GB 5009.270-2023 English PDF

GB 5009.270-2023: National food safety standard - Determination of inositol in foods
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GB 5009.270-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of Inositol
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 - Gas 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... 8
8 Others... 8
Method II - Microbiological Method... 9
9 Principle... 9
10 Reagents and Materials... 9
11 Culture Media and Reagents... 10
12 Analytical Procedures... 11
13 Expression of Analysis Results... 14
14 Precision... 15
15 Others... 15
Appendix A Gas Chromatogram... 16
Appendix B Culture Media and Reagents... 17
Appendix C Four-parameter Logistic Curve Fitting Equation... 20
National Food Safety Standard - Determination of Inositol
in Foods
1 Scope
This Standard specifies the methods for the determination of inositol (myo-inositol) in foods.
Method 1 - gas chromatography is applicable to the determination of inositol in infant
formulas, formulas for special medical purposes, milk and dairy products, and beverages.
Method 2 - microbiological method is applicable to the determination of inositol in foods.
Method I - Gas Chromatography
2 Principle
The inositol in the specimen is extracted with water and precipitated with ethanol. After the
supernatant is centrifuged and dried, it is derivatized with a silanization reagent. The
derivative is extracted with n-hexane, separated by gas chromatography and detected by a
hydrogen flame ionization detector. Adopt the external standard method for quantitative
determination.
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 Absolute ethanol (C2H6O).
3.1.2 95% ethanol (C2H6O).
3.1.3 Acetonitrile (C2H3N).
3.1.4 n-Hexane (C6H14).
3.1.5 Trimethylchlorosilane (C3H9ClSi).
3.1.6 Hexamethyldisilazane (C6H19NSi2).
3.1.7 N, N-dimethylformamide (C3H7NO).
3.1.8 Anhydrous sodium sulfate (Na2SO4).
3.2 Preparation of Reagents
3.2.1 70% ethanol. measure-take 700 mL of absolute ethanol, use water to reach a constant
volume of 1,000 mL, and evenly mix it.
3.2.2 Silanization reagent. respectively draw-take trimethylchlorosilane,
hexamethyldisilazane and N, N-dimethylformamide, mix them in a volume ratio of 1. 2. 8,
and conduct ultrasonic mixing. Prepare it right before use.
NOTE. if the silanization reagent appears white and turbid, it needs to be prepared again.
3.3 Reference Material
Inositol reference material (C6H12O6, CAS. 87-89-8). purity  99%, or a standard substance
certified by the state and awarded a reference material certificate.
3.4 Preparation of Standard Solutions
3.4.1 Inositol standard stock solution (1.00 mg/mL). weigh-take 100 mg (accurate to 0.1 mg)
of inositol reference material that has been dried at 105 C  2 C to a constant mass, use 25
mL of water to dissolve it, and use 95% ethanol to reach a constant volume of 100 mL, and
evenly mix it. Store it at 2 C ~ 8 C. It shall remain valid for 1 month.
3.4.2 Inositol standard working solution (0.100 mg/mL). accurately transfer-take 5.00 mL of
inositol standard stock solution, use 70% ethanol to reach a constant volume of 50 mL, and
evenly mix it. Prepare it right before use.
4 Instruments and Equipment
4.1 Gas chromatograph. equipped with a hydrogen flame ionization detector.
4.2 Analytical balance. with a division value of 0.1 mg and 1 mg.
4.3 Centrifuge. with a speed  4,000 r/min.
4.4 Oven. with a temperature accuracy of  2 C.
4.5 Constant-temperature water bath. with a temperature accuracy of  2 C.
4.6 Rotary evaporator.
4.7 Vortex oscillator.
4.8 Ultrasonoscope.
4.9 Nitrogen blower.
5 Analytical Procedures
5.1 Specimen Preparation
5.1.1 Dissolution
Weigh-take 1 g of solid specimen or 12 g of liquid specimen (accurate to 1 mg) that has been
evenly mixed into a 100 mL conical flask. For solid specimen, use 12 mL of 40 C ~ 45 C
warm water to dissolve it, and perform ultrasonic extraction for 10 minutes. Transfer the
above treated specimen solution into a 50 mL volumetric flask, use 95% ethanol to reach a
constant volume to the scale, and evenly mix it; let it stand to precipitate for 20 minutes. If
the specimen has lumpy, rather than flocculent precipitate, re-weigh the sample, and use 30
mL of 40 C ~ 45 C warm water to re-dissolve the sample, add acetonitrile to reach a
constant volume to the scale and evenly mix it; let it stand to precipitate for 20 minutes.
After the precipitation is completed, draw-take 10 mL of the supernatant, at a speed not
lower than 4,000 r/min, centrifuge it for 5 min, then, accurately transfer-take 5.00 mL of the
supernatant to a 25 mL rotary evaporation bottle or screw-top glass bottle and reserve it for
drying.
5.1.2 Drying
Add an appropriate amount of absolute ethanol to the specimen to be dried, at a temperature
not higher than 80 C, use the rotary evaporator or nitrogen blower to concentrate it to near
dryness. At 100 C, bake it for 1 h. Take it out to cool to room temperature and reserve it for
derivatization.
5.1.3 Derivatization
Add 10 mL of the silanization reagent to the dried specimen, conduct ultrasound for 5
minutes, seal and evenly mix it in a 25 mL screw-top glass bottle. In 80 C water bath, react
for 75 minutes, during which, take it out and oscillate once every 20 minutes. After it is over,
cool to room temperature, add 5 mL of n-hexane and vortex for 2 minutes. Let it stand for
stratification, then, take 3 mL of n-hexane extracting solution into a centrifuge tube that has
been pre-added with a little anhydrous sodium sulfate, vortex, then, at a speed not lower than
4,000 r/min, centrifuge for 5 min. Then, transfer the solution into a sample injection bottle to
obtain the specimen determination solution to be determined by the gas chromatograph.
5.2 Preparation of Inositol Standard Determination Solutions
Respectively draw-take 0.200 mL, 0.400 mL, 0.600 mL, 0.800 mL, 1.00 mL and 2.00 mL of
inositol standard working solution (0.100 mg/mL) into rotary evaporation bottles or screw-
top glass bottles. The other analytical procedures are the same as 5.1.2 and 5.1.3.The
inositol content in the obtained standard determination solutions is respectively. 0.020 mg,
0.040 mg, 0.060 mg, 0.080 mg, 0.100 mg and 0.200 mg.
NOTE. the concentration range of the inositol standard determination solutions can be adjusted
Method II - Microbiological Method
9 Principle
Inositol is an essential nutrient for the growth of Saccharomyces cerevisiae. Under certain
conditions, there is a corresponding relation between the growth of Saccharomyces
cerevisiae and the inositol content. Taking the standard working curve as a reference, in
accordance with the absorbance value of the solution to be tested, the inositol content in the
specimen to be tested can be calculated.
10 Reagents and Materials
10.1 Equipment
10.1.1 Balance. with a division value of 0.1 mg, 1 mg and 0.1 g.
10.1.2 pH meter. with an accuracy of  0.01.
10.1.3 Spectrophotometer (at a wavelength of 550 nm).
10.1.4 Constant-temperature incubator. 30 C  1 C.
10.1.5 Oscillation incubator. 30 C  1 C, with an oscillation frequency of 140 r/min ~ 160
r/min.
10.1.6 High-pressure steam sterilizer. 121 C (0.10 MPa ~ 0.12 MPa); 125 C (0.13 MPa ~
0.15 MPa).
10.1.7 Thermostat (or water bath). 100 C  1 C.
10.1.8 Centrifuge. with a speed  2,000 r/min.
10.1.9 Refrigerator. 2 C ~ 5 C.
10.1.10 Vortex oscillator.
10.1.11 Homogenizer.
10.2 Materials
10.2.1 Glass beads. with a diameter of about 5 mm.
10.2.2 Test tube. 18 mm  180 mm.
10.2.3 Sterile pipette. 10 mL (with a scale of 0.1 mL) or 10 mL micropipette and tip.
10.2.4 Conical flask. 200 mL or 250 mL.
10.2.5 Volumetric flask (Type A). 50 mL, 100 mL and 250 mL.
10.2.6 Funnel. with a diameter of 90 mm.
10.2.7 Quantitative filter paper. with a diameter of 90 mm.
NOTE. before using the glass instrument, use an active agent (add sodium laurel sulfonate or
household detergent to the washing water) to clean the hard glass measuring tube and
other necessary glassware. After cleaning, dry heat at 200 C for 2 hours.
11 Culture Media and Reagents
Unless it is otherwise specified, the reagents used in this Method are all analytically pure,
and the water is Grade-2 water specified in GB/T 6682.
11.1 Culture Media
11.1.1 Malt extract agar culture medium. see B.1 in Appendix B.
11.1.2 Malt extract liquid culture medium. see B.2 in Appendix B.
11.1.3 Culture medium for inositol determination. see B.3 in Appendix B.
NOTE. commercial synthetic media can be prepared in accordance with the instructions.
11.2 Reagents and Strain
11.2.1 Sodium chloride (NaCl).
11.2.2 Sodium hydroxide (NaOH).
11.2.3 Hydrochloric acid (HCl).
11.2.4 Phosphorus pentoxide (P2O5).
11.2.5 Saccharomyces cerevisiae ATCC 9080, or other validated equivalent standard strains.
11.3 Preparation of Reagents
11.3.1 Sterile sodium chloride solution (0.85%). weigh-take 8.5 g of sodium chloride and
dissolve it in 1,000 mL of water, and divide it into test tubes, with 10 mL in each tube. At
121 C, sterilize for 15 minutes.
11.3.2 Hydrochloric acid solution (1 mol/L). measure-take 90 mL of concentrated
hydrochloric acid, reach a constant volume of 1,000 mL and evenly mix it.
11.3.3 Hydrochloric acid solution (0.44 mol/L). measure-take 39.6 mL of concentrated
hydrochloric acid, reach a constant volume of 1,000 mL and evenly mix it.
11.3.4 Sodium hydroxide solution (15 mol/L). weigh-take 300 g of sodium hydroxide and
dissolve it in water. After cooling, reach a constant volume of 500 mL and evenly mix it.
11.3.5 Sodium hydroxide solution (1 mol/L). weigh-take 40 g of sodium hydroxide and
dissolve it in water. After cooling, reach a constant volume of 1,000 mL and evenly mix it.
11.4 Reference Material
Inositol reference material (C6H12O6, CAS. 87-89-8). purity  99%, or a standard substance
certified by the state and awarded a reference material certificate.
11.5 Preparation of Standard Solutions
11.5.1 Inositol standard stock solution (0.2 mg/mL). place inositol reference material in a
phosphorus pentoxide desiccator to dry for more than 24 hours, weigh-take 50 mg (accurate
to 0.1 mg) of the above-mentioned inositol reference material into a 100 mL beaker, use
water to dissolve it, then, transfer it to a 250 mL brown volumetric flask. Dilute to the scale
and evenly mix it. Store it at 2 C ~ 8 C. It shall remain valid for 1 month.
11.5.2 Inositol standard intermediate solution (10 g/mL). accurately transfer-take 5.00 mL
of inositol standard stock solution, use water to reach a constant volume in a 100 mL brown
volumetric flask. Store it at 2 C ~ 8 C. Prepare it right before use.
11.5.3 Inositol standard working solution (1 g/mL and 2 g/mL). accurately transfer-take
10.00 mL of inositol standard intermediate solution twice, respectively use water to reach a
constant volume in a 100 mL brown volumetric flask and a 50 mL brown volumetric flask.
Prepare it right before use.
12 Analytical Procedures
12.1 Preparation of Strain
12.1.1 Strain recovery
Inoculate Saccharomyces cerevisiae onto the slant of the malt extract agar culture medium,
at 30 C  1 C, culture it for 16 h ~ 24 h. Then, transplant 2 ~ 3 generations to enhance the
activity and prepare a stock strain. Store it in the refrigerator at 4 C. The storage period
shall not exceed 2 weeks.
12.1.2 Preparation of bacterial suspension
Before use, inoculate the stock strain onto the slant of a new malt extract agar culture
medium, at 30 C  1 C, culture it for 16 h ~ 24 h. Then, transplant one ring of the slant
culture into 10 mL of malt extract liquid culture medium, at 30 C  1 C, culture it for 20 h
~ 24 h. Thoroughly oscillate and mix the above-mentioned 10 mL fresh culture, transfer it to
a centrifuge tube, at 2,000 r/min, centrifuge it for 15 min, and discard the supernatant. Add
10 mL of sterile 0.85% sodium chloride solution, mix and re-suspend it. Repeat the
centrifugation and re-suspension steps twice to prepare a 10 mL bacterial suspension and
reserve it for later use.
Take sterile 0.85% sodium chloride solution as a blank, use a spectrophotometer to
determine the light transmittance of the bacterial suspension at a wavelength of 550 nm.
Adjust the concentration of the bacterial suspension, so that the light transmittance is 60% ~
80%, and use it within 1 hour.
12.2 Preparation and Extraction of Specimens
Solid specimens, for example, cereals, need to be crushed, ground and sieved (the sieve plate
has an aperture of 0.3 mm ~ 0.5 mm); for specimens such as meat and meat products, use a
homogenizer to make them into chyme; specimens such as fruits and vegetables need to be
homogenized and evenly mixed; for liquid specimens, shake and mix them before
determination. Specimens are prepared right before use.
Accurately weigh-take an appropriate amount of specimen, preferably containing 0.5 mg ~
2.0 mg of inositol. Generally, for foods with a relatively high inositol content, such as. fresh
fruits and vegetables, offal and raw meat, weigh-take 1 g (accurate to 0.001 g); for foods
with a relatively low inositol content, such as. cereals and beans, weigh-take 5 g (accurate to
0.001 g); for general nutrient supplements and compound nutritional fortifiers, weigh-take
0.1 g ~ 0.5 g (accurate to 0.001 g); for liquid beverages or liquid or semi-liquid specimens,
weigh-take 5 g ~ 10 g (accurate to 0.001 g), and place it in a 250 mL conical flask. For solid
specimens, add 80 mL of hydrochloric acid solution (0.44 mol/L); for liquid or semi-solid
specimen...