GB/T 5413.27-1997 Determination of DHA and EPA in infant formula and milk powder

This standard specifies the method for determining DHA and EPA by gas chromatography. This standard is applicable to the determination of DHA and EPA content in infant formula and milk powder. GB/T 5413.27-1997 Determination of DHA and EPA in infant formula and milk powder GB/T5413.27-1997 Standard download decompression password: www.bzxz.net

GB/T5413.27—1997wwW.bzxz.Net
Adding DHA and EPA to infant formula is a scientific and technological achievement in the past two years, and the detection method of its content is still in the exploratory stage. The method given in this standard is determined by repeated experiments and verification based on a large number of domestic and foreign literatures, and has the characteristics of simple operation, rapidity and high sensitivity.
This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry Federation.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. Participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestlé (China) Investment Services Co., Ltd. The main drafters of this standard: Liu Bo, Yang Jinbao, Wang Yun. 330
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of DHA and EPA
Milk powder and formula foods for infant and young children-Determination of DHA and EPA Contents1 Scope
This standard specifies the method for determining DHA and EPA by gas chromatography. This standard applies to the determination of DHA and EPA in infant formula foods and milk powder. 2 Method Summary
GB/T 5413.27—1997
Replaces GB 5413—85
Extract the fat in the sample, and after methylation reaction, use gas chromatography and flame ionization detector to quantitatively determine the content of DHA and EPA in the sample.
3 Reagents
All reagents, if no specifications are specified, are of analytical grade; all experimental water, if no other requirements are specified, is grade tertiary water. 3.1 Taka-Diastase. 3.2 Ammonia: volume fraction ≤ 25%.
3.3 Ethanol: volume fraction ≥ 95%.
3.4 ​​Ether,
3.5 Petroleum ether: boiling range, 30-60°C.
3.6 N-hexane: chromatographic grade.
3.7 Methanol solution of potassium hydroxide: c(KOH) is 4 mol/L, use anhydrous methanol. 3.8 Standard solution
3.8.1 Standard stock solution: accurately weigh 1.00 g of standard fish oil (EPA 163.9 mg/g, DHA 135.30 mg/g), dissolve it in n-hexane and make up to 50 mL (EPA concentration is 3.2678 mg/ml, DHA concentration is 2.7060 mg/mL), and store in a freezer. 3.8.2 Standard working solution: Take 1.00 mL of the standard stock solution, dilute it with n-hexane and make it up to 10 mL (the concentration of EPA is 0.3268 mg/mL, and the concentration of DHA is 0.2706 mg/mL). Prepare it before use to prevent the concentration from changing due to the evaporation of the solvent. 4
Instruments and glassware
Common laboratory instruments and:
4.1 Mao's liposuction flask.
4.2 Centrifuge.
4.3 Water bath.
4.4 Shaker.
4.5 Gas chromatograph.
Approved by the State Administration of Technical Supervision on May 28, 1997 and implemented on September 1, 1998
4.6 Flame ionization detector.
GB/T 5413. 27-—1997
4.7 Chromatographic column: 2m, 10% EGSS, GAS-CHROMQM/100～200 mesh, stainless steel column. 5 Operation steps
5.1 Sample treatment
5.1.1 Starch-containing sample
Accurately weigh 1.0000g sample and put it into a Mao's liposuction bottle, add 0.1g Taka amylase, and then add 10mL45~50℃ distilled water. After mixing evenly, use nitrogen to remove the air in the bottle, cover the bottle stopper, place it in a 45℃ oven for 30min, take it out and cool it to room temperature. 5.1.2 Starch-free sample
Accurately weigh 1.0000g sample. Put it into a Mao's liposuction bottle (4.1), dissolve it with 10mL65℃ hot water, shake it to make the sample completely dispersed, and cool it to room temperature.
5.2 Preparation of test solution
5.2.1 Add 2 mL of 25% ammonia water (3.2) to the above sample solution, place in a 65℃ water bath (3.3) for 15 min, take out, shake gently, and cool to room temperature.
5.2.2 Add 10 mL of ethanol (3.3) and mix well. Add 25 mL of ether (3.4), plug the stopper and shake for 1 min. Add 25 mL of petroleum ether (3.5), plug the stopper and shake for 1 min. Add another 25 mL of petroleum ether, plug the stopper and shake for 1 min. Centrifuge or let stand to separate the ether layer and the water layer. Transfer the organic layer to a flask. The reagents added for the second extraction are 5 mL of ethanol, 15 mL of ether, and 15 mL of petroleum ether, and the operation is the same as before. For the third extraction, no ethanol is added, only 15 mL of ether and 15 mL of petroleum ether are used, and the operation is the same as above. 5.2.3 Combine the extracted ether solution, concentrate to near dryness under reduced pressure, dissolve the residue with n-hexane and make up to 10 mL, shake well. Accurately pipette 2 mL of this solution into a 10 mL centrifuge tube with a cap, add 0.2 mL of potassium hydroxide methanol solution (3.7), shake for more than 1 minute, and leave for 10 minutes (methylation reaction). If the organic layer is turbid, centrifuge it to clarify it. This organic layer is the sample solution to be tested. 5.3 Determination
5.3.1 Determination conditions
a) Injector temperature: 230℃;
b) Detector FID temperature: 250℃;
c) Injection volume. 2.0μL;
d) Sensitivity: 10\,
e) Attenuation: S;
f) Nitrogen flow rate: 50mL/min, Hydrogen flow rate: 25mL/min; Air flow rate: 300mL/min; g) Column temperature: 190℃.
5.3.2 Qualitative determination
Qualitative determination based on retention time. Change the chromatographic conditions, such as column temperature and carrier gas flow rate, to change the retention time of the standard components EPA and DHA. If the retention time of a peak in the sample changes in the same way as that of a peak in the standard, it proves that the sample contains the same component as the standard.
5.3.3 Quantitative determination
External standard quantitative method.
Inject a certain amount of standard working solution into the gas chromatograph to obtain the peak area A of EPA and DHA; inject an equal volume of the sample solution into the chromatograph to obtain the peak area B of EPA and DHA in the sample. 6 Expression of analytical results
The content of EPA or DHA in the sample (mg/100g) = Where: B-the corresponding area of ​​component i (EPA or DHA) in the sample; 332
GB/T 5413.27--1997
-the mass concentration of component i in the standard sample, mg/mL; V-the total volume of the sample to be tested, mL;
A, the peak area of ​​component i in the standard working solution; m-the mass of the sample·g.
Allowable difference
The difference between the two measured values ​​of the same sample shall not exceed 10% of the average value of the two measurements. 333
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