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GB 5009.265-2016 PDF in English


GB 5009.265-2016 (GB5009.265-2016) PDF English
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GB 5009.265-2016: PDF in English

GB 5009.265-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of
polycyclic aromatic hydrocarbons in foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
Table of Contents
1 Scope ... 3 
2 Principle ... 3 
3 Reagents and materials ... 3 
4 Instruments and equipment... 5 
5 Analytical procedures ... 5 
6 Expression of analytical results ... 9 
7 Precision ... 9 
8 Others ... 9 
9 Principle ... 10 
10 Reagents and materials ... 10 
11 Instruments and equipment ... 10 
12 Analytical procedures ... 10 
13 Expression of analytical results ... 13 
14 Precision ... 13 
15 Others ... 13 
Appendix A Liquid chromatogram of polycyclic aromatic hydrocarbon standard
solution ... 15 
Appendix B Gas chromatography-mass spectrometry total ion chromatogram
of polycyclic aromatic hydrocarbon standard solution ... 16 
National food safety standard - Determination of
polycyclic aromatic hydrocarbons in foods
1 Scope
This standard specifies the liquid chromatography for determining polycyclic
aromatic hydrocarbons in food (naphthalene, acenaphthene, fluorene,
phenanthrene, anthracene, fluoranthene, pyrene, benzo[α]anthracene,
chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[α]pyrene,
indeno[1,2,3-c,d]pyrene, dibenzo[α,h]anthracene and benzo[g,h,i]perylene), as
well as the gas chromatography-mass spectrometry for determining the
polycyclic aromatic hydrocarbons in foods (naphthalene, acenaphthylene,
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene,
benzo[α]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene,
benzo[α]pyrene, indeno[1,2,3-c,d]pyrene, dibenzo[α,h]anthracene and
benzo[g,h,i]perylene).
This standard applies to the determination of polycyclic aromatic hydrocarbons
in foods.
Method I - High-performance liquid chromatography
2 Principle
The polycyclic aromatic hydrocarbons in the specimen are extracted by the use
of an organic solvent. The extract is concentrated to near dryness, dissolved in
a solvent, purified by the use of PSA (N-propylethylenediamine) and C18 solid-
phase extraction filler or by the use of Florisil solid-phase extraction column.
After concentration and making it reach to a certain volume, it is separated by
the high-performance liquid chromatography. The fluorescence intensity of
various polycyclic aromatic hydrocarbons at different excitation wavelengths
and emission wavelengths is determined. It is quantified by external standard
method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical
grade, the water is the grade I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN). Chromatographically pure.
3.1.2 n-Hexane (C6H14). Chromatographically pure.
3.1.3 Dichloromethane (CH2Cl2). Chromatographically pure.
3.1.4 Diatomaceous earth. Chromatographically pure.
3.1.5 Magnesium sulfate (MgSO4). Excellent grade.
3.1.6 N-propylethylenediamine (PSA). Particle size 40 μm.
3.1.7 Tai-sealed C18 solid-phase extraction packing. Particle size 40 μm ~ 63
μm.
3.1.8 Florisil solid-phase extraction column. 500 mg, 3 mL.
3.1.9 Organic phase microporous membrane. 0.22 μm.
3.2 Preparation of reagent
3.2.1 n-hexane-dichloromethane mixed solution (1 + 1). TAKE 500 mL of n-
hexane; ADD 500 mL of dichloromethane; MIX it uniformly.
3.2.2 Acetonitrile-saturated n-hexane. TAKE 800 mL of n-hexane; ADD 200 mL
of acetonitrile; SHAKE to mix it uniformly; LET it be standing for layering. The
upper n-hexane layer is acetonitrile-saturated n-hexane.
3.3 Standard substance
Polycyclic aromatic hydrocarbons (naphthalene, acenaphthylene,
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene,
benzo[α]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene,
benzo[α]pyrene, indeno [1,2,3-c,d]pyrene, dibenzo[α,h]anthracene and
benzo[g,h,i]perylene) certified standard solution (200 μg/mL), which is
preserved at -18 °C.
Warning - Polycyclic aromatic hydrocarbons are known to be
carcinogenic, teratogenic, mutagenic. The carcinogenicity increases with
increasing number of benzene rings. Special attention shall be paid to
safety protection during the determination. The determination shall be
carried out in a fume hood, it shall wear gloves, to minimize exposure.
3.4 Preparation of standard solution
3.4.1 Polycyclic aromatic hydrocarbon standard intermediate solution (1000
ng/mL). PIPETTE 0.5 mL of polycyclic aromatic hydrocarbon standard solution;
USE acetonitrile to make its volume reach to 100 mL. PRESERVE it at -18 °C.
a) ADD 10 mL of n-hexane; VORTEX it for 30 s; then PLACE it in a 40 °C
water bath for ultrasonic for 30 min; CENTRIFUGE it at 4500 r/min for 5
min; TAKE the supernatant in a glass centrifuge tube B; USE 10 mL of n-
hexane to repeat extraction one time for the lower part of the centrifuge
tube A; COMBINE the extract into the centrifuge tube B; PERFORM
nitrogen blowing (temperature control below 35 °C) to remove the reagent;
BLOW it almost dry.
b) In the centrifuge tube B, ADD 4 mL of acetonitrile; VORTEX it for 30 s;
then ADD 900 mg of magnesium sulfate, 100 mg of PSA and 100 mg of
C18 packing; VORTEX it mix it for 30 s; CENTRIFUGE at 4500 r/min for
3 min; TAKE the supernatant into a 10 mL glass graduated centrifuge tube
C; USE 2 mL of acetonitrile to repeat extraction once for the lower part of
the centrifuge tube B; COMBINE the extract into the centrifuge tube C;
PERFORM nitrogen blowing to the reagent to make it reach to about 1 mL;
USE acetonitrile to make its volume reach to about 1 mL; MIX it uniformly;
MAKE it pass the 0.22 μm organic-phase microporous membrane, to
obtain the sample solution to be determined.
5.2.2 Foods such as aquatic products, meat and vegetables
WEIGH 2 g ~ 5 g (accurate to 0.01 g) of specimen in a 50 mL stoppered glass
centrifuge tube A; ADD 1 g ~ 5 g of diatomaceous earth; USE a glass rod to stir
it uniformly; MAKE treatment according to steps a) and b) in 5.2.1 subsequently,
to obtain the sample solution to be determined.
5.2.3 High-fat foods or animal & vegetable oils
WEIGH 1 g ~ 4 g (accurate to 0.01 g) of specimen in a 50 mL stoppered glass
centrifuge tube A; USE the procedures below to make treatment.
a) ADD 20 mL of acetonitrile and 10 mL of acetonitrile-saturated n-hexane;
VORTEX it for 30 s; PLACE it in a 40 °C water bath for ultrasonic for 30
min; after shaking it uniformly, FREEZE and CENTRIFUGE it at 4500
r/min (-4 °C) for 5 min; PIPETTE the lower layer of acetonitrile in a 100 mL
heart-shaped bottle; USE 20 mL of acetonitrile to make repeated
extraction once for the solution in the centrifuge tube A; COMBINE the
extract in the heart-shaped bottle; MAKE it subject to 35 °C rotary
evaporation under reduced pressure to near dryness. ADD 5 mL of n-
hexane; VORTEX it for 30 s to dissolve it.
b) Sequentially USE 5 mL of dichloromethane and 10 mL of n-hexane to
activate the Florisil solid-phase extraction column; TRANSFER all the 5
mL extracted sample which is obtained from a) into the Florisil solid-phase
extraction column; then USE 5 mL of n-hexane to wash the heart-shaped
bottle; COMBINE the washing solution into the column. USE 8 mL of n-
12.2 Extraction of specimen
Same as 5.2.
12.3 Blank test
The blank test uses the same analytical procedure as the test except that no
specimen is added.
12.4 Reference conditions for gas chromatography-mass spectrometry
a) Column. DB-5 MS, the column length is 30 m, the inner diameter is 0.25
mm, the film thickness is 0.25 μm, or column of equivalent performance.
b) Column’s temperature program. The initial temperature is 90 °C, rise the
temperature at 20 °C/min to 320 °C, maintain this temperature for 2 min;
c) Sample inlet’s temperature. 250 °C;
d) Chromatography-mass interface’s temperature. 280 °C;
e) Ion source temperature. 230 °C;
f) Carrier gas. helium, purity ≥ 99.999%, 1.0 mL/min;
g) Ionization mode. EI;
h) Ionization energy. 70 eV;
i) Mass scanning range. 50 amu ~ 450 amu;
j) Method of determination. Selective ion monitoring mode;
k) Injection method. non-split injection; open the valve after 2.0 min;
l) Sample injection volume. 1.0 μL;
m) Solvent delay. 3 min.
12.5 Production of standard curves
Respectively INJECT the standard series working solution into the gas
chromatography-mass spectrometer, to determine the corresponding peak
area. USE the mass concentration of the standard working solution as the
abscissa and the peak area as the ordinate, to draw the standard curve.
12.6 Determination of sample solution
INJECT the sample solution to be determined into the gas chromatograph-
mass spectrometer, to determine the corresponding peak area. According to
13 Expression of analytical results
The content of polycyclic aromatic hydrocarbons in the sample, Xi, is calculated
according to formula (2).
Where.
Xi - The content of polycyclic aromatic hydrocarbons in the specimen, in
micrograms per kilogram (μg/kg);
ρi - The concentration of polycyclic aromatic hydrocarbons i in the sample
solution to be determined which is calculated according to the standard
curve, in nanograms per milliliter (ng/mL);
V - The final constant volume of the sample solution to be determined, in
milliliters (mL);
1000 - Unit conversion;
m - The mass of the specimen, in grams (g).
When the content is greater than or equal to 10 μg/kg, it retains three significant
figures; when the content is less than 10 μg/kg, it retains two significant figures.
Note. The calculation result shall be deducted from the blank value.
14 Precision
The absolute difference between two independent determinations obtained
under repeatability conditions shall not exceed 20% of the arithmetic mean.
15 Others
When the sample amount is 4 k and the constant volume is 1 mL, the detection
limit and limit of quantification of the method are as shown in Table 5.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.