GB 5009.272-2016 PDF in English
GB 5009.272-2016 (GB5009.272-2016) PDF English
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Determination of phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine of soybean phospholipids
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Standards related to (historical): GB 5009.272-2016
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GB 5009.272-2016: PDF in English GB 5009.272-2016
NATIONAL STANDARD OF THE
PEOPLE’S 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 μm 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 μm, 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 μL;
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);
ρ -- 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);
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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