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GB 5009.89-2023 PDF in English


GB 5009.89-2023 (GB5009.89-2023) PDF English
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GB 5009.89-2023: PDF in English

GB 5009.89-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of Niacin
and Niacinamide in Foods
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. MARCH 6, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
Method I - High Performance Liquid Chromatography... 4
2 Principle... 4
3 Reagents and Materials... 4
4 Instruments and Equipment... 5
5 Analytical Procedures... 6
6 Expression of Analysis Results... 8
7 Precision... 9
8 Others... 9
Method II - Microbiological Method... 9
9 Principle... 9
10 Reagents and Materials... 9
11 Instruments and Equipment... 11
12 Analytical Procedures... 12
13 Expression of Analysis Results... 17
14 Precision... 18
15 Others... 18
Appendix A Liquid Chromatogram of Niacin and Niacinamide Standard Solution... 19
Appendix B Preparation of Culture Medium... 20
National Food Safety Standard - Determination of Niacin
and Niacinamide in Foods
1 Scope
This Standard specifies the methods for the determination of niacin and niacinamide in foods.
In this Standard, Method 1 is applicable to the determination of niacin and niacinamide in
prepared milk powder, special dietary foods (excluding partially hydrolyzed milk protein
formulas, deeply hydrolyzed milk protein formulas or amino acid formulas, and infant formulas
for special medical purposes for amino acid metabolism disorders) and special-purpose
beverages. Method 2 is applicable to the determination of niacin (or niacinamide) in foods.
Method I - High Performance Liquid Chromatography
2 Principle
After pre-treatment, such as. enzymatic hydrolysis and protein precipitation, the sample is
extracted through ultrasonic oscillation in a weakly acidic environment, separated by C18
chromatographic column and detected by a UV detector. In accordance with the retention time
of the chromatographic peak, conduct qualitative determination, and adopt the external standard
method for quantitative determination. Calculate the content of niacin and niacinamide in the
specimen.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl). guaranteed reagent.
3.1.2 Sodium hydroxide (NaOH). guaranteed reagent.
3.1.3 Methanol (CH3OH). chromatographically pure.
3.1.4 Isopropyl alcohol (C3H8O). chromatographically pure.
3.1.5 Sodium heptane sulfonate (C7H15NaO3S). chromatographically pure.
3.1.6 Amylase. enzyme activity  1.5 U/mg.
3.2 Preparation of Reagents
3.2.1 Hydrochloric acid solution (5.0 mol/L). measure-take 415 mL of hydrochloric acid and
add water to reach a constant volume of 1,000 mL.
3.2.2 Hydrochloric acid solution (0.1 mol/L). draw-take 8.3 mL of hydrochloric acid and add
water to reach a constant volume of 1,000 mL.
3.2.3 Sodium hydroxide solution (5.0 mol/L). weigh-take 200 g of sodium hydroxide and add
water to reach a constant volume of 1,000 mL.
3.2.4 Sodium hydroxide solution (0.1 mol/L). draw-take 20 mL of sodium hydroxide solution
(5.0 mol/L) and add water to reach a constant volume of 1,000 mL.
3.3 Reference Materials
3.3.1 Niacin (C6H5NO2, CAS. 59-67-6). purity  98%, or a standard substance certified by the
state and awarded a reference material certificate.
3.3.2 Niacinamide (C6H6N2O, CAS. 98-92-0). purity  98%, or a standard substance certified
by the state and awarded a reference material certificate.
3.4 Preparation of Standard Solutions
3.4.1 Niacin and niacinamide standard stock solution (1.000 mg/mL). place niacin (or
niacinamide) reference material into a desiccator containing phosphorus pentoxide and dry it
overnight. Respectively and accurately weigh-take 0.1 g (accurate to 1 mg), use 0.1 mol/L
hydrochloric acid to dissolve it, and reach a constant volume of 100 mL. It can be stored for 1
month when refrigerated at 2 C ~ 8 C.
3.4.2 Niacin and niacinamide standard mixed intermediate solution (100.0 g/mL). respectively
and accurately draw-take 10.0 mL of niacin and niacinamide standard stock solution into a 100
mL volumetric flask, add water to reach a constant volume to the scale, and evenly mix it. It
can be stored for 1 month when refrigerated at 2 C ~ 8 C.
3.4.3 Niacin and niacinamide standard mixed working solutions. respectively and accurately
draw-take 1.0 mL, 2.0 mL, 5.0 mL, 10.0 mL and 20.0 mL of the standard mixed intermediate
solution into 100 mL volumetric flasks, add water to reach a constant volume to the scale and
evenly mix them. Thus, standard mixed working solutions with a mass concentration of 1.0
g/mL, 2.0 g/mL, 5.0 g/mL, 10.0 g/mL and 20.0 g/mL are obtained. Prepare them right
before use.
4 Instruments and Equipment
4.1 High performance liquid chromatograph. equipped with UV detector or diode array detector.
4.2 Balance. with a division value of 0.1 mg and 0.01 g, respectively.
4.3 Constant-temperature incubator. 30 C ~ 80 C.
4.4 Ultrasonic equipment.
4.5 pH meter. with an accuracy of 0.1.
4.6 Pulverizer.
5 Analytical Procedures
5.1 Sample Pre-treatment
5.1.1 Specimen preparation
Sample pre-treatment. take at least 200 g of representative sample. For lumpy or granular
samples, use a pulverizer to pulverize them; for powdery, pasty or liquid samples, thoroughly
mix them and place them in a closed container.
5.1.2 Starches and starch-containing foods
5.1.2.1 Weigh-take about 5.0 g (accurate to 0.01 g) of evenly mixed solid specimen, place it in
a 150 mL conical flask, add about 25 mL of 45 C ~ 50 C water, add about 0.5 g of amylase,
shake it well, then, fill the conical flask with nitrogen, use a stopper to cap it, and place it in an
incubator at 50 C ~ 60 C for enzymatic hydrolysis for about 30 minutes. Take it out and cool
to room temperature.
5.1.2.2 Weigh-take about 20.0 g (accurate to 0.01 g) of evenly mixed liquid specimen, place it
in a 150 mL conical flask, add about 0.5 g of amylase, shake it well, then, fill the conical flask
with nitrogen, use a stopper to cap it, and place it in an incubator at 50 C ~ 60 C for enzymatic
hydrolysis for about 30 minutes. Take it out and cool to room temperature.
5.1.3 Starch-free foods
5.1.3.1 Weigh-take about 5.0 g (accurate to 0.01 g) of evenly mixed solid specimen, place it in
a 150 mL conical flask, and add about 25 mL of 45 C ~ 50 C water. Place it in ultrasonic
equipment and oscillate for more than 10 minutes to thoroughly dissolve it, let it stand for 5
minutes ~ 10 minutes, then, cool it to room temperature.
5.1.3.2 Weigh-take about 20.0 g (accurate to 0.01 g) of evenly mixed liquid specimen and place
it in a 150 mL conical flask. Place it in ultrasonic equipment and oscillate for more than 10
minutes to thoroughly dissolve it, let it stand for 5 minutes ~ 10 minutes, then, cool it to room
temperature.
5.1.4 Specimen extraction
After the specimen solution drops to room temperature, use 5.0 mol/L hydrochloric acid
solution and 0.1 mol/L hydrochloric acid solution to adjust the pH of the specimen solution to
factor 1.008.
7 Precision
The absolute difference between the results of two independent determinations obtained under
repeatability conditions shall not exceed 10% of the arithmetic mean.
8 Others
For solid samples. when the sampling size is 5 g, the detection limit of niacin is 30 g/100 g,
and the quantitation limit is 100 g/100 g; the detection limit of niacinamide is 40 g/100 g,
and the quantitation limit is 120 g/100 g.
For liquid samples. when the sampling size is 20 g, the detection limit of niacin is 7.5 g/100
g, and the quantitation limit is 25 g/100 g; the detection limit of niacinamide is 10 g/100 g,
and the quantitation limit is 30 g/100 g.
Method II - Microbiological Method
9 Principle
Niacin and niacinamide are essential nutrients for the growth of Lactiplantibacillus plantarum.
In the niacin determination culture medium, the growth of Lactiplantibacillus plantarum is
correlated with the niacin (or niacinamide) content. In accordance with the standard curve of
niacin (or niacinamide) content and light transmittance (or absorbance), calculate the niacin (or
niacinamide) content in the specimen.
10 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 or Grade-2 water specified in GB/T 6682.
10.1 Strain
Lactiplantibacillus plantarum (the former Lactobacillus plantarum) ATCC 8014, or equivalent
strain.
10.2 Culture Media
10.2.1 Lactobacillus agar culture medium. see B.1 in Appendix B.
10.2.2 Lactobacillus broth culture medium. see B.2 in Appendix B.
10.2.3 Medium for niacin determination. see B.3 in Appendix B.
NOTE. commercially available synthetic media can be used and operated in accordance with the
instructions.
10.3 Reagents
10.3.1 Absolute ethanol (C2H5OH).
10.3.2 Sulfuric acid (H2SO4). 95% ~ 98%.
10.3.3 Sodium hydroxide (NaOH).
10.3.4 Sodium chloride (NaCl).
10.4 Preparation of Reagents
10.4.1 Ethanol solution (with a volume fraction of 25%). measure-take 250 mL of absolute
ethanol, add water to reach a constant volume of 1,000 mL.
10.4.2 Sulfuric acid solution A (10 mol/L). measure-take 560 mL of sulfuric acid, add it to water
and dilute it to 1,000 mL.
10.4.3 Sulfuric acid solution B (0.5 mol/L). measure-take 50 mL of sulfuric acid A (10 mol/L),
add it to water and dilute it to 1,000 mL.
10.4.4 Sodium hydroxide solution A (10 mol/L). weigh-take 400 g of sodium hydroxide, add
water to dissolve it and dilute to 1,000 mL.
10.4.5 Sodium hydroxide solution B (0.1 mol/L). draw-take 10 mL of sodium hydroxide
solution A (10 mol/L), add water to dilute it to 1,000 mL.
10.4.6 Sterile physiological saline (8.5 g/L). weigh-take 8.5 g of sodium chloride and dissolve
it in 1,000 mL of distilled water, divide it into stoppered test tubes, with 10 mL in each tube. At
121 C, perform autoclaved sterilization for 15 min.
10.5 Reference Materials
10.5.1 Niacin (C6H5NO2, CAS. 59-67-6). purity  98%, or a standard substance certified by the
state and awarded a reference material certificate.
10.5.2 Niacinamide (C6H6N2O, CAS. 98-92-0). purity  98%, or a standard substance certified
by the state and awarded a reference material certificate.
10.6 Preparation of Standard Solutions
10.6.1 Niacin (or niacinamide) standard stock solution (50 g/mL). place niacin (or
niacinamide) reference material into a desiccator containing phosphorus pentoxide and dry it
overnight. In accordance with purity, weigh it, so that the niacin (or niacinamide) content is
11.14 Graduated pipette. 5 mL (with a scale of 0.1 mL).
11.15 Glass funnel. with a diameter of 100 mm.
11.16 Conical flask. with a capacity of 250 mL.
11.17 Beaker. with a capacity of 100 mL.
11.18 Dispenser. 0 mL ~ 10 mL.
11.19 Micropipette. 1,000 L and 200 L.
11.20 Sterile centrifuge tube. 1.5 mL.
11.21 Syringe filter. with an aperture of 0.22 m.
NOTE. the cleaned glassware and metal appliances shall be dry-heated at 250 C for 1 h ~ 2 h.
12 Analytical Procedures
12.1 Preparation of Test Bacterial Suspension
12.1.1 After the Lactiplantibacillus plantarum strain is activated, use an inoculating needle to
pierce and inoculate it onto the lactobacillus agar culture medium, at 36 C  1 C, culture for
16 h ~ 24 h. Transplant 2 ~ 3 generations to enhance the vitality. Store it in the refrigerator as a
slant culture. It shall remain valid for 1 month.
12.1.2 Transplant the strain activated within 24 hours to sterilized lactobacillus broth, at 36 C
 1 C, culture for 16 h ~ 24 h. At 3,000 r/min ~ 5,000 r/min, centrifuge it for 5 min, discard
the supernatant, add 10 mL of physiological saline, and use a vortex mixer to oscillate the
suspension, then, centrifuge it for about 5 minutes and discard the supernatant. After washing
2 ~ 3 times as before, add 10 mL of physiological saline and evenly oscillate it. Draw-taken an
appropriate amount of the bacterial suspension into 10 mL of physiological saline and evenly
mix it to prepare a test bacterial suspension.
12.1.3 Use physiological saline as a blank, use a spectrophotometer to determine the
transmittance (% T) of the test bacterial suspension at a wavelength of 550 nm, and adjust the
amount of the above-mentioned bacterial solution added, so that the transmittance of the test
bacterial suspension is between 60% T and 80% T.
12.2 Specimen Extraction
Lumpy and granular specimens need to be crushed; powdered specimens, such as. milk powder
and rice flour, need to be evenly mixed; fruits and vegetables, meat, eggs, fish and animal offal,
etc., need to be made into chyme; semi-solid foods need to be homogenized and evenly mixed;
liquid specimens shall be shaken and mixed before use.
12.2.1 Solid specimens. accurately weigh-take the specimen (accurate to 0.001 g) and place it
in a conical flask. Specifically speaking, 2 g ~ 5 g of fresh fruit and vegetable specimens; 0.2 g
~ 1 g of cereals, beans, nuts, offal, raw meat and dried specimens; 2 g ~ 3 g of milk powder and
rice flour specimens; 0.1 g ~ 0.5 g of general nutrient supplements and compound nutritional
supplements; 0.2 g ~ 1 g of other foods.
12.2.2 Liquid beverages or liquid and semi-liquid specimens. weigh-take 5 g ~ 10 g (accurate
to 0.001 g) of specimen (or use a one-mark pipette to draw-take an appropriate volume) and
place it in a conical flask. Special-purpose beverages do not need to be processed in accordance
with 12.2.3 after sample weighing. After weighing the sample, directly reach a constant volume
of 100 mL (V); in accordance with 12.2.4, dilute it.
If the niacin (or niacinamide) content in the specimen is excessively low, the sampling size can
be appropriately increased.
12.2.3 Add sulfuric acid solution B (in milliliters) that is 10 times the specimen mass (in grams),
put the above-mentioned mixture into a pressure steam sterilizer, at 121 C, hydrolyze it for 30
minutes, then, take it out and quickly cool to room temperature in a water bath. Use sodium
hydroxide solution A and sodium hydroxide solution B to adjust pH to 4.5  0.2, transfer it into
a 250 mL (V1) volumetric flask, and use water to reach a constant volume to the scale.
Use a quantitative filter paper to filter it. The first 10 mL of filtrate shall be discarded. Draw-
take 5 mL (V2) of the filtrate into a 100 mL beaker, add about 20 mL of water, and use sodium
hydroxide solution B to adjust pH to 6.8  0.2.Transfer it into a 100 mL (V) volumetric flask
and add water to reach a constant volume to the scale.
12.2.4 Dilution. in accordance with the niacin (or niacinamide) content in the specimen, use
water to appropriately dilute the extracting solution, so that the mass concentration of niacin
(or niacinamide) in the specimen extracting solution after dilution is 5 ng/mL ~ 12 ng/mL.
12.3 Specimen Determination
12.3.1 Test tube culture method
12.3.1.1 Standard curve series of tubes
In accordance with the sequence listed in Table 1, add water, standard curve working solution
and culture medium for niacin determination to the culture tube. Prepare 3 tubes for each
number in Table 1.In uninoculated blank test tube (UN), inoculated blank test tube (IN) and
standard series of tubes S1 ~ S8, the mass concentration of niacin (or niacinamide) is
respectively. 0 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 40
ng/mL and 50 ng/mL.
12.3.1.6 Determination
After the culture is completed, visually inspect each test tube. The culture solution in the
uninoculated blank test tube (UN) shall be clear. There shall be a gradient difference in the
absorbance of the culture solution in the standard curve series of tubes and the specimen series
of tubes. If the uninoculated blank test tube (UN) is turbid, then, the determination shall be
deemed as invalid.
12.3.1.6.1 Use the uninoculated blank test tube (UN) as a blank, adjust the spectrophotometer
transmittance to 100% T, and read the reading of the inoculated blank test tube (IN). Then, use
the inoculated blank test tube (IN) as the blank, adjust the transmittance to 100% T, and
successively read the transmittance (% T) (or absorbance A) of other test tubes.
12.3.1.6.2 Use a vortex mixer to thoroughly mix each test tube (a drop of antifoaming agent
can also be added), then, immediately transfer the culture solution into a cuvette, at a
wavelength of 550 nm, perform colorimetric determination. After the reading is stable, read the
light transmittance. The stabilization time of each test tube shall be the same. Successively read
the transmittance of other test tubes. Culture tubes whose transmittance exceeds the
concentration range of standard curve series of tubes S1 ~ S8 shall be discarded. Take the mass
concentration of the niacin or niacinamide reference material as the x-coordinate and the
transmittance as the y-coordinate to draw a standard curve.
NOTE. to draw a standard curve, absorbance A can also be used as the y-coordinate.
12.3.1.6.3 For the test tubes of each numbered test solution, use the transmittance or absorbance
of each test tube to calculate the concentration of niacin (or niacinamide) in each milliliter of
the specimen extracting solution, and calculate the average concentration of niacin (or
niacinamide) in the numbered extracting solution. The concentration measured of each test tube
shall not exceed 15% of the average value, and any excess shall be discarded. If the number
of tubes that comply with this requirement is less than 2/3 of the total number of tubes of all 4
numbered extracting solutions, the data used to calculate the specimen content is insufficient
and the test needs to be re-performed. If the number of tubes that comply with this requirement
is less than 2/3 of the original number of tubes, re-calculate the average value of niacin (or
niacinamide) content per milliliter of extracting solution in each numbered valid specimen tube,
and use this average value to calculate the total average value of all numbered specimen tubes
as ρ. In accordance with Formula (3) and the dilution factor and sampling size, calculate the
content of niacin (or niacinamide) in the specimen.
12.3.2 Microplate culture method
12.3.2.1 Standard curve series of centrifuge tubes
Filter and sterilize the niacin standard curve working solution into a sterile centrifuge tube under
sterile conditions. In accordance with Table 3, prepare 3 sets of standard curve series of
centrifuge tubes. The niacin (or niacinamide) mass concentration in uninoculated blank pore
(UN), inoculated blank pore (IN) and S1 ~ S8 is the same as that in the test tube method.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.