GB 5009.168-2016National Food Safety Standard — Determination of Fatty Acids in Foods (English PDF)
食品安全国家标准 食品中脂肪酸的测定
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Issued by
National Health and Family Planning Commission / CFDA
Level / Type
National · Mandatory
Issue date
December 23, 2016
Implementation date
June 23, 2017
Scope
GB 5009.168-2016 is the English-translated version of 食品安全国家标准 食品中脂肪酸的测定.
Specifies analytical methods for determining total fats, saturated, monounsaturated, and polyunsaturated fatty acids in food products using capillary column gas chromatography via fatty acid methyl ester derivatization.
Document preview — GB 5009.168-2016
National Standard of the People's Republic of China
Issued by: National Health and Family Planning Commission of the People’s Republic of China; China Food and Drug Administration.
Contents
- Foreword2
- 1 Scope3
- 2 Principle
- 3 Reagents and materials
- 4 Apparatus and equipment
- 5 Analysis procedure
- 6 Expression of analysis results
- 7 Principle
- 8 Reagents and materials
- 9 Apparatus and equipment
- 10 Analysis procedure
- 11 Expression of analysis results
- 12 Principle
- 13 Reagents and materials
- 14 Apparatus and equipment
- 15 Analysis procedure
- 16 Expression of analysis results
- 17 Precision20
- 18 Quantitation limits20
- Annex A Molecular formula and CAS number of single fatty acid methyl ester standards
- Annex B Typical reference chromatogram of 37 kinds of fatty acid methyl ester standard solutions
- Annex C Retention time and relative retention time reference for fatty acid methyl esters
- Annex D Coefficients for conversion between fatty
- Annex E Quantitation limits for fatty acids
Foreword
This Standard replaces GB/T 5009.168-2003 “Determination of eicosapentaenoic acid and docosahexaenoic acid in foods”, GB/T 22223-2008 “Determination of total fat, saturated fat, and unsaturated fat in foods - Hydrolytic Extraction - Gas Chromatography”, GB 5413.27-2010 “National food safety standard - Determination of fatty acids in foods for infants and young children milk and milk products”, GB/T 9695.2- 2008 “Meat and meat products - Determination of fatty acids”, GB/T 17376- 2008 “Animal and vegetable fats and oils - Preparation of methyl esters of fatty acids”, GB/T 17377-2008 “Animal and vegetable fats and oils - Analysis by gas chromatography of methyl esters of fatty acids”, SN/T 2922-2011 “Determination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in foods for export - Gas chromatography”, NY/T 91-1988 “Determination of erucic acid in the oil of rapeseed - Gas chromatographic method”.
Compared with GB/T 5009.168-2003, the main changes of this Standard are as follows:
the standard name is changed to “National food safety standard - Determination of fatty acids in foods”;
- ADD the internal standard method and the normalization method;
1 Scope
This Standard specifies the determination method for the content of fatty acids in foods.
This Standard applies to the determination of total fats, saturated fats (fatty acids) and unsaturated fats (fatty acids) in foods.
In this Standard, the hydrolysis-extraction method applies to the determination of the content of fatty acids in foods; the transesterification method applies to the determination of the content of fatty acids in fat samples with free fatty acid content of not more than 2 %; the acetyl chloride-methanol method applies to the determination of the content of fatty acids in milk powder and anhydrous cream samples with water content less than 5 %.
2 Principle
2.1 Hydrolysis-extraction method: after the fats in the sample that has added with internal standard are extracted with hydrolysis-ether solution, saponify and methylesterify the sample under alkaline conditions to produce fatty acid methyl esters. After analyzed by capillary column gas chromatography, use internal standard method to quantitatively determine the content of fatty acid methyl esters. According to the content and conversion coefficients of various fatty acid methyl esters, calculate the content of total fats, saturated fats (fatty acids), monounsaturated fats (fatty acids) and polyunsaturated fats (fatty acids).
Animal and vegetable fat samples are directly saponified and fatty-acid-methyl esterified after adding with internal standard, without fat extraction.
2.2 Transesterification method (applies to fats with free fatty acid content of not more than 2 %): DISSOLVE fats in isooctane; after adding internal standard, ADD potassium hydroxide methanol solution to make the sample methyl-esterified through transesterification. After the reaction is complete, USE sodium sulfate to neutralize the residual potassium hydroxide to avoid saponification of methyl esters.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytical regents and the water is the Grade 1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Ammonia (NH3 · H2O).
3.1.3 Pyrogallic acid (C6H6O3).
3.1.4 Ether (C4H10O).
3.1.6 Ethanol (C2H6O) (95 %).
3.1.7 Methanol (CH3OH): chromatographically pure.
3.1.9 N-heptane [CH3(CH2)5CH3]: chromatographically pure.
3.1.10 Boron trifluoride methanol solution, with a concentration of 15 %.
3.1.12 Sodium chloride (NaCl).
3.1.13 Isooctane [(CH3)2CHCH2C(CH3)3]: chromatographically pure.
3.1.14 Sodium bisulfate (NaHSO4).
3.1.15 Potassium hydroxide (KOH).
3.2 Preparation of reagents
3.2.1 Hydrochloric acid solution (8.3 mol/L): WEIGH 250 mL of hydrochloric acid; DILUTE with 110 mL of water; MIX well. It can be placed for 2 months at room temperature.
3.2.2 Ether-petroleum ether mixture (1 + 1): TAKE the same volume of ether and petroleum ether; MIX well for further use.
3.2.3 Sodium hydroxide methanol solution (2 %): TAKE 2 g of sodium hydroxide and DISSOLVE in 100 mL of methanol, MIX well.
3.2.4 Saturated sodium chloride solution: WEIGH 360 g of sodium chloride and DISSOLVE in 1.0 L of water; STIR to dissolve; Stand until clear for further use.
3.2.5 Potassium hydroxide methanol solution (2 mol/L): DISSOLVE 13.1 g of potassium hydroxide in 100 mL of anhydrous methanol; it may slightly heat; ADD anhydrous sodium sulfate; DRY; FILTRATE to obtain clear solution.
3.3 Standards
3.3.1 Triundecanoin (C36H68O6, CAS number: 13552-80-2).
4 Apparatus and equipment
4.1 Homogenizer or laboratory tissue pulverizer or grinder.
4.2 Gas chromatograph: with a hydrogen flame ion detector (FID).
4.3 Capillary chromatographic column: with polydipyldiphenyl siloxane strong polar stationary phase, a column length of 100 m, an inner diameter of 0.25 mm and a film thickness of 0.2 μm.
4.5 Analytical balance: with a division of 0.1 mg.
4.6 Rotary evaporator.
5 Analysis procedure
5.1 Preparation of samples
During sampling and preparation, it shall avoid sample contamination. Solid or semisolid samples are pulverized using a tissue pulverizer or grinder. Liquid samples are homogenized using a homogenizer, stored at -18 °C or below, and used for analysis after thawed.
5.2 Pretreatment of samples
5.2.1 Hydrolysis-extraction method
5.2.1.1 Weighing of samples
WEIGH 0.1 g ~ 10 g (accurate to 0.1 mg, containing about 100 mg ~ 200 mg of fats) of uniform sample into a 250-mL flat-bottom flask; accurately ADD 2.0 mL of triundecanoin internal standard solution. ADD about 100 mg of pyrogallic acid; ADD several zeolites; ADD 2 mL of 95 % ethanol and 4 mL of water; MIX well. According to the type of sample, select appropriate hydrolysis method. For dairy products, use alkali hydrolysis method; for cheeses, use acid and alkali hydrolysis method; for animal and vegetable fats, directly carry out the procedure 5.2.1.4; for the rest of foods, use acid hydrolysis method. NOTE: SELECT internal standard according to the need of actual work. […]
5.2.2 Transesterification method
Applies to fat samples with free fatty acid content of not more than 2 %.
5.3 Determination
5.3.2 Determination of samples
5.3.2.1 Determination of sample solutions
6 Expression of analysis results
The content of single fatty acid methyl ester in the sample is calculated according to equation (1). The response factor of fatty acid methyl ester i is calculated according to equation (2). The content of saturated fats (fatty acids) in the sample is calculated according to equation (3), and the content of single saturated fatty acids in the sample is calculated according to equation (4). XSaturated Fat the content of saturated fats (fatty acids), in grams per gram (g/100 g); The coefficient of conversion from fatty acid methyl esters to fatty acids FFAMEi-FAi is shown in Annex D. The coefficient of conversion from fatty acid methyl ester i to fatty acids is calculated according to equation (5). The content of monounsaturated fats (fatty acids) in the sample is calculated according to equation (6), and the content of each monounsaturated fatty acid methyl ester in the sample is calculated according to equation (7). XMono-Unsaturated Fat the content of monounsaturated fats (fatty acids) in the sample, in grams per gram (g/100 g); […]
7 Principle
7.1 Hydrolysis-extraction method: after the fats in the sample are extracted with hydrolysis-ether solution, saponify and methyl-esterify the sample under alkaline conditions to produce fatty acid methyl esters. After analyzed by capillary column gas chromatography, use external standard method to quantitatively determine the content of fatty acids. Animal and vegetable pure fat samples are directly saponified and fatty-acid-methyl esterified without fat extraction. 7.2 Acetyl chloride-methanol method (applies to milk powder and anhydrous cream samples with water content of less than 5 %): acetyl chloride reacts with methanol to produce hydrochloric acid-methanol and make the fats and free fatty acids methyl esterified. After extracted with toluene, separately tested with gas chromatography, and quantify with external standard method. 7.3 Transesterification method (applies to fats with free fatty acid content of not more than 2 %): DISSOLVE the fat sample in isooctane; ADD potassium hydroxide methanol solution to make the sample methyl-esterified through transesterification. […]
8 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytical regents and the water is the Grade 1 water specified in GB/T 6682.
8.1 Reagents
8.1.2 Ammonia (NH3 · H2O).
8.1.3 Pyrogallic acid (C6H6O3).
8.1.4 Ethyl ether (C4H10O).
8.1.6 Ethanol (C2H6O) (95 %).
8.1.7 Methanol (CH3OH): chromatographically pure.
8.1.8 Sodium hydroxide (NaOH).
8.1.9 N-heptane [CH3(CH2)5CH3]: chromatographically pure.
8.1.10 Boron trifluoride methanol solution: with a concentration of 15 %.
8.1.11 Anhydrous sodium sulfate (Na2SO4).
8.1.14 Toluene (C7H8): chromatographically pure.
8.1.15 Acetyl chloride (C2H3ClO).
8.1.16 Isooctane [(CH3)2CHCH2C(CH3)3]: chromatographically pure.
8.1.17 Sodium bisulfate (NaHSO4).
8.1.18 Potassium hydroxide (KOH).
8.2 Preparation of reagents
8.2.3 Sodium hydroxide methanol solution (2 %): the same as 3.2.3. 8.2.4 Saturated sodium chloride solution: the same as 3.2.4. 8.2.5 Acetyl chloride methanol solution (with a volume fraction of 10 %): MEASURE 40 mL of methanol in a 100-mL dry flask; accurately PIPETTE 5.0 mL of acetyl chloride and slowly add dropwise; KEEP stirring; COOL to room temperature; […]
8.3 Standards
8.3.1 Mixed fatty acid methyl ester standard: the same as 3.3.2.
8.3.2 Single fatty acid methyl ester standard: the same as 3.3.3.
8.4 Preparation of standard solutions
8.4.1 Single fatty acid methyl ester standard solution: the same as 3.4.3.
8.4.2 Fatty acid triglyceride standard working solution: SELECT the corresponding triglyceride standard according to the type of fatty acid to be analyzed in the sample.
9 Apparatus and equipment
9.1 Homogenizer or laboratory tissue pulverizer or grinder.
9.2 Gas chromatograph: with a hydrogen flame ion detector (FID).
9.3 Capillary chromatographic column: with polydipyldiphenyl siloxane strong polar stationary phase, a column length of 100 m, an inner diameter of 0.25 mm and a film thickness of 0.2 μm.
9.4 Constant temperature water bath: with a temperature range of 40 °C ~ 100 °C, temperature control ± 1 °C.
9.5 Analytical balance: with a division of 0.1 mg.
9.7 Rotary evaporator.
9.8 Screw glass tube (with a screw cap with inner lining made of PTFE): 15 mL.
9.9 Centrifuge tube: 50 mL.
10 Analysis procedure
10.1 Preparation of samples
The procedure is the same as 5.1.
10.2 Pretreatment of samples
10.2.1 Hydrolysis-extraction method
10.2.1.1 Weighing of samples
WEIGH 0.1 g ~ 10 g (accurate to 0.1 mg, containing100 mg ~ 200 mg of fat) of uniform sample into a 250-mL flat-bottomed flask; […]
10.2.1.3 Fat extraction
The procedure is the same as 5.2.1.3.
The procedure is the same as 5.2.1.4.
Animal and vegetable fat samples are directly saponified and fatty-acid-methylesterified without fat extraction (the same as 5.2.1.4).
10.2.2 Acetyl chloride-methanol method
10.2.2.1 Weighing of samples
Accurately WEIGH 0.5 g of milk powder sample or 0.2 g of anhydrous cream sample (all accurate to 0.1 mg) in a 15-mL dry screw glass tube; ADD 5.0 mL of toluene.
10.2.2.2 Preparation of test solutions
ADD 6 mL of 10 % acetyl chloride solution to the sample; FILL with nitrogen; SCREW the screw cap. SHAKE to mix well; PLACE at 80 °C ± 1 °C water batch for 2 h; REMOVE to shake once every 20 min; REMOVE and cool to room temperature after water bath. TRANSFER the solution after reaction to a 50-mL centrifuge tube; […]
10.2.3 Transesterification method
10.2.3.1 Weighing of samples
WEIGH 60.0 mg of sample into a test tube with a stopper, accurate to 0.1 mg.
10.2.3.2 Preparation of methyl esters
11 Expression of analysis results
11.1 Content of each fatty acids in samples
QUANTIFY by the peak area of chromatographic peaks. The content of each fatty acids in the sample is calculated according to equation (12).
The content of total fatty acids in the sample is calculated according to equation (13).
XTotal FA the content of total fatty acids in the sample, in grams per gram (g/100 g);
The result retains 3 significant digits.
12 Principle
12.1 Hydrolysis-extraction method: after the fats in the sample are extracted with hydrolysis-ether solution, saponify and methyl-esterify the sample under alkaline conditions to produce fatty acid methyl esters. After analyzed by capillary column gas chromatography, use area normalization method to quantitatively determine the percentage content of fatty acids.
Animal and vegetable pure fat samples are directly saponified and fatty-acid-methyl esterified without fat extraction.
12.2 Transesterification method (applies to fats with free fatty acid content of not more than 2 %): DISSOLVE the fat sample in isooctane; ADD potassium hydroxide methanol solution to make the sample methyl-esterified through transesterification. After the reaction is complete, use sodium sulfate to neutralize the residual potassium hydroxide, and use area normalization method to quantitatively determine the percentage content of fatty acids.
13 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytical regents and the water is the Grade 1 water specified in GB/T 6682.
13.1 Reagents
13.3 Standards
13.3.1 Mixed fatty acid methyl ester standard solution: the same as 3.3.2.
13.3.2 Single fatty acid methyl ester standard: the same as 3.3.3.
13.4 Preparation of standard solutions
Single fatty acid methyl ester standard solution: the same as 3.4.3.
14 Apparatus and equipment
The apparatus and equipment are the same as Clause 4.
15 Analysis procedure
15.1 Preparation of samples
The procedure is the same as 5.1.
15.2 Hydrolysis-extraction method
15.2.1 Weighing of samples
WEIGH 0.1 g ~ 10 g (accurate to 0.1 mg, containing100 mg ~ 200 mg of fat) of uniform sample into a 250-mL flat-bottomed flask; […]
15.2.2 Hydrolysis of samples
The procedure is the same as 5.2.1.2.
15.2.3 Fat extraction
The procedure is the same as 5.2.1.3.
The procedure is the same as 5.2.1.4.
15.3 Transesterification method
15.3.1 Weighing of samples
WEIGH 60.0 mg of sample into a test tube with a stopper, accurate to 0.1 mg.
15.3.2 Preparation of methyl ester
16 Expression of analysis results
The percentage ratio of a fatty acid in the sample to total fatty acids Yi is calculated according to equation (14).
The result retains 3 significant digits.
17 Precision
The absolute difference between the two independent determination results obtained under repeatability conditions shall not exceed 10 % of the arithmetic mean.
18 Quantitation limits
CAS number Molecular formula Abbr. of fatty acid No.
Annex A Molecular formula and CAS number of single fatty acid methyl ester standards
The molecular formula and CAS number of single fatty acid methyl ester standards are shown in Table A.1.
Annex B Typical reference chromatogram of 37 kinds of fatty acid methyl ester standard solutions
The reference chromatogram of 37 kinds of fatty acid methyl ester standard solutions is shown in Figure B.1.
Annex C Retention time and relative retention time reference for fatty acid methyl esters
The retention time and relative retention time reference for fatty acid methyl esters are given in Table C.1.
B A Abbr. of fatty acid No.
C - Cis-10-pentadecenoic acid methyl ester;
D - Cis-8,11,14-Eicosatrienoic acid methyl ester.
E - Cis-5,8,11,14,17-eicosapentaenoic acid methyl ester.
Remaining clauses in the full document
- Annex D Coefficients for conversion between fatty
- Annex E Quantitation limits for fatty acids
......
This preview omits tables, figures, formulas and parts of the technical clauses. The complete document — 30 pages — is available in the English PDF.
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