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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 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, and NY/T 91-1988 Determination of Erucic Acid in the Oil of Rapeseed — Gas Chromatographic Method.
The following changes have been made with respect to GB/T 5009.168-2003.
— The title is revised as "National Food Safety Standard — Determination of Fatty Acid in Foods";
— Internal standard method and normalization method are added;
— Chromatographic column of the original standard is modified: glass column is changed to capillary chromatographic column.
National Food Safety Standard —
Determination of Fatty Acid in Foods
1 Scope
This standard specifies the methods for determination of fatty acid in foods.
This standard is applicable to the determination of total fat, saturated fat (acid) and unsaturated fat (acid) in foods.
In this standard, hydrolysis-extraction method is applicable to the determination of fatty acid content in foods; trans-esterification method is applicable to the determination of fatty acid content of grease sample with free fatty acid content not greater than 2%; acetyl chloride-methanol method is applicable to the determination of fatty acid content of milk powder and anhydrous butter sample with moisture content less than 5%.
Method I Internal Standard Method
2 Principle
2.1 Hydrolysis-extraction method: after hydrolysis of specimen with internal standard substance, fat in the specimen is extracted by diethyl ether solution, and then saponification and methyl esterification can be carried out under alkaline conditions to produce fatty acid methyl ester; after gas chromatography with capillary column, the quantitative determination of fatty acid methyl ester content may be carried out by internal standard method. The content of total fat, saturated fat (acid), monounsaturated fat (acid) and polyunsaturated fat (acid) can be calculated according to the content of all kinds of fatty acid methyl ester and conversion factor.
Fat extraction may be omitted for specimen of animal and vegetable fats and oils, instead saponification and methyl esterification of fatty acid may be directly carried out with internal standard substance.
2.2 Trans-esterification method (applicable to grease with free fatty acid content not greater than 2%): dissolve grease in isooctane, and add internal standard substance; add potassium hydroxide and methanol solution for methyl esterification via trans-esterification; for the purpose of avoiding saponification of methyl ester, neutralize the residual potassium hydroxide with sodium hydrogen sulfate after complete reaction.
3 Reagents and materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 1 water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Ammonia water (NH3·H2O).
3.1.3 Pyrogallic acid (C6H6O3).
3.1.4 Diethyl ether (C4H10O).
3.1.5 Petroleum ether: boiling range of 30℃ to 60℃.
3.1.6 Ethyl alcohol (C2H6O) (95%).
3.1.7 Methanol (CH3OH): chromatographically pure.
3.1.8 Sodium hydroxide (NaOH ).
3.1.9 Normal heptane [CH3(CH2)5CH3]: chromatographically pure.
3.1.10 Boron trifluoride - methanol solution, concentration of 15%.
3.1.11 Anhydrous sodium sulfate (Na2SO4).
3.1.12 Sodium chloride (NaCl).
3.1.13 Isooctane [(CH3)2CHCH2C(CH3)3]: chromatographically pure.
3.1.14 Sodium hydrogen sulfate (NaHSO4).
3.1.15 Potassium hydroxide (KOH).
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (8.3mol/L): measure 250mL of hydrochloric acid and dilute with 110mL water, mix well; its storage life at room temperature is 2 months.
3.2.2 Ethyl ether-petroleum ether mixed solution (1+1): take equivalent diethyl ether and petroleum ether, mix well for use.
3.2.3 Sodium hydroxide-methanol solution (2%): take 2g sodium hydroxide and dissolve in 100 mL methanol, mix well.
3.2.4 Saturated sodium chloride solution: weigh 360g sodium chloride and dissolve in 1.0 L water by stirring, take clear solution for use.
3.2.5 Potassium hydroxide-methanol solution (2mol/L): dissolve 13.1g potassium hydroxide in 100mL absolute methanol, slight heating is allowable; add anhydrous sodium sulfate, dry and filter, take clear solution for use.
3.3 Standards
3.3.1 Triundecanoin (C36H68O6, CAS No.: 13552-80-2).
3.3.2 Mixed fatty acid methyl ester standard.
3.3.3 Individual fatty acid methyl ester standard: see Annex A.
3.4 Preparation of standard solution
3.4.1 Triundecanoin internal standard solution (5.00mg/mL): accurately weigh 2.5g (to the nearest 0.1mg) triundecanoin in a beaker and dissolve it with methanol, transfer it into a 500mL volumetric flask and scale the volume with methanol; it may be preserved in refrigerator for a month.
3.4.2 Mixed fatty acid methyl ester standard solution: take moderate volume of mixed fatty acid methyl ester standard solution and transfer it into a 10mL volumetric flask, dilute and scale the volume with normal heptane; it may be preserved for 3 months in refrigerator of -10℃ or below.
3.4.3 Individual fatty acid methyl ester standard solution: take individual fatty acid methyl ester respectively from ampoule bottles and transfer them to 10mL volumetric flasks, wash the ampoule bottles with normal heptane; scale the volume with normal heptane, individual standard solutions of different fatty acid methyl ester are obtained respectively; they may be preserved for 3 months in refrigerator of -10℃ or below.
4 Apparatus
4.1 Refiner or tissue pulverizer or grinder for laboratory use.
4.2 Gas chromatograph: equipped with hydrogen flame ionization detector (FID).
4.3 Capillary chromatographic column: cyanopropyl phenyl polysiloxane with strong polar stationary phase, 100m in column length, 0.25mm in inner diameter and 0.2μm in film thickness.
4.4 Thermostatic water bath: temperature control range 40℃ to 100℃; temperature control ±1℃.
4.5 Analytical balance: with sensitivity of 0.1mg.
4.6 Rotary evaporator.
5 Analysis procedure
5.1 Specimen preparation
Specimen shall be avoided from contamination during the sampling and preparation. Solid or semi-solid specimen shall be pulverized with tissue pulverizer or grinder; liquid specimen is made into homogenate with refiner and preserved by freezing at -18℃ or below, and used after defrosting during analysis.
5.2 Pretreatment of specimen
5.2.1 Hydrolysis-extraction method
5.2.1.1 Weighing of specimen
Weigh uniform specimen 0.1g to 10g (to the nearest 0.1mg, fat content about 100mg to 200mg) and transfer into a 250mL florence flask, accurately add 2.0mL triundecanoin internal standard solution. Add approximately 100mg pyrogallic acid and several zeolite pellets, and then add 2mL ethyl alcohol (95%) and 4mL water, mix well. Select corresponding hydrolysis method according to the type of specimen: alkali hydrolysis for milk products; acid and alkali hydrolysis for cheese; method as specified in 5.2.1.4 for animal and vegetable fats and oils; acid hydrolysis for other foods.
Note: internal standard shall be selected according to the requirements of practical work; internal standard substance shall not be added for the first determination of the specimen with uncertain components. Observe whether there is interference peak present at the peak position of internal standard substance, and, if any, select successively tritridecanoin or trinonadecanoin or tridocosahexaenoin served as internal standard.
5.2.1.2 Hydrolysis of the specimen
Acid hydrolysis: foods (except milk products and cheese): add 10 mL hydrochloric acid solution and mix well. Place the flask into water bath at 70℃ to 80℃ where hydrolysis lasts for 40 min. Shake contents of flask every 10 min to incorporate particulates adhering to sides of flask into solution. After hydrolysis, remove flask from bath and allow to cool to room temperature.
Alkali hydrolysis: milk products (milk powder, liquid milk and other specimens): add 5mL ammonia water and mix well. Place the flask into water bath at 70℃ to 80℃ where hydrolysis lasts for 20 min. Shake contents of flask every 5 min to incorporate particulates adhering to sides of flask into solution. After hydrolysis, remove flask from bath and allow to cool to room temperature.
Acid and alkali hydrolysis: cheese: add 5mL ammonia water, mix well. Place the flask into water bath at 70℃ to 80℃ where hydrolysis lasts for 20 min. Shake contents of flask every 10 min to incorporate particulates adhering to sides of flask into solution. Subsequently, add 10mL hydrochloric acid and continue hydrolysis for 20 min; shake contents of flask every 10 min to incorporate particulates adhering to sides of flask into solution. After hydrolysis, remove flask from bath and allow to cool to room temperature.
5.2.1.3 Fat extraction
After hydrolysis, add 10mL ethyl alcohol (95%) into the specimen, mix well. Transfer the hydrolysate of flask to a separating funnel, wash the flask and stopper with 50mL ethyl ether-petroleum ether mixed solution, collect washing fluid and transfer to the funnel and cover it. Shake 5 min and keep it still for 10 min. Collect extracting solution of ether layer into a 250 mL flask. Extract hydrolysate 3 times according to aforesaid procedures, finally, wash the separating funnel with ethyl ether-petroleum ether mixed solution and collect into the flask. Concentrate it with rotary evaporator to dry, the residues are fat extract.
