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GB 5413.15-2010: National food safety standard Determination of vitamin niacin and niacinamide in foods for infants and young children, milk and milk products
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National food safety standard Determination of vitamin niacin and niacinamide in foods for infants and young children, milk and milk products
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GB 5413.15-2010
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| GB/T 5413.15-1997 | English | 239 |
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Milk powder and formula foods for infant and young children--Determination of vitamin PP content
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Basic data | Standard ID | GB 5413.15-2010 (GB5413.15-2010) | | Description (Translated English) | National food safety standard Determination of vitamin niacin and niacinamide in foods for infants and young children, milk and milk products | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53;X82 | | Classification of International Standard | 67.100.10 | | Word Count Estimation | 10,179 | | Date of Issue | 2010-03-26 | | Date of Implementation | 2010-06-01 | | Older Standard (superseded by this standard) | GB/T 5413.15-1997 | | Adopted Standard | AOAC 944.13, IDT | | Regulation (derived from) | Circular of the Ministry of Health (2010)7 | | Issuing agency(ies) | Ministry of Health of the People's Republic of China | | Summary | This Chinese standard specifies the infant foods and milk products Determination of niacin and niacinamide method. This standard applies to infant foods and milk products Determination of nicotinic acid and nicotinamide. | GB 5413.15-2010: National food safety standard Determination of vitamin niacin and niacinamide in foods for infants and young children, milk and milk products
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National food safety standard Determination of vitamin niacin and niacinamide in foods for infans and young children, milk and milk products
National Standards of People's Republic of China
People's Republic of China Ministry of Health issued
Issued on. 2010-03-26
2010-06-01 implementation
National Food Safety Standard
Determination of infant foods and dairy products nicotinic acid and nicotinamide
National food safety standard
Determination of vitamin niacin and niacinamide in foods for infants and
young children, milk and milk products
Foreword
This standard is equivalent to the second law of international analysts Institute (AOAC) 944.13 Niacin and Niacinamide (Nicotinic Acid and
Nicotinamide) in Vitamin Preparations.
This standard replaces GB/T 5413.15-1997 "infant formula milk powder and nicotinic acid and nicotinamide determination."
The second method compared with the standard GB/T 5413.15-1997, main changes are as follows.
- Increasing the optical density determination;
- Added Determination of starch sample;
- To increase the standard curve drawing text description.
Appendix A of this standard is an informative annex.
This standard replaces the standard for the previous editions.
--GB 5413-1985, GB/T 5413.15-1997.
National Food Safety Standard
Determination of infant foods and dairy products nicotinic acid and nicotinamide
1 Scope
This standard specifies the method for determination of infant foods and dairy products nicotinic acid and nicotinamide.
This standard applies to food and dairy products Determination of nicotinic acid and nicotinamide infants.
2 Normative documents
The standard file referenced in the application of this standard is essential. For dated references, only the edition date of the note
Apply to this standard. For undated references, the latest edition (including any amendments) applies to this standard.
The first method HPLC
Principle 3
The sample was extracted with hot water, after acidic protein precipitation to C18 column, using a UV detector.
4 Reagents and materials
Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provided a water.
4.1 Amylase. enzyme activity ≥1.5 U/mg.
4.2 hydrochloric acid.
4.3 sodium hydroxide.
4.4 hydrochloric acid (2.4 mol/L). accurate Pipette 10 mL of hydrochloric acid (4.2) in 50 mL volumetric flask, and water volume.
4.5 sodium hydroxide solution (2.5 mol/L). Weigh 5.0g of sodium hydroxide (4.3) in 50 mL volumetric flask, and water volume.
4.6 perchloric acid (HClO4). volume fraction of 60%.
4.7 methanol (CH4O). chromatography.
4.8 isopropanol (C3H8O). chromatography.
4.9 heptane sulfonate (C7H15NaO3S). pure class distinctions.
4.10 standard solution
4.10.1 Niacin and Niacinamide standard stock solution (1.0 mg/mL). Weigh nicotinic acid and nicotinamide standards for each 0.1 g (accurate to 0.0001 g),
They were placed in 100 mL volumetric flask and dissolve to volume with water.
4.10.2 Niacin and Niacinamide intermediate standard solution (40 μg/mL). Imbibe respectively nicotinic acid and nicotinamide stock standard solution (4.10.1)
2 mL to 50 mL quantitative flask to volume with water. Pro preparation before use.
4.10.3 Niacin and Niacinamide standard series measured liquid. Imbibe nicotinic acid and nicotinamide mixed intermediate standard solution (4.10.2) 0.0, respectively
mL, 1.0 mL, 2.0 mL, 5.0 mL, 10.0 mL, 50 mL volumetric flask to volume with water. The standard series of concentrations of 0.00 μg/mL,
0.80 μg/mL, 1.60 μg/mL, 4.00 μg/mL, 8.00 μg/mL. Pro preparation before use.
5. Apparatus
5.1 high performance liquid chromatography with UV detection.
5.2 pH meter. accuracy of 0.01.
5.3 ultrasonic oscillator.
5.4 Balance. a sense of the amount of 0.1 mg.
5.5 Incubator. 30 ℃ ~ 80 ℃.
Step 6 Analysis
6.1 Pretreatment of the sample
6.1.1 starch-containing sample. Weigh mixed solid sample of about 5.0 g (accurate to 0.0001 g) was added about 25 mL 45 ℃ ~ 50 ℃
Water or mixed liquid sample weighed about 20.0 g (accurate to 0.0001 g) in 150 mL Erlenmeyer flask, then add about 0.5 g of starch
Enzyme (4.1), after shaking the Erlenmeyer flask nitrogen, covered with cork and placed in culture for about 30 min in the incubator of 50 ℃ ~ 60 ℃, remove the cold
To room temperature.
6.1.2 No Starch. Weigh mixed solid sample of about 5.0 g (accurate to 0.0001 g) was added about 25 mL 45 ℃ ~ 50 ℃
Water or mixed liquid sample weighed about 20.0 g (accurate to 0.0001 g) in 150 mL conical flask, shake and let stand 5 min ~
10 min, fully dissolved, and cooled to room temperature.
6.1.3 Extraction. The above conical flask placed in an ultrasonic oscillator oscillation about 10 min.
6.1.4 sedimentation and volume. to be a sample solution was cooled to room temperature, with hydrochloric acid (4.4) adjusting the pH of the sample solution to 1.7 ± 0.1, for about
After 2 min, then sodium hydroxide solution (4.5) to adjust the pH value of the sample solution to 4.5 ± 0.1. The sample solution was transferred 50 mL volumetric flask
, Washed with water repeatedly washed conical flask washings were combined in 50 mL volumetric flask with water to volume, and mix through a filter paper, and the filtrate
Then by 0.45 μm microporous membrane pressure filtration with test tubes and collect the sample test solution.
6.2 Reference chromatographic conditions
Column. C18 column (particle size 5 μm, 150 mm × 4.6 mm) or equivalent in performance to the column.
Mobile phase. methanol (4.7) 70 mL, isopropanol (4.8) 20 mL, heptane sulfonate (4.9) 1 g, with 910 mL of water to dissolve and mix
After uniform, with perchloric acid (4.6) adjusted to pH 2.1 ± 0.1, through 0.45 μm membrane filter.
Flow rate. 1.0 mL/min.
Detection wavelength. 261 nm.
Column temperature. 25 ℃.
Injection volume. 10 μL.
6.3 Quantitative Analysis
6.3.1 Standard curve drawing
The nicotinic acid and nicotinamide series of mixed standard assay solution (4.10.3) sequentially (standard sample chromatogram see Appendix A Determination
Figure A.1.1). Record each component peak area or peak height, peak area or peak height for the vertical axis, the concentration of the standard solution measured cross
Axis, the standard curve.
6.3.2 Sample Determination
A sample test solution (6.1.4) in chromatography. Record each component peak area or peak height, according to the standard curve to calculate the sample to be
Measuring liquid niacin and nicotinamide concentration ci of each component.
7 expression analysis
7.1 Calculation sample of nicotinic acid or nicotinamide
Sample niacin or nicotinamide in press (1) calculated as follows.
Vc
X i
twenty one
×× = or (1)
Where.
2-- sample content X1 or nicotinic acid or nicotinamide, in micrograms per hundred grams (μg/100 g);
m-- sample mass, in grams (g);
The concentration of the sample test solution ci-- niacin or nicotinamide, in micrograms per milliliter (μg/mL);
V-- volume of the sample solution, in milliliters (mL).
7.2 Calculation of total sample vitamin niacin content
The total content of the sample by the vitamin niacin (2) calculated as follows.
X = 1 2X X (2)
Where.
The total content of the sample X-- vitamin niacin, in micrograms per hundred grams (μg/100 g);
X1-- niacin content of the sample, in micrograms per hundred grams (μg/100 g);
Content X2-- sample nicotinamide, in micrograms per hundred grams (μg/100 g).
The arithmetic mean of two under the same condition of independent determination results indicated that the results to two significant figures.
8 Precision
Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean.
The second method microbiological method
Principle 9
Use of Lactobacillus (Lactobacillus plantarum) ATCC 8014 niacin and niacinamide specificity, containing niacin and smoke
Amide acidity samples generated growth and the formation of the optical density to determine the content of niacin and niacinamide.
10 Reagents and materials
Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provisions of secondary water.
10.1 sulfuric acid solution A (10 mol/L). mass fraction of 95% to 98% of 560 mL of concentrated sulfuric acid was slowly added to 600 mL of water,
With stirring. After cooling dilute to 1000 mL.
10.2 sulfuric acid solution B (1 mol/L). draw 100 mL 10 mol/sulfuric acid solution A L of (10.1), transferred to a 1000 mL volumetric flask
Volume with water.
10.3 sodium hydroxide solution A (3.75 mol/L). Weigh 150 g of sodium hydroxide in 1000 mL beaker, dissolved in 400 mL of water, cold
After cooling to room temperature, transferred to a 1000 mL volumetric flask, and water volume.
Sodium hydroxide solution 10.4 B (0.375 mol/L). lessons 100 mL of sodium hydroxide solution A (10.3) was transferred to a 1000 mL volumetric flask
Volume with water.
10.5 sodium hydroxide standard titration solution (0.1 mol/L ± 0.0002 mol/L). Weigh 4g (accurate to 0.0001 g) of sodium hydroxide with water
Diluted to 1000 mL, calibrated with potassium hydrogen phthalate. Save this solution containers should be sealed to prevent the penetration of carbon dioxide.
Calibration 10.5.1 sodium hydroxide standard solution. Weigh about 0.18 g (accurate to 0.0001 g) at 105 ℃ ~ 110 ℃ drying to constant weight o
Potassium hydrogen phthalate with 50 mL of aqueous addition to carbon dioxide in the conical flask, add two drops of 5 g/L of phenolphthalein indicator, with a good hydroxide
Sodium titration to pink, while for blank experiments. Sodium hydroxide standard solution is.
c =
2042.0) (
21 × -VV
(3)
Where.
c-- the sodium hydroxide concentration, in units of moles per liter (mol/L);
Quality m-- potassium hydrogen phthalate, in grams (g);
V1-- amount of sodium hydroxide solution, in milliliters (mL);
The amount of sodium hydroxide solution V2-- blank test, in milliliters (mL).
10.5.2 phenolphthalein solution. Weigh 0.5 g phenolphthalein dissolved in 75 mL volume fraction of 95% ethanol and added 20mL of water, hydroxide
Sodium Standard Solution (10.5), until one drop immediately turned pink, then add water volume to 100 mL.
10.6 hydrochloric acid (0.1 mol/L). draw 8.3 mL of hydrochloric acid, diluted with water to 1000 mL.
10.7 ethanol solution. volume fraction of 25%. Measure 250 mL of absolute ethanol was added 750 mL of water, and mix.
10.8 strains. Lactobacillus (Lactobacillus plantarum) ATCC 8014.
10.9 medium (or using commercially available synthetic medium)
10.9.1 Lactobacillus agar culture medium. light tryptone 15 g, glucose 10 g, tomato juice 100 mL, monopotassium phosphate 2 g, polysorbate
Monooleate 1 g, agar 10 g, add distilled water to 1000 mL, pH6.8 ± 0.2 (20 ℃ ~ 25 ℃). 121 ℃ autoclave 15min,
spare.
10.9.2 Lactobacillus broth medium. light tryptone 15g, glucose 10 g, tomato juice 100 mL, monopotassium phosphate 2 g, polysorbate
Monooleate 1 g, add distilled water to 1000 mL, pH 6.8 ± 0.2 (20 ℃ ~ 25 ℃). 121 ℃ autoclave 15min, standby.
10.9.3 niacin measurement medium. Vitamin determined by acid hydrolysis of casein protein amino acids (Casamino Acids) 12 g, glucose 40 g,
Sodium acetate 20 g, L- cystine 0.4 g, DL- tryptophan 0.2 g, adenine hydrochloride 20 mg, guanine hydrochloride 20 mg, uracil 20 mg,
Thiamine hydrochloride 200 μg, calcium pantothenate 200 μg, pyridoxine hydrochloride 400 μg, riboflavin 400 μg, p- amino acid 100 μg, biotin
0.8 μg, dipotassium phosphate 1 g, potassium dihydrogen phosphate 1 g, magnesium sulfate 0.4 g, sodium chloride 20 mg, ferrous sulfate 20 mg, manganese sulfate 20 mg.
Add distilled water to 1000 mL, adjust pH to 6.7 ± 0.2 (20 ℃ ~ 25 ℃).
10.10 standard solution
10.10.1 nicotinic acid standard stock solution (100 μg/mL). Remove dried niacin standards in phosphorus pentoxide desiccator, weighed 50.0
mg (accurate to 0.1 mg), dissolved in ethanol (10.7) and set the volume to 500 mL, 2 ℃ ~ 4 ℃ refrigerator, storage period of four
month.
10.10.2 niacin intermediate standard solution (10 μg/mL). 10 mL to 100 mL draw content from nicotinic acid stock standard solution (10.10.1) in
Flask with ethanol (10.7) constant volume, 2 ℃ ~ 4 ℃ refrigerator, shelf life of one month.
10.10.3 niacin working standard solution (100 ng/mL). 5.0 mL lessons from intermediate standard solution (10.10.2), diluted with water to 500 mL.
Pro preparation before use.
10.11 0.9% saline solution. Weigh 0.9 g of sodium chloride in 100 mL volumetric flask, dilute to the mark, shaking dissolved. Packed in a stoppered test tube
, Each tube 10 mL, 121 ℃ sterilization 15 min, to prepare once a week.
10.12 bromothymol blue indicator. Weigh 0.1 g bromothymol blue in a mortar, was added 1.6 mL of sodium hydroxide standard titration solution
(10.5) grinding, add a little water until completely dissolved, transferred to a 250 mL volumetric flask to volume with water.
11 instruments and equipment
11.1 spectrophotometer.
11.2 pH meter. accuracy of 0.01.
11.3 Vortex.
11.4 balance. a sense of the amount of 0.1 mg.
11.5 Incubator. 36 ℃ ± 1 ℃.
11.6 Centrifuge. ≥2000 rpm rev/min.
11.7 Buret. sub-scale value is 0.1 mL.
12 analysis steps
12.1 Preparation of test broth
12.1.1 The Lactobacillus (Lactobacillus plantarum) ATCC 8014 lyophilized powder into Lactobacillus broth (10.9.2)
Tubes, 36 ℃ ± 1 ℃ cultured 24 h, transferred to Lactobacillus agar medium (10.9.1) tube, 36 ℃ ± 1 ℃ incubated for 24 h.
Develop good Lactobacillus agar (10.9.1) in vitro cultures of bacteria as a reserve.
12.1.2 from the stock transferred to three medium strain Lactobacillus agar (10.9.1) in test tubes were placed in an incubator 36
℃ ± 1 ℃ cultured 24 h. Subculture monthly, as the monthly tube stored in the refrigerator. Monthly from monthly tube in turn reseeded 3
Took to save the new strain.
12.1.3 from January inoculated culture tube in a revaccination one Lactobacillus agar (10.9.1) tube, 36 ℃ ± 1 ℃ culture
24 h, as measured by daily inoculation tube daily.
12.1.4 inoculated a Lactobacillus broth (10.9.2) from the date of inoculation tube, 36 ℃ ± 1 ℃ cultured 24 h. Under sterile conditions
The culture broth was centrifuged 10 min (2000 r/min), the supernatant was discarded. Oscillation cells washed and centrifuged with 10 mL of normal saline (10.11)
10 min (2000 r/min), the supernatant was discarded, then 10 mL of physiological saline (10.11) oscillating wash. As before centrifugation, discard
Supernatant. Coupled with 10 mL of normal saline (10.11), and mix. Draw an appropriate amount of bacterial suspension in 10 mL of normal saline (10.11), the
Mix broth made test.
12.1.5 saline (10.11) as control, using a spectrophotometer at 550 nm wavelength, the measured test bacteria (12.1.4) transmittance
Rate, this value should be between 60% to 80%.
12.2 Preparation of samples
Weigh 2 g (accurate to 0.0001 g) or solid sample 5 g (accurate to 0.0001 g) a liquid sample (containing about niacin 0.1 mg) in 250
mL conical flask, add 20 mL of sulfuric acid solution B (10.2) The sample was dissolved into 121 ℃ autoclave autoclave kept 30 min, taking
The cooled to room temperature. Adjusted with sodium hydroxide solution A (10.3) and sodium hydroxide solution B (10.4) pH to 6.0 to 6.5, and then hydrochloric acid (10.6),
PH was adjusted to 4.5 ± 0.1, water volume to 100 mL, and filtered. Draw 25 mL filtrate in 100 mL beaker, drops sodium hydroxide standard
Fixed solution (10.5) adjusted to pH 6.8 ± 0.1, into 250 mL volumetric flask.
12.3 standard curve tube production
Add distilled water in Table 1, the standard solution and the medium in tubes, Table 1, each of the numbers required to make three. Tube S2 to S7
, A considerable amount of niacin 0 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng.
Table 1 Standard curve tube production
Tube No. S1 S2 S3 S4 S5 S6 S7
Distilled water (mL) 5 5 4 3 2 1 0
Standard solution (mL) 0 0 1 2 3 4 5
Medium (mL) 5 5 5 5 5 5 5
12.4 sample tube production
Add distilled water in Table 2, the sample and the medium in tubes, making the need for each number in the table 3.
Table 2 Sample tubes production
Tube No. 1234
Distilled water (mL) 4 3 2 1
Sample (mL) 1 2 3 4
Medium (mL) 5 5 5 5
12.5 Sterilization
The standard curve tubes and sterilized sample tubes 121 ℃ 5 min, rapidly cooled to room temperature (Commercially available media according to label directions sterilized).
Note. to ensure that the heating and cooling process conditions uniform, too sterile or too close to the number of tubes, can adversely affect the sterilization pot.
12.6 inoculation
Under sterile conditions, each of the above to each tube was added one drop (about 50 μL) test bacteria (12.1.4), capped, thoroughly shaken by mixing
Test tube (standard curve unvaccinated except blank tube S1).
12.7 Training
12.7.1 acidity method. 36 ℃ ± 1 ℃, cultured 72 h. By visual inspection of each tube, the inner tube unvaccinated broth should be
Clarification of the standard curve and the sample tube tube broth turbidity due gradient. Unvaccinated if turbidity tube, the result is invalid.
12.7.2 densitometry. at 36 ℃ ± 1 ℃, cultured 16 h ~ 24 h. The other with 12.7.1.
12.8 Determination
12.8.1 acidity law
12.8.1.1 with 10 mL water unvaccinated inoculated blank blank S1 and S2 of the tube culture was transferred Erlenmeyer flask with bromothymol
Blue (10.12) as an indicator or pH meter to pH 6.8 ± 0.2 for the titration endpoint titration with standard sodium hydroxide solution (10.5) standard titration
Standard curve tube blank tube unvaccinated and vaccinated blank tube S 1 S 2. Sodium hydroxide standard titration solution volume recorded under consumption.
Note. If the volume of the number of sodium hydroxide standard titration solution was inoculated blank titration consumption is equal to or higher than the level of non-vaccinated blank 1.5 mL, the measurement results
invalid.
12.8.1.2 with 10 mL of water to the standard curve and the sample tube in tube cultures go Erlenmeyer flask with bromothymol blue (10.12)
As an indicator or pH meter to pH 6.8 ± 0.2 for the titration end point (10.5) standard titration curve of the test tube and titration with standard sodium hydroxide solution
Sample tube culture. Sodium hydroxide standard titration solution volume recorded under consumption.
NOTE. Volume Number sodium hydroxide standard titration solution is usually a standard curve tube S7 consumed between 8 mL ~ 12 mL.
12.8.2 densitometry
Blank inoculate tube (Table 1 tube No. S 2) as a control, remove the highest concentration of the standard curve tube S 7, oscillation 5 s, at a wavelength of 550 nm
Read the optical density conditions, culture back again. 2 h after the same conditions to re-measure the optical density of the tube, if the optical density of the absolute difference between the two
Results ≤2%, then remove all the tubes measuring optical density of the sample and standard solutions.
12.9 Draw the standard curve
A standard curve for the horizontal pipe niacin content to consume sodium hydroxide standard titration solution titration milliliters or optical density value for the vertical
The standard curve.
12.10 calculate sample tube niacin content
Milliliters or optical density measured consumption of sodium hydroxide standard titration solution, Richard from the standard curve for each sample tube in accordance with 12.8
Corresponding niacin content. Three sample tube should be calculated for each number of content per milliliter of liquid measuring tube niacin, and its average value is compared.
Tube relative deviation less than 15% of valid test tube, sample tube should be discarded invalid, the total number of valid sample tube should be greater than the sum of all sample tubes
2/3. Recalculation of the average number of valid sample tube of each solution was measured per ml niacin content, calculated as the average of all the numbers again
The total average of the sample tube of Cx.
Note. The value of the sample tube niacin content of less than 100 ng, 500 ng of the above should be discarded.
13 analysis results presentation
Sample niacin (nicotinamide) content according to formula (4) Calculated.
X = 100
×× f
Cx (4)
Where.
X-- sample niacin (nicotinamide) content in micrograms per hundred grams (μg/100 g);
Cx-- 12.10 overall average calculated in units of nanograms (ng);
f-- dilution;
M-- mass or volume of the sample in grams (g).
The arithmetic mean of two under the same condition of independent determination results indicated that the results to two significant figures.
14 Precision
Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean.
Other 15
This standard is the first method detection limit is. nicotinic acid 30 μg/100 g, nicotinamide 40 μg/100 g.
Appendix A
(Informative)
Liquid chromatogram niacin and niacinamide standard solution
LC Figure A.1 niacin and niacinamide standard solution
Liquid chromatogram niacin and niacinamide standard solution is shown in Figure A.1.
Figure A.1 liquid chromatogram nicotinic acid and nicotinamide standard solution
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