GB/T 22388-2008 English PDF (GBT22388-2008)

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GB/T 22388-2008: Determination of melamine in raw milk and dairy products
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...