GB/T 22338-2008 PDF English
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Determination of multi-residues of chloramphenicol in animal-original food
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GB/T 22338-2008: PDF in English (GBT 22338-2008) GB/T 22338-2008
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.120
X 22
Determination of multi-residues of chloramphenicol in
animal-original food
ISSUED ON: SEPTEMBER 01, 2008
IMPLEMENTED ON: DECEMBER 01, 2008
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine of the People's Republic of China;
Standardization Administration of the People's Republic of China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Gas chromatography-mass spectrometry ... 4
3 Liquid chromatography-mass spectrometry/mass spectrometry ... 9
Annex A (informative) Gas chromatography-mass spectrometry total ion current
chromatogram and mass spectrum of chloramphenicol derivatives ... 16
Annex B (informative) Average recovery rate and precision of chloramphenicol in
different matrices (GC/MS method) ... 17
Annex C (informative) Reference conditions for liquid chromatography-mass
spectrometry/mass spectrometry ... 18
Annex D (informative) Liquid chromatography-mass spectrometry/mass spectrometry
total ion chromatogram and reconstructed ion chromatogram of chloramphenicol ... 19
Annex E (informative) Average recovery rate and precision of chloramphenicol drugs
in different matrices (LC-MS/MS method) ... 21
Determination of multi-residues of chloramphenicol in
animal-original food
1 Scope
This Standard specifies the methods of gas chromatography-mass spectrometry and
liquid chromatography-mass spectrometry/mass spectrometry to determine the multi-
residues of chloramphenicol in animal-original food.
This Standard is applicable to the qualitative confirmation and quantitative
determination of chloramphenicol, florfenicol and thiamphenicol residues in aquatic
products, livestock and poultry products and livestock and poultry by-products.
2 Gas chromatography-mass spectrometry
2.1 Principle
The sample is extracted by ethyl acetate. It is distributed and purified by 4% sodium
chloride solution and n-hexane solution. After purification by Florisil column, take
toluene as the reaction medium. Use N,O bis(trimethylsilyl)trifluoroacetamide-
trimethylchlorosilane (BSTFA+TMCS, 99+1) to silanize at 70°C. Use gas
chromatography/negative chemical ionization source mass spectrometry to determine.
Use internal standard working curve method for quantification.
2.2 Reagents and materials
Unless otherwise stated, it shall only use the confirmed analytically-pure reagents and
secondary deionized water or water of equivalent purity in the analysis.
2.2.1 Methanol: Chromatographically pure.
2.2.2 Toluene: Pesticide residue level.
2.2.3 N-hexane: Pesticide residue level.
2.2.4 Ethyl acetate.
2.2.5 Ether.
2.2.6 Sodium chloride.
2.2.7 Chloramphenicol (CAP), florfenicol (FF), thiamphenicol (TAP) standard material:
2.4 Determination steps
2.4.1 Extraction
Weigh 10g (accurate to 0.01g) of the crushed tissue sample into a 50mL stoppered
centrifuge tube. Add 1.0mL of internal standard solution (2.2.11) and 30mL of ethyl
acetate. Shake for 30min. Centrifuge at 4000r/min for 2min. Transfer the supernatant
to a round bottom flask. Use 30mL of ethyl acetate to extract the residue one more time.
Combine the extracts. Conduct rotary evaporation at 35 ℃ to 1mL ~ 2mL, for
purification.
2.4.2 Purification
2.4.2.1 Liquid-liquid extraction
Extract the concentrate (2.4.1). Add 1mL of methanol to dissolve. Use 20mL of sodium
chloride solution (2.2.9) and 20mL of n-hexane to conduct liquid-liquid extraction.
Discard the n-hexane layer. Use 40mL of ethyl acetate to extract the aqueous phase
twice. Combine the ethyl acetate phases in a heart-shaped flask. Rotate and evaporate
to near dryness at 35°C. Use nitrogen to dry slowly.
2.4.2.2 Florey silica column purification
Use 5mL of methanol, 5mL of methanol-diethyl ether (3+7) solution and 5mL of diethyl
ether successively to rinse Flory silica column, for future use. Use 5.0mL of ether to
dissolve the residue (2.4.2.1) and load the sample. Use 5.0mL of ether to rinse the
Florisil column. Use 5.0mL of methanol-diethyl ether solution (3+7) to elute. Use
nitrogen to slowly blow dry the nitrogen, for silanization.
2.4.3 Silanization
Use 0.2mL of toluene to dissolve the purified specimen (2.4.2.2). Add 0.1mL of
silanization reagent (2.2.13) to mix. Derivatize at 70°C for 60min. Use nitrogen to blow
dry slowly. Use 1.0mL of n-hexane to set volume constant, for determination.
2.4.4 Determination
2.4.4.1 Gas chromatography-mass spectrometry conditions
a) Chromatographic column: DB-5MS capillary column, 30m×0.25mm (inner
diameter)×0.25μm, or its equivalent;
b) Column temperature: at 50°C for 1min; 25°C/min to 280°C; hold for 5 min;
c) Inlet temperature: 250°C;
d) Injection method: splitless injection; splitless time is 0.75min;
e) Carrier gas: high-purity helium; purity ≥99.999%;
2.7 Recovery rate and precision
See Annex B.
3 Liquid chromatography-mass spectrometry/mass
spectrometry
3.1 Principle
For residues of chloramphenicol, thiamphenicol and florfenicol in different animal-
original food, use acetonitrile, ethyl acetate-ether or ethyl acetate to respectively extract.
The extract is purified with a solid phase extraction cartridge. Use liquid
chromatography-mass spectrometry/mass spectrometer to determine. Chloramphenicol
is quantified by internal standard method. Thiamphenicol and florfenicol are quantified
by external standard method.
3.2 Reagents and materials
Unless otherwise stated, it shall only use the confirmed analytically-pure reagents and
secondary deionized water or water of equivalent purity in the analysis.
3.2.1 Methanol: liquid chromatography level.
3.2.2 Acetonitrile: liquid chromatography level.
3.2.3 Acetone: liquid chromatography level.
3.2.4 n-propanol: liquid chromatography level.
3.2.5 n-hexane: liquid chromatography level.
3.2.6 Ethyl acetate: liquid chromatography level.
3.2.7 Diethyl ether.
3.2.8 Sodium acetate.
3.2.9 Ammonium acetate.
3.2.10 β-glucuronidase: about 40,000 activity units.
3.2.11 Acetonitrile saturated n-hexane: Take 200mL of n-hexane (3.2.5) into a 250mL
separatory funnel. Add a small amount of acetonitrile (3.2.2). Shake vigorously. After
standing for stratification, discard the lower acetonitrile layer.
3.2.12 Acetone-n-hexane (1+9): Mix acetone (3.2.3) and n-hexane (3.2.5) well in a
volume ratio of 1:9.
3.2.13 Acetone-n-hexane (6+4): Mix acetone (3.2.3) and n-hexane (3.2.5) well in a
volume ratio of 6:4.
3.2.14 Ethyl acetate-diethyl ether (75+25): Mix 75mL of ethyl acetate (3.2.6) and 25mL
of ether (3.2.7) solution well.
3.2.15 Sodium acetate buffer (0.1mol/L): Weigh 13.6g of sodium acetate (3.2.8) into a
1000mL volumetric flask. Add 980mL of water to dissolve and mix well. Use acetic
acid to adjust the pH to 5.0. Set the volume to the scale and mix well.
3.2.16 Ammonium acetate solution (10mmol/L): Weigh 0.77g of ammonium acetate
(3.2.9) into a 1000mL volumetric flask. Use water to set the volume to the scale. Mix
well.
3.2.17 Chloramphenicol, thiamphenicol and florfenicol standard substances: purity is
≥99.0%.
3.2.18 Chloramphenicol deuterated internal standard (chloramphenicol-D5) substance:
purity is ≥99.0%.
3.2.19 Standard stock solution: Accurately and respectively weigh an appropriate
amount of chloramphenicol, thiamphenicol and florfenicol standard substances (3.2.17)
(accurate to 0.1mg). Use acetonitrile to prepare 500μg/mL standard stock solution (it
can be used for 6 months when stored in the dark at 4°C).
3.2.20 Standard intermediate solutions of chloramphenicol, thiamphenicol and
florfenicol: Accurately and respectively pipette appropriate amounts of standard stock
solutions of chloramphenicol, thiamphenicol and florfenicol (3.2.19). Use acetonitrile
to dilute to 50μg/mL standard intermediate solution of chloramphenicol, thiamphenicol
and florfenicol (it can be used for 3 months when stored in the dark at 4°C).
3.2.21 Mixed standard working solution of chloramphenicol, thiamphenicol and
florfenicol: Accurately and respectively pipette an appropriate amount of standard
intermediate solutions of chloramphenicol, thiamphenicol and florfenicol (3.2.20). Use
mobile phase to dilute to a suitable mixed standard working solution (prepare when it
is required).
3.2.22 Chloramphenicol deuterated internal standard (chloramphenicol-D5) stock
solution: Accurately weigh an appropriate amount of chloramphenicol-D5 standard
substance (3.2.18) (accurate to 0.1mg). Use acetonitrile to prepare 100μg/mL standard
stock solution (it can be used for 12 months when stored in the dark at 4°C).
3.2.23 Chloramphenicol deuterated internal standard (chloramphenicol-D5)
intermediate solution: Accurately pipette an appropriate amount of chloramphenicol-
D5 stock solution (3.2.22). Use acetonitrile to prepare 1μg/mL internal standard
intermediate solution (it can be used for 6 months when stored in the dark at 4°C).
The specimen shall be stored at -20°C.
3.5 Determination steps
3.5.1 Extraction
3.5.1.1 Animal tissues (except liver and kidney) and aquatic products
Weigh 5g of specimen (accurate to 0.01g). Place it in a 50mL centrifuge tube. Add
100μL of chloramphenicol deuterated internal standard (chloramphenicol-D5) working
solution (3.2.24) and 30mL of acetonitrile. Homogenate. Centrifuge for 5min. Transfer
the supernatant to a 250mL separatory funnel. Add 15mL of acetonitrile-saturated n-
hexane (3.2.11). Shake for 5min. Let it still for layering. Transfer the acetonitrile layer
to a 100 mL brown heart-shaped bottle. Add 30mL of acetonitrile to the residue. Shake
for 3min. Centrifuge for 5min. Transfer the supernatant to the same separatory funnel.
Shake for 5min. Let it still for layering. Transfer the acetonitrile layer to the same brown
heart-shaped bottle. Add 5mL of n-propanol to the heart-shaped bottle. Evaporate to
dryness in a water bath at 40°C. Blow dry with nitrogen. Add 5mL of acetone-n-hexane
(3.2.12) to dissolve the residue.
3.5.1.2 Animal liver and kidney tissue
Weigh 5g of specimen (accurate to 0.01g). Place in a 50mL centrifuge tube. Add 30mL
of sodium acetate buffer (3.2.15). Homogenize for 2min. Add 300μL of β-glucuronidase
(3.2.10). Incubate overnight at 37°C. Add 100μL of chloramphenicol deuterated
internal standard (chloramphenicol-D5) working solution (3.2.24) and 20mL of ethyl
acetate-diethyl ether (3.2.14) to the digested sample. Shake for 2min. Centrifuge for
5min. Take the upper organic layer into a heart-shaped bottle. Make it nearly dry by
rotary evaporation in a water bath at 40°C. Blow dry with nitrogen. Add 5mL of
acetone-n-hexane (3.2.12) to dissolve the residue.
3.5.1.3 Honey
Weigh 5g of honey specimen (accurate to 0.01g). Place in a 50mL centrifuge tube. Add
100μL of chloramphenicol deuterated internal standard (chloramphenicol-D5) working
solution (3.2.24), 5mL of water. Mix well. Add 20mL of ethyl acetate. Shake for 2min.
Centrifuge for 5min. Pipette the organic layer into a 100mL brown heart-shaped bottle.
Add 20mL of ethyl acetate to the centrifuge tube. Shake for 2min. Centrifuge for 5min.
Combine the organic layers in a brown heart-shaped bottle. Conduct rotary evaporation
to dryness in a 40°C water bath. Use 3mL of water to dissolve the residue. Mix well.
3.5.2 Purification
3.5.2.1 Animal tissue and aquatic products
Use 5mL of acetone-n-hexane (3.2.12) to rinse the LC-Si silica gel cartridge. Discard
the eluent. Transfer the residue solution (3.5.1.1, 3.5.1.2) to the solid phase extraction
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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