipitation: After the sample solution cools to room temperature, adjust the pH value of the sample solution to 1.70 with hydrochloric acid solution (10.1), let it stand for 2 minutes, and then adjust the pH value of the sample solution to 4.50 with sodium hydroxide solution (10.2). 12.1.4 Volume adjustment: Transfer the sample solution to a 50mL volumetric flask, rinse the conical flask repeatedly with distilled water, combine the washings in a 50mL volumetric flask, adjust the volume to the mark with water, and pour into a funnel for natural filtration. Take about 10mL of this filtrate, filter it under pressure through a 0.45μm microporous filter membrane, and collect this filtrate in a 10mL test tube, which is the sample reserve solution. 12.2 Instrument working conditions
Detector sensitivity: 0.002AU/mV.
Detector wavelength: 261nm.
Column temperature: 25℃.
Flow rate: 1.00mL/min.
Mobile phase: 7.0% methanol by volume, 2.0% isopropanol by volume, 1g/L sodium octane sulfonate in water, adjusted to pH=2.10 with perchloric acid.
12.3 Quantitative analysis
12.3.1 Preparation of sample solution for the machine: Accurately pipette 1.00mL of sample reserve solution and 1.00mL of distilled water, and combine them in test tube A, which is solution A; accurately pipette 1.00mL of sample reserve solution and 1.00mL of standard working solution, and combine them in test tube B, which is solution B. 12.3.2 Determination on the machine: Inject a certain amount of solution A into the liquid chromatograph to obtain the peak area A:; inject the same volume of solution B as solution A into the chromatograph to obtain the peak area B:.
13 Expression of analysis results
The content of vitamin PP in the sample (mg/100g) = X, + X, where: X, —- the content of niacin in the sample, mg/100g; Xz the content of niacinamide in the sample, mg/100g. Where X or X2:
In the formula: m-
-mass of the sample, g:
GB/T5413.15—1997
X1±2(mg/100g）=
-peak area of solution A obtained in 12.3.2; B,
peak area of solution B obtained in 12.3.2;
A, X c. × V × 100
m(B - A,) × 1000
-concentration of nicotinic acid or nicotinamide in the standard working solution, μg/mL; volume of sample solution, mL.
14 Allowable difference
The difference between two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 286
(3)00mL standard working solution, combined in test tube B, this is solution B. 12.3.2 On-machine determination: Inject a certain amount of solution A into the liquid chromatograph to obtain the peak area A:; inject the same volume of solution B as solution A into the chromatograph to obtain the peak area B:.
13 Expression of analysis results
The content of vitamin PP in the sample (mg/100g) = X, + X, where: X, —- the content of niacin in the sample, mg/100g; Xz the content of niacinamide in the sample, mg/100g. Where X or X2:
In the formula: m-
-mass of the sample, g:
GB/T5413.15—1997
X1±2(mg/100g）=
-peak area of solution A obtained in 12.3.2; B,
peak area of solution B obtained in 12.3.2;
A, X c. × V × 100
m(B - A,) × 1000
-concentration of nicotinic acid or nicotinamide in the standard working solution, μg/mL; volume of sample solution, mL.
14 Allowable difference
The difference between two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 286
(3)00mL standard working solution, combined in test tube B, this is solution B. 12.3.2 On-machine determination: Inject a certain amount of solution A into the liquid chromatograph to obtain the peak area A:; inject the same volume of solution B as solution A into the chromatograph to obtain the peak area B:.
13 Expression of analysis results
The content of vitamin PP in the sample (mg/100g) = X, + X, where: X, —- the content of niacin in the sample, mg/100g; Xz the content of niacinamide in the sample, mg/100g. Where X or X2:
In the formula: m-
-mass of the sample, g:
GB/T5413.15—1997
X1±2(mg/100g）=
-peak area of solution A obtained in 12.3.2; B,
peak area of solution B obtained in 12.3.2;
A, X c. × V × 100
m(B - A,) × 1000
-concentration of nicotinic acid or nicotinamide in the standard working solution, μg/mL; volume of sample solution, mL.
14 Allowable difference
The difference between two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 286
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