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GB 5009.272-2016 English PDF (GB5009.272-2016)

GB 5009.272-2016 English PDF (GB5009.272-2016)

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GB 5009.272-2016: Determination of phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine of soybean phospholipids

This Standard specifies the method for determination of the content of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in soybean lecithin, soybean oil, rapeseed oil, peanut oil and sunflower oil by high performance liquid chromatography. This Standard applies to the determination of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in oily soybean lecithin, de-oiled soybean lecithin, soybean oil, rapeseed oil, peanut oil and sunflower oil. This Standard does not apply to the determination of soy lysophosphatidylcholine and soybean lysophosphatidylethanolamine.
GB 5009.272-2016
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National Food Safety Standard - Determination of
Phosphatidylcholine, Phosphatidylethanolamine and
Phosphatidylinositol in Foods
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the PRC;
State Food and Drug Administration
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 5
5 Analysis steps ... 6
6 Description of the analysis result ... 7
7 Precision ... 8
8 Others ... 8
Appendix A Liquid chromatogram of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol standard solution ... 9
National Food Safety Standard - Determination of
Phosphatidylcholine, Phosphatidylethanolamine and
Phosphatidylinositol
1 Scope
This Standard specifies the method for determination of the content of
phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in soybean lecithin, soybean oil, rapeseed oil, peanut oil and sunflower oil by high performance liquid chromatography.
This Standard applies to the determination of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in oily soybean lecithin, de-oiled soybean lecithin, soybean oil, rapeseed oil, peanut oil and sunflower oil. This Standard does not apply to the determination of soy lysophosphatidylcholine and soybean lysophosphatidylethanolamine.
2 Principle
The sample is directly dissolved or extracted by trichloromethane, and purified by amino solid phase extraction column, separated by high performance liquid chromatography, detected by ultraviolet detector, and quantified by external standard method.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, the water is grade-1 water specified by GB/T 6682.
3.1 Reagents
3.1.1 n-hexane [CH3(CH2)4CH3]: chromatographic pure.
3.1.2 Isopropanol [(CH3)2CHOH]: chromatographic pure.
3.1.3 Acetic acid (CH3COOH): chromatographic pure.
3.1.4 Trichloromethane (CHC13).
3.1.5 Diethyl ether (CH3CH2OCH2CH3).
4.2 Analytical balance: sensitivity of 1 mg and 0.1 mg.
4.3 stoppered test tube: 100 mL.
4.4 Vortex oscillator.
4.5 Rotary evaporator: rotating speed of 10 r/min ~ 120 r/min.
4.6 Centrifuge: with the speed larger than 8 000 r/min.
4.7 Nitrogen concentrator.
5 Analysis steps
5.1 Sample preparation
5.1.1 Preparation and pretreatment of soybean lecithin samples
The sample shall be protected from light in a closed and moisture-proof container. The sample shall be thoroughly mixed before use.
According to the content of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the sample, weigh 15 mg ~ 50 mg (accurate to 0.1mg) of sample; use the mixed solution of n-hexane-isopropanol-acetic acid aqueous solution to dissolve and fix-volume to 5 mL. Filter through a 0.45 ??m microporous membrane. Store at a temperature below -16??C after sealing.
5.1.2 Preparation and pretreatment of oil sample
For the sample, separate the sample according to GB/T 5524; take 50 g and place in a sample bottle for use.
Accurately weigh 4 g of the oil sample (accurate to 1 mg); place it in a 100 mL stoppered test tube; add 50.0 mL of trichloromethane; rotate and mix. Firstly, use 1.0 mL of trichloromethane to activate the amino solid phase extraction column; transfer 10.0 mL of the oil trichloromethane solution to the amino silica solid phase extraction column; then, sequentially use 2.0 mL of mixed solution of trichloromethane-isopropyl alcohol and 3.0 mL of mixed solution of acetic acid-diethyl ether to rinse the column; then, use 3.0 mL of methanol to elute the phospholipid; repeat it for 4 times more; collect the eluate. Use the rotary evaporator to evaporate the eluate to near dryness at 45??C; then transfer to nitrogen blowing; after drying, add 10.0 mL of mixed solution of n-hexane-isopropanol-acetic acid aqueous solution; centrifuge at 4 000 r/min for 5 min; take the supernatant for liquid chromatography analysis.
5.2 Apparatus reference conditions
5.2.1 Chromatographic column: silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 ??m, or equivalent columns; 5.2.2 Mobile phase: mixed solution of n-hexane-isopropanol-acetic acid aqueous solution (8+8+1).
5.2.3 Detection wavelength: 205 nm.
5.2.4 Flow velocity: 1 mL/min.
5.2.5 Column temperature: 30??C.
5.2.6 Injection volume: 10 ??L;
5.3 Preparation of the standard curve
Inject standard series working solution into liquid chromatograph separately to test the corresponding peak areas; use the concentration of standard working solution as the abscissa and the peak area as the ordinate to draw the standard curve. For the liquid chromatogram of phosphatidylcholine, phosphatidylethanolamine and
phosphatidylinositol, see Figure A.1.
5.4 Determination of sample solution
Inject the sample solution into the liquid chromatography to get the peak areas; get the concentration of phosphatidylcholine, phosphatidylethanolamine and
phosphatidylinositol in the to-be-test solution according to the standard curve. 6 Description of the analysis result
6.1 Calculation of ciguatoxin in the sample
Calculate the content of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the sample according to Formula (1):
Where:
w -- the content of phosphatidylcholine (phosphatidylethanolamine,
phosphatidylinositol) in the sample, in milligrams per gram (mg/g);
?? -- the concentration of the to-be-test component (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol) that is obtained from the standard working curve, in milligrams per milliliter (mg/mL);
V ?€? the volume of the sample solution, in milliliter (mL);

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