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

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

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.
GB/T 22388-2008
GB
NATIONAL STANDARD OF THE
PEOPLE 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 ??m, or equivalent. C18 column, 250mm x 4.6mm [internal diameter (i.d.)], 5 ??m, 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 ??m, 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 ??L.
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 ??g 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 ??g/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 ??g/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

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