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GB/T 22388-2008 (GB/T22388-2008)

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GB/T 22388-2008: PDF in English (GBT 22388-2008)

GB/T 22388-2008
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.100
C 53
Determination of melamine
in raw milk and dairy products
ISSUED ON. OCTOBER 7, 2008
IMPLEMENTED ON. OCTOBER 7, 2008
Issued by. General Administration of Quality Supervision, Inspection and
Quarantine;
Standardization Administration Committee.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Method One -- High performance liquid chromatography ... 4 
4 Method Two -- Liquid chromatography-mass spectrometry/mass
spectrometry (LC-MS/MS) ... 9 
5 Method Three -- Gas chromatography-mass spectrometry (GC-MS and GC-
MS/MS) ... 12 
Annex A (Informative) Melamine standard product chromatogram ... 18 
Determination of melamine
in raw milk and dairy products
1 Scope
This Standard specifies three determination methods for melamine in raw milk,
dairy product as well as products containing dairy, i.e., high performance liquid
chromatography (HPLC), liquid chromatography - mass spectrometry/mass
spectrometry (LC-MS/MS) and gas chromatography - mass spectrometry
[including gas chromatography-mass spectrometry (GC-MS), gas
chromatography - mass spectrometry/mass spectrometry (GC-MS/MS).
This Standard applies to the quantitative determination of melamine in raw milk,
dairy products and products containing dairy. Liquid chromatography - mass
spectrometry/mass spectrometry and gas chromatography - mass
spectrometry (including gas chromatography - mass spectrometry/mass
spectrometry) are applicable simultaneously to the qualitative confirmation of
melamine in raw milk, dairy products, and products containing dairy.
The limit of quantitation of HPLC of this Standard is 2 mg/kg. The limit of
quantitation of liquid chromatography - mass spectrometry/mass spectrometry
is 0.01 mg/kg. The limit of quantitation of gas chromatography - mass
spectrometry is 0.05 mg/kg (the limit of quantitation of gas chromatography -
mass spectrometry/mass spectrometry is 0.005 mg/kg).
2 Normative references
The following standards contain the provisions which, through reference in this
Standard, constitute the provisions of this Standard. For dated references,
subsequent amendments (excluding corrections) or revisions do not apply to
this Standard. However, the parties who enter into agreement based on this
Standard are encouraged to investigate whether the latest versions of these
documents are applicable. For undated reference documents, the latest
versions apply to this Standard.
GB/T 6682, Water for analytical laboratory use - Specification and test
methods (GB/T 6682-2008, ISO 3696.1987, MOD)
3 Method One -- High performance liquid
3.4.1.2 Cheese, cream and chocolate, etc.
Weigh 2 g (to the nearest of 0.01 g) of sample in the mortar. Add appropriate
amount of sea sand (4 to 6 times the mass of the sample), grind into dry powder.
Transfer to a 50 mL plugged plastic centrifuge tube. Clean the mortar several
times with 15mL of trichloroacetic acid solution (3.2.8). Transfer the cleaning
solution to the centrifuge tube. Add 5 mL of acetonitrile to the centrifuge tube.
The remaining operation is the same as "perform ultrasonic extraction for
10min...mix with 5 mL of water to make the solution to be purified" in 3.4.1.1.
NOTE. If the fat content of the sample is high, it can be purified by SPE column after
degreasing with a saturated liquid-liquid distribution of n-hexane saturated with trichloroacetic
acid solution.
3.4.2 Purification
Transfer the liquid to be purified in 3.4.1 to the solid phase extraction column
(3.2.13). Wash sequentially with 3 mL of water and 3 mL of methanol. After
pumping to near dryness, elute with 6 mL of ammoniated methanol solution
(3.2.9). The flow rate of entire solid phase extraction process does not exceed
1 mL/min. Eluent dried with nitrogen at 50°C. Residue (equivalent to 0.4 g of
sample) is set to volume with 1mL of mobile phase. Perform vortex mixing for 1
min. After going through microporous membrane (3.2.16), leave it for HPLC
determination.
3.5 High performance liquid chromatography determination
3.5.1 HPLC reference conditions
a) Chromatographic column.
C8 column, 250mm x 4.6mm [internal diameter (i.d.)], 5 μm, or equivalent.
C18 column, 250mm x 4.6mm [internal diameter (i.d.)], 5 μm, or equivalent.
b) Mobile phase.
C8 column, ion pair reagent buffer (3.2.10)-acetonitrile (85+15, volume
ratio), mixing well.
C18 column, ion pair reagent buffer (3.2.10)-acetonitrile (90+10, volume
ratio), mixing well.
c) Flow rate. 1.0 mL/min.
d) Column temperature. 40°C.
e) Wavelength. 240 nm.
Weigh 1 g (to the nearest of 0.01 g) of sample in 50mL plugged plastic
centrifuge tube. Add 8 mL of trichloroacetic acid solution (3.2.8) and 2 mL of
acetonitrile. Perform ultrasonic extraction for 10 min. Then oscillate to extract
for 10 min, centrifuge at no less than 4000 r/min for 10min. Filter the
supernatant through a filter paper moistened with a solution of trichloroacetic
acid as the solution to be purified.
4.4.1.2 Cheese, cream and chocolate, etc.
Weigh 1 g (to the nearest of 0.01 g) of sample in the mortar. Add appropriate
amount of sea sand (4 to 6 times the mass of the sample), grind into dry powder.
Transfer to a 50 mL plugged plastic centrifuge tube. Clean the mortar several
times with 15mL of trichloroacetic acid solution (3.2.8). Transfer the cleaning
solution to the centrifuge tube. Add 2 mL of acetonitrile. The remaining
operation is the same as "perform ultrasonic extraction for 10min...make the
solution to be purified" in 4.4.1.1.
NOTE. If the fat content of the sample is high, it can be purified by SPE column after
degreasing with a saturated liquid-liquid distribution of n-hexane saturated with trichloroacetic
acid solution.
4.4.2 Purification
Transfer the liquid to be purified in 4.4.1 to the solid phase extraction column
(3.2.13). Wash sequentially with 3 mL of water and 3 mL of methanol. After
pumping to near dryness, elute with 6 mL of ammoniated methanol solution
(3.2.9). The flow rate of entire solid phase extraction process does not exceed
1 mL/min. Eluent dried with nitrogen at 50°C. Residue (equivalent to 1 g of
sample) is set to volume with 1mL of mobile phase. Perform vortex mixing for 1
min. After going through microporous membrane (3.2.16), leave it for LC-
MS/MS determination.
4.5 Liquid chromatography-mass spectrometry/mass spectrometry
determination
4.5.1 LC reference conditions
a) Chromatographic column. strong cation exchange and reversed-phase
C18 mixed packing, mixing ratio (1.4), 150mm×2.0mm [inner diameter
(i.d.)], 5 μm, or equivalent.
b) Mobile phase. equal volume of ammonium acetate solution (4.2.3) and
acetonitrile are mixed well and adjusted with acetic acid until pH=3.0 for
use.
c) Injection volume. 10 μL.
Unless otherwise specified, all reagents are analytically pure, and water is
grade one according to GB/T 6682.
5.2.1 Pyridine. premium pure.
5.2.2 Lead acetate.
5.2.3 Derivatization reagent. N, O-bistrimethylsilyl trifluoroacetamide (BSTFA)
+ trimethylchlorosilane (TMCS) (99+1), chromatographic pure.
5.2.4 Lead acetate solution (22 g/L). take 22 g of lead acetate; dissolve with
about 300 mL of water and set to volume of 1 L.
5.2.5 Melamine standard solution. accurately pipet 1 mL of melamine
standard stock solution (3.2.12) into 100 mL volumetric flask; set to volume with
methanol; 1 mL of this standard solution is equivalent to 10 μg of melamine
standard product; store in a 4°C refrigerator; it shall be valid for 3 months.
5.2.6 Argon. purity is greater than or equal to 99.999%.
5.2.7 Helium. purity is greater than or equal to 99.999%.
5.2.8 Same with 3.2.
5.3 Instruments and equipment
5.3.1 Gas chromatography-mass spectrometry (GC-MS) instrument.
equipped with electron impact ionization (EI).
5.3.2 Gas chromatography-mass spectrometry/mass spectrometry (GC-
MS/MS) instrument. equipped with electron impact ionization (EI).
5.3.3 Electronic incubator.
5.3.4 Same with 3.3.
5.4 Sample processing
5.4.1 GC-MS method
5.4.1.1 Extraction
5.4.1.1.1 Liquid milk, milk powder, yogurt, ice cream and toffee, etc.
Weigh 5 g of (to the nearest of 0.01 g) sample to a 50 mL plugged colorimetric
tube. Add 25 mL of trichloroacetic acid solution (3.2.8). Perform vortex
oscillation for 30 s. Then add 15 mL of trichloroacetic acid solution. Perform
ultrasonic extraction for 15 min. Add 2 mL of lead acetate solution (5.2.4). Set
to the volume with trichloroacetic acid solution. After mixing well, transfer 30 mL
j) Ion source temperature. 220°C.
k) Quadrupole temperature. 150°C.
l) Gas collision. argon, 0.2394 Pa (1.8 mTorr).
m) Collision energy. 15 V.
n) Scanning method. multiple reaction monitoring (MRM), quantitative ion
m/z 342>327, qualitative ion m/z 342>327, 342>171.
5.5.2 Drawing of standard curve
5.5.2.1 GC-MS method
Accurately pipet 0, 0.4, 0.8, 1.6, 4, 8, 16 mL of melamine standard solution
(5.2.5). Respectively place them in 7 100-mL volumetric flasks. Dilute to the
scale with methanol. Respectively take 1 mL . Dry with nitrogen. Derivatize
according to 5.4.3. Prepare standard solutions of which the derivatized product
concentrations are 0, 0.05, 0.1, 0.2, 0.5, 1, 2 μg/mL, respectively. The reaction
liquid is for GC-MS determination. The concentration of the standard working
solution is plotted on the abscissa, and the area of the quantified ion mass
chromatogram on the ordinate. Draw the standard working curve. See Figure
A.3 in Annex A for GC-MS selection ion mass chromatograms of standard
solution. See Figure A.4 in Annex A for melamine derivative selective ion mass
spectra.
5.5.2.2 GC-MS/MS method
Accurately pipet 0, 0.04, 0.08, 0.4, 0.8, 4, 8 mL of melamine standard solution
(5.2.5). Respectively place them in 7 100-mL volumetric flasks. Dilute to the
scale with methanol. Respectively take 1 mL . Dry with nitrogen. Derivatize
according to 5.4.3. Prepare standard solutions of which the derivatized product
concentrations are 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1 μg/mL, respectively. The
reaction liquid is for GC-MS/MS determination. The concentration of the
standard working solution is plotted on the abscissa, and the area of the
quantified ion mass chromatogram on the ordinate. Draw the standard working
curve. See Figure A.5 in Annex A for GC-MS/MS multiple reaction monitoring
mass chromatograms of standard solution.
5.5.3 Quantitative determination
The melamine response in the sample to be tested shall be within the linear
range of the standard curve. When it exceeds the linear range, it shall dilute the
purification liquid. Re-derivatization is followed by injection analysis.
5.5.4 Qualitative determination
......
 
(Above excerpt was released on 2018-06-02, modified on 2021-06-07, translated/reviewed by: Wayne Zheng et al.)
Source: https://www.chinesestandard.net/PDF.aspx/GBT22388-2008