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GB 5009.83-2016 English PDF (GB5009.83-2016)

GB 5009.83-2016 English PDF (GB5009.83-2016)

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GB 5009.83-2016: National Food Safety Standard -- Determination of Carotene in Foods

This Standard specifies the method for determination of carotene in foods. This Standard chromatographic condition I is applicable to the determination of carotene and total carotene in foods. The chromatographic condition II is applicable to the determination of carotene in foods.
GB 5009.83-2016
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Carotene in Foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principles ... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 6
5 Analytical procedures ... 6
6 Analysis results expression ... 11
7 Precision ... 13
8 Others ... 13
Appendix A Calibration method for concentration of standard solution ... 15 Appendix B Confirmation of retention time of ??-carotene isomers and
calculation of chromatographic purity of all-E-??-carotene ... 17
Appendix C Liquid chromatogram of carotene ... 19
Appendix D Percentile absorption coefficients of carotene ... 21
Foreword
This Standard replaces GB 5413.35-2010 ?€?National Food Safety Standard - Determination of ??-carotene in foods for infants and young children, milk and milk products?€?, GB/T 5009.83-2003 ?€?Determination of carotene in foods?€?, and NY/T 82.15-1988 ?€?Method for determination of fruit juice - Determination of ??- carotene?€?.
As compared with GB 5413.35-2010, the main changes of this Standard are as follows.
- The standard?€?s name is changed to ?€?National Food Safety Standard -
Determination of Carotene in Foods?€?;
- ADD pretreatment methods for common foods;
- ADD the chromatographic conditions where ??-carotene and ??-carotene
need to be differentiated;
- MODIFY the results expression of carotene.
National Food Safety Standard -
Determination of Carotene in Foods
1 Scope
This Standard specifies the method for determination of carotene in foods. This Standard?€?s chromatographic condition I is applicable to the determination of ??-carotene, ??-carotene, and total carotene in foods. The chromatographic condition II is applicable to the determination of ??-carotene in foods. 2 Principles
The sample is saponified to release the carotene to a free state. After using petroleum ether to extract dichloromethane and dilute, ADOPT reversed phase chromatography to separate and external standard method to quantify.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure. The water is the Grade I water specified in GB/T 6682.
3.1 Reagents
3.1.1 ??-amylase. Enzyme activity???1.5 U/mg.
3.1.2 Papain. Enzyme activity???5 U/mg.
3.1.3 Potassium hydroxide (KOH).
3.1.4 Anhydrous sodium sulfate (Na2SO4).
3.1.5 Ascorbic acid (C6H8O6).
3.1.6 Petroleum ether. The boiling range is 30 ??C~60 ??C.
3.1.7 Methanol (CH4O). chromatographically pure.
3.1.8 Acetonitrile (C2H3N). chromatographically pure.
3.1.9 Chloroform (CHCl3). chromatographically pure.
3.1.10 Methyl tert-butyl ether [CH3OC(CH3)3]. chromatographically pure. 3.1.11 Dichloromethane (CH2Cl2). chromatographically pure.
3.1.12 Anhydrous ethanol (C2H6O). guaranteed reagent.
3.1.13 N-hexane (C6H14). chromatographically pure.
3.1.14 2,6-di-tert-butyl-4-methylphenol (C15H24O, BHT).
3.2 Preparation of reagent
Potassium hydroxide solution. WEIGH 500 g of solid potassium hydroxide; ADD 500 mL of water to dissolve. Prepared before use.
3.3 Standards
3.3.1 ??-carotene (C40H56, CAS number. 7488-99-5). Purity???95%. Or a
reference material which has been certified by the state and awarded a
reference material certificate.
3.3.2 ??-carotene (C40H56, CAS number. 7235-40-7). Purity???95%. Or a reference material which has been certified by the state and awarded a reference material certificate.
3.4 Preparation of standard solutions
3.4.1 Standard stock solution of ??-carotene (500 ??g/mL). Accurately WEIGH 50 mg (accurate to 0.1 mg) of ??-carotene standard; ADD 0.25 g of BHT; USE
dichloromethane to dissolve; TRANSFER to a 100 mL brown volumetric flask and DILUTE to volume. PROTECT it from light at below -20 ??C. The service life shall not exceed 3 months. The standard stock solution, before use, needs to be calibrated. For specific operations, SEE Appendix A.
3.4.2 Standard intermediate solution of ??-carotene (100 ??g/mL). Accurately PIPETTE 10.0 mL of standard stock solution of ??-carotene into a 50 mL brown volumetric flask; and USE dichloromethane to dilute to volume.
3.4.3 Standard stock solution of ??-carotene (500 ??g/mL). Accurately WEIGH 50 mg (accurate to 0.1 mg) of ??-carotene standard; ADD 0.25 g of BHT; USE
dichloromethane to dissolve; TRANSFER to a 100 mL brown volumetric flask and DILUTE to volume. PROTECT it from light at below -20 ??C. The service life shall not exceed 3 months. The standard stock solution, before use, needs to be calibrated. For specific operations, SEE Appendix A.
Note. The ??-carotene standard is mainly all-E-??-carotene. During storage, affected by temperature, oxidation, and other factors, some all-E-??-carotene isomerizes to cis- Liquid sample. WEIGH accurately 5 g~10 g (accurate to 0.001 g); PLACE in a 250 mL conical flask; and ADD 1 g of ascorbic acid.
5.2.2.2 Saponification
TAKE the pretreated sample; ADD 75 mL of anhydrous ethanol, SHAKE well; ADD 25 mL of potassium hydroxide solution; and CAP the bottle stopper.
PLACE in a constant temperature oscillating water bath which has been
preheated to 53 ??C??2 ??C to saponify for 30 min. REMOVE, LET stand, and
COOL to room temperature.
Note. If saponification is not complete, the saponification time may be appropriately extended to 1 h.
5.3 Sample extraction
TRANSFER the saponification solution to a 500 mL separatory funnel; ADD 100 mL of petroleum ether, gently SHAKE, EXHAUST, and CAP the stopper. After oscillating at room temperature for 10 min, LET it stand for stratification; TRANSFER the aqueous phase to another separatory funnel, to perform
second extraction according to the above method. COMBINE the organic
phases; and USE water to wash to near-neutral. DISCARD the aqueous phase; and the organic phase is dehydrated by filtration over anhydrous sodium sulfate. The filtrate is collected in a 500 mL evaporating flask and concentrated under reduced pressure at 40 ??C??2 ??C on a rotary evaporator until nearly dry. USE nitrogen to blow-dry; USE a pipette to accurately add 5.0 mL of
dichloromethane; CAP the stopper to fully dissolve the extract. After filtering it through a 0.45 ??m membrane and discarding approximately 1 mL of the initial filtrate, COLLECT in a sample injection bottle for use.
Note. If necessary, to concentrate or dilute according to the carotene content in the sample solution to be determined, so that the concentration of ??-carotene and/or ??-carotene in the sample solution to be determined is in the range of 0.5 ??g/mL~10 ??g/mL. 5.4 Chromatographic determination
5.4.1 Chromatographic condition I (applicable to the determination of ??- carotene, ??-carotene, and total carotene in foods)
5.4.1.1 Reference chromatographic conditions
Reference chromatographic conditions are listed below.
a) Chromatographic column. C30 column; the column length is 150 mm; the inner diameter is 4.6 mm; the particle size is 5 ??m; or equivalent column; ?? - Calibration concentration of ??-carotene standard working solution, in micrograms per milliliter (??g/mL);
CP - Chromatographic purity of all-E-??-carotene, %.
5.4.1.3 Sample determination
Under the same chromatographic conditions, INJECT the solution to be
determined into liquid chromatograph; qualitative based on the retention time; based on the peak area, USE the external standard method to quantify. The concentration of ??-carotene in the solution to be determined shall be calculated according to the standard curve regression equation. ??-carotene shall be calculated based on the all-E-??-carotene response factor.
5.4.2 Chromatographic condition II (applicable to the determination of ??- carotene in foods)
5.4.2.1 Reference chromatographic conditions
Reference chromatographic conditions are listed below.
a) Chromatographic column. C18 column; the column length is 250 mm; the inner diameter is 4.6 mm; the particle size is 5 ??m; or equivalent column; b) Mobile phase. chloroform. acetonitrile. methanol=3.12.85, containing 0.4 g/L of ascorbic acid, filtered through a 0.45 ??m membrane for use;
c) Flow velocity. 2.0 mL/min;
d) Detection wavelength. 450 nm;
e) Column temperature. 35 ??C??1 ??C;
f) Injection volume. 20 ??L.
5.4.2.2 Making of standard curve
INJECT the standard working solution of ??-carotene into HPLC (for
chromatogram, SEE Figure C.2); qualitative based on the retention time; DETERMINE the peak area. TAKE the concentration of standard series working solution as the abscissa, the peak area as the ordinate, to plot standard curve; and CALCULATE regression equation.
5.4.2.3 Sample determination
Under the same chromatographic conditions, INJECT the solutions to be
determined into liquid chromatograph separately, to perform HPLC analysis; in peak area (AU);
A9Z - The peak area of 9-cis-??-carotene in the sample solution to be determined, in peak area (AU);
A13Z - The peak area of 13-cis-??-carotene in the sample solution to be
determined, in peak area (AU);
1.2 - Relative correction factor of 13-cis-??-carotene;
A15Z - The peak area of 15-cis-??-carotene in the sample solution to be
determined, in peak area (AU);
1.4 - Relative correction factor of 15-cis-??-carotene;
AxZ - The peak area of other cis-??-carotene in the sample solution to be determined, in peak area (AU);
V - Constant volume of sample solution, in milliliters (mL);
100 - The coefficient which expresses the result in micrograms per hundred grams (??g/100 g);
RF - All-E-??-carotene response factor, in peak area milliliters per microgram (AU ?? mL/??g);
m - Sample mass, in grams (g).
Note 1. Due to the different percentile absorption coefficients of the isomers of ??-carotene (SEE Appendix D), in the calculation for ??-carotene, a relative correction factor needs to be used to correct the results.
Note 2. If the content of other cis-??-carotene in the sample is low, the calculation may not be performed.
The total carotene content in the sample shall be calculated according to the equation (4).
Where.
Xtotal - Total carotene content in the sample, in micrograms per hundred grams (??g/100 g);
X?? - The content of ??-carotene in the sample, in micrograms per hundred grams (??g/100 g);
total
Appendix B
Confirmation of retention time of ??-carotene isomers and calculation of chromatographic purity of all-E-??-carotene
Note. When chromatographic condition I is used for the determination of ??-carotene, the retention time of ??-carotene isomers needs to be determined, and the
chromatographic purity of ??-carotene standard solution shall be corrected. B.1 Reagent
Iodine solution (I2). 0.5 mol/L.
B.2 Preparation of reagents
B.2.1 Iodohydrin solution (0.05 mol/L). PIPETTE 5 mL of iodine solution; USE ethanol to dilute to 50 mL, and MIX well.
B.2.2 Isomerized ??-carotene solution. TAKE 10 mL of standard stock solution of ??-carotene in a beaker; ADD 20 ??L of iodohydrin solution; SHAKE well and IRRADIATE it in sunlight or 30 cm away from 40 W fluorescent lamp for 15 min; USE dichloromethane to dilute to 50 mL. SHAKE well and FILTER by a 0.45 ??m membrane for HPLC chromatographic analysis.
B.3 Confirmation of retention time of ??-carotene isomers
Separately TAKE standard intermediate solution of ??-carotene (100 ??g/mL) and isomerized ??-carotene solution; according to chromatographic condition I, INJECT them into HPLC for chromatographic analysis. According to the
chromatogram of standard intermediate solution of ??-carotene, CONFIRM the retention time of all-E-??-carotene. COMPARE the peak area changes in the chromatograms of standard intermediate solution of ??-carotene and isomerized ??-carotene solution, and the positional relationship with all-E-??-carotene, to confirm the retention time of cis-??-carotene isomers. A larger chromatographic peak before all-E-??-carotene is 13-cis-??-carotene; a larger chromatographic peak immediately after all-E-??-carotene is 9-cis-??-carotene; 15-cis-??-carotene is followed by 13-cis-??-carotene; and there may be other smaller cis-structure chromatographic peaks. The chromatogram is shown in Figure C.1.
B.4 Calculation of chromatographic purity of all-E-??-carotene standard
solution
TAKE standard working solution of ??-carotene (3 ??g/mL); according to

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