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GB 5009.270-2016 English PDF (GB5009.270-2016)

GB 5009.270-2016 English PDF (GB5009.270-2016)

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GB 5009.270-2016: National Food Safety Standard -- Determination of Inositol in Foods

This Standard specifies the method of determining inositol in foods. Method I in this Standard is applicable to the determination of inositol in foods; Method II in this Standard is applicable to the determination of inositol in modulated dairy products and beverages.
GB 5009.270-2016
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Inositol in Foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
PEOPLE Republic of China;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
Method I Microbiological Method ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Equipment ... 6
5 Analytical Procedures ... 6
6 Expression of Analytical Result ... 9
7 Precision ... 10
8 Others ... 10
Method II Gas Chromatography ... 10
9 Principle ... 10
10 Reagents and Materials ... 10
11 Instruments and Equipment ... 11
12 Analytical Procedures ... 12
13 Expression of Analytical Result ... 13
14 Precision ... 14
15 Others ... 14
Appendix A Culture Medium and Reagent ... 15
Appendix B Gas Chromatogram of Inositol Standard Derivative ... 17
National Food Safety Standard -
Determination of Inositol in Foods
1 Scope
This Standard specifies the method of determining inositol in foods.
Method I in this Standard is applicable to the determination of inositol in foods; Method II in this Standard is applicable to the determination of inositol in modulated dairy products and beverages.
Method I -- Microbiological Method
2 Principle
Utilize saccharomyces uvarum?€?s specificity and sensitivity to inositol to quantitatively determine the content of substances to be tested in the sample. In the culture medium that contains all the nutritional ingredients other than the substances to be tested, the growth of microorganism and the content of substances to be tested are in a linear relation. Compare in accordance with transmittance and standard working curve, then, the content of substances to be tested in the sample can be calculated. 3 Reagents and Materials
Unless it is otherwise stipulated, reagents that are used in this method are all analytical purity; use Grade-2 water stipulated in GB/T 6682.
3.1 Reagents
3.1.1 Sodium chloride (NaCl).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Phosphorus pentoxide (P2O5).
3.1.5 Saccharomyces uvarum, ATCC 9080, or other equivalent standard bacterial strains.
3.2 Reagent Preparation
3.2.1 Sodium chloride solution (9 g/L). weigh-take 9.0 g of sodium chloride, then, dissolve it in 1,000 mL of water. Divide it into test tubes; each test tube shall have 10 mL. Start 121 ??C sterilization for 15 min.
3.2.2 Hydrochloric acid solution (1 mol/L). measure-take 82 mL of hydrochloric acid, cool it down, then, dilute to the constant volume of 1,000 mL.
3.2.3 Hydrochloric acid solution (0.44 mol/L). measure-take 36.6 mL of hydrochloric acid, cool it down, then, dilute to the constant volume of 1,000 mL.
3.2.4 Sodium hydroxide solution (600 g/L). weigh-take 300 g of sodium hydroxide, dissolve it in water; cool it down, then, dilute to the constant volume of 500 mL. 3.2.5 Sodium hydroxide solution (1 mol/L). weigh-take 40 g of sodium hydroxide, dissolve it in water; cool it down, then, dilute to the constant volume of 1,000 mL. 3.3 Standards
Inositol standards (C6H12O6). purity ??? 99.9%, or standard substances that are nationally authenticated and awarded the certificate of standard substance.
3.4 Preparation of Standard Solution
3.4.1 Inositol standard stock solution (0.2 mg/mL). place inositol standards in a dryer of phosphorus pentoxide, then, start drying for over 24 h. Weigh-take 50 mg of inositol standards (accurate to 0.1 mg); use water to thoroughly dissolve it, then, dilute to constant volume in a 250 mL brown volumetric flask. Store it in the refrigerator at 4 ??C. 3.4.2 Inositol standard intermediate solution (10 ??g/mL). absorb-take 5.00 mL of inositol standard stock solution, then, use water to dilute in a 100 mL brown volumetric flask. Store it in the refrigerator at 4 ??C.
3.4.3 Inositol standard working solution (1 ??g/mL and 2 ??g/mL). absorb-take 10 mL of inositol standard intermediate solution twice. Respectively use water to dilute in a 100 mL volumetric flask and a 50 mL volumetric flask. This working solution needs to be prepared every time before usage.
3.5 Materials
3.5.1 Culture medium
3.5.1.1 Malt Extract Agar. it can be prepared in accordance with Appendix A. 3.5.1.2 Culture medium for inositol determination. it can be prepared in accordance with Appendix A.
NOTE. some commercial synthetic culture mediums have satisfying effect, commercial synthetic culture mediums shall be prepared in accordance with the instruction on Extract Agar slant culture medium. Culture it at 30 ??C ?? 1 ??C for 20 h ~ 24 h. Use inoculating loop to scrape bacterial lawn to a test tube that holds sodium chloride solution (9 g/L). Start centrifugation at 2,000 r/min for 15 min; rinse for 3 times ~ 4 times. Absorb-take a certain amount of the bacterial solution, then, transfer it into a test tube that holds 10 mL of sodium chloride solution (9 g/L); prepare inoculum suspension. Use spectrophotometer; take sodium chloride solution as the blank solution; determine the transmittance of the inoculum suspension at the wavelength of 550 nm. Adjust the amount of bacterial solution, or add a certain amount of sodium chloride solution, so that the transmittance of the inoculum suspension can be at 60% ~ 80%.
5.2 Sample Preparation
In terms of sample like cereals and milk powder, grind it, then, sieve it (sieve plate aperture. 0.3 mm ~ 0.5 mm). In terms of sample like meat and meat products, use a grinder to make it into chime. In terms of sample like fruits and vegetables, homogenize it, mix it up. In terms of liquid sample, shake it up before usage. Store in the refrigerator at 4 ??C. Determine within 1 week.
5.3 Sample Extraction
Accurately weigh-take 0.5 mg ~ 2.0 mg of inositol sample. General milk powder, fresh fruits and vegetables, internal organs and raw meat sample shall take 1 g (accurate to 0.01 g); cereals, beans and natural food with a low content shall take 5 g (accurate to 0.01 g); general nutrient supplements and compound nutrition enhancers shall take 0.1 g ~ 0.5 g; liquid beverages or fluid and semi-fluid sample shall take 5 g ~ 10 g. Place the sample into a 250 mL conical flask. In terms of dry powder sample, add 80 mL of hydrochloric acid solution (0.44 mol/L); in terms of liquid sample, add 100 mL of hydrochloric acid solution (0.44 mol/L), then, mix it up.
Cover the conical flask with aluminum foil. Start hydrolysis at 125 ??C in an autoclave for 1 h. Take it out, then, cool it down to room temperature. Add around 2 mL of sodium hydroxide solution (600 g/L), then, cool it down. Use sodium hydroxide solution (1 mol/L) or hydrochloric acid solution (1 mol/L) to adjust pH value to 5.2; transfer it into a 250 mL volumetric flask, then, dilute to the constant volume. Mix it up, filter it, then, collect the filtrate. This filtrate shall be considered as the solution to be tested. Adjust the extent of dilution, so that the concentration of inositol in the solution to be tested is within the range of 1 ??g/mL ~ 10 ??g/mL.
5.4 Standard Curve Drawing
In accordance with the sequence in Table 1, add water, inositol standard working solution and the culture medium for inositol determination into culture tubes in triplicate. Cx---average value that is calculated in 5.8.5, expressed in (??g);
m---mass of sample, expressed in (g);
f---dilution factor of test solution;
1,000---conversion coefficient;
100---conversion coefficient.
The calculation result shall retain 3 significant figures.
7 Precision
In terms of natural food. the absolute difference of two independent determination results that are obtained under repeatability conditions shall not exceed 15% of arithmetic mean value.
In terms of fortified food. the absolute difference of two independent determination results that are obtained under repeatability conditions shall not exceed 8% of arithmetic mean value.
8 Others
In terms of natural food. when the sampling size is 5 g, the detection limit of this method is 2.5 mg/ 100 g; the limit of quantitation is 5 mg/100 g. In terms of fortified food. when the sampling size is 1 g, the detection limit of this method is 12.5 mg/ 100 g; the limit of quantitation is 25 mg/100 g.
Method II -- Gas Chromatography
9 Principle
After using water and ethanol to extract inositol in the sample, derive with silanization reagent; extract with hexane. Separate with gas chromatography; quantify with the external standard method.
10 Reagents and Materials
Unless it is otherwise stipulated, reagents that are used in this method are all analytical purity; use Grade-1 water stipulated in GB/T 6682.
10.1 Reagents
12 Analytical Procedures
12.1 Sample Processing and Derivation
12.1.1 Sample processing. grind solid and powder sample, then, mix it up. Weigh-take 1 g (accurate to 0.1 mg), place it in a 50 mL volumetric flask; add 12 mL of lukewarm water at 40 ??C to dissolve the sample; directly weigh-take 12 g (accurate to 0.1 mg) of liquid sample, then, place it in a 50 mL volumetric flask. Conduct ultrasonic extraction of the above sample for 10 min; use 95% ethanol to dilute to the constant volume, then, mix it up. After placing it still for 20 min, take 10 mL and place in a 15 mL centrifuge tube; start centrifugation at not lower than 4,000 r/min for 5 min. Take 5 mL of the supernatant and place it in a rotary evaporation concentration bottle.
12.1.2 Drying and derivation. add 10 mL of absolute ethanol to the concentration bottle. At 80 ??C ?? 5 ??C, rotate and concentrate it till it reaches near dry, then, add 5 mL of absolute ethanol to continue the concentration, till it reaches dry. Transfer the concentration bottle to the oven, start drying at 100 ??C ?? 5 ??C for 1 h. Add 10.0 mL of N, N-dimethylformamide; start ultrasonic dissolution for 5 min, then, transfer it into a 25 mL centrifuge tube that has a screw cap. Add 3.0 mL of silanization reagent and place it in water bath at 80 ??C ?? 5 ??C for 75 min of derivative reaction. During this period, take it out every 20 min for oscillation, then, take it out, cool down to the room temperature. Add 5 mL of hexane; after oscillating mixing, keep it still for stratification. Take 3 mL of the supernatant; add a little anhydrous sodium sulfate to the centrifuge tube in advance; place the supernatant into the centrifuge tube that has a screw cap. After oscillation, start centrifugation at not lower than 4,000 r/min. Take it as the test solution.
12.2 Preparation of Inositol Standard Test Solution
Respectively absorb 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL and 10.0 mL of inositol standard solution; place it into a concentration bottle. Operate in accordance with the steps in 12.1.2.
12.3 Determination
12.3.1 Reference chromatographic conditions
Please see reference chromatographic conditions as follows.
a) Chromatographic column. filling is 50% cyanopropyl-methyl polysiloxane capillary column. Column length. 60 m; internal diameter. 0.25 mm; film thickness. 0.25 ??m; or chromatographic column that has equivalent
performance;
b) Inlet temperature. 280 ??C;

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