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GB/T 20756-2006 (GBT20756-2006)

GB/T 20756-2006_English: PDF (GBT 20756-2006, GBT20756-2006)
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GB/T 20756-2006English230 Add to Cart 0--9 seconds. Auto-delivery Method for the determination of chloramphenicol, thiamphenicol, and florfenicol residues in edible animal muscles, liver and aquatic products -- LC-MS-MS method Valid GB/T 20756-2006

BASIC DATA
Standard ID GB/T 20756-2006 (GB/T20756-2006)
Description (Translated English) Method for the determination of chloramphenicol, thiamphenicol, and florfenicol residues in edible animal muscles, liver and aquatic products. LC-MS-MS method
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard X04
Classification of International Standard 67.050
Word Count Estimation 10,155
Date of Issue 2006-12-31
Date of Implementation 2007-03-01
Quoted Standard GB/T 6379.1; GB/T 6379.2; GB/T 6682
Drafting Organization QingHuangdao Exit Inspection and Quarantine of the PRC
Administrative Organization State Administration of Quality Supervision, Inspection and Quarantine of the PRC
Regulation (derived from) China National Standard Approval Announcement 2006 No.13 (Total No.100)
Proposing organization People Republic of China Qinhuangdao Entry - Exit Inspection and Quarantine Bureau
Issuing agency(ies) General Administration of Quality Supervision, Inspection and Quarantine of PRC, China National Standardization Administration Committee
Summary This standard specifies the edible animal muscle, liver, fish and shrimp chloramphenicol, thiamphenicol and florfenicol residues in liquid chromatography-tandem mass spectrometry. This standard applies to edible animal muscle, liver, fish and shrimp chloramphenicol, determination of thiamphenicol and florfenicol residues. The detection limit of the standard methods: chloramphenicol is 0. 1��g/kg, thiamphenicol and florfenicol is 1. 0��g/kg.

Standards related to: GB/T 20756-2006

GB/T 20756-2006
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.050
X 04
Method for the Determination of Chloramphenicol,
Thiamphenicol, and Florfenicol Residues in Edible Animal
Muscles, Liver and Aquatic Products – LC-MS-MS Method
ISSUED ON: DECEMBER 31, 2006
IMPLEMENTED ON: MARCH 01, 2007
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine;
Standardization Administration of the People’s Republic of China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Principle ... 4
4 Reagents ... 5
5 Instruments ... 6
6 Preparation and Storage of Specimen ... 6
7 Test Procedures ... 7
8 Calculation of Results ... 9
9 Precision ... 10
Appendix A (Informative) Multiple Reaction Monitoring (MRM) Chromatograms of
Chloramphenicol, Thiamphenicol, and Florfenicol Standard Substances and Internal
Standard Substance of Chloramphenicol-D5 ... 12
Appendix B (Informative) Recovery Rate ... 13
Method for the Determination of Chloramphenicol,
Thiamphenicol, and Florfenicol Residues in Edible Animal
Muscles, Liver and Aquatic Products – LC-MS-MS Method
1 Scope
This Standard specifies the LC-MS-MS method for the determination of chloramphenicol,
thiamphenicol and florfenicol residues in edible animal muscle, liver, fish and shrimp.
This Standard applies to the determination of chloramphenicol, thiamphenicol and florfenicol
residues in edible animal muscle, liver, fish and shrimp.
The detection limit of this Standard method: 0.1μg/kg for chloramphenicol, and 1.0μg/kg for
thiamphenicol and florfenicol.
2 Normative References
The provisions in following documents become the provisions of this Standard through
reference in this Standard. For dated references, the subsequent amendments (excluding
corrigendum) or revisions do not apply to this Standard, however, parties who reach an
agreement based on this Standard are encouraged to study if the latest versions of these
documents are applicable. For undated references, the latest edition of the referenced document
applies.
GB/T 6379.1 Accuracy (trueness and precision) of measurement methods and results -
Part 1: General principles and definitions (GB/T 6379.1-2004, ISO 5725-1:1994, IDT)
GB/T 6379.2 Accuracy (trueness and precision) of measurement methods and results - Part
2: Basic method for the determination of repeatability and reproducibility of a standard
measurement method (GB/T 6379.2-2004, ISO 5725-2:1994, IDT)
GB/T 6682 Water for laboratory use – Specifications (GB/T 6682-1992, neq ISO
3696:1987)
3 Principle
Chloramphenicol, thiamphenicol and florfenicol in the sample are extracted with ethyl acetate
under alkaline conditions. After the extract was spun to dryness; the residue is dissolved in
water and degreased by n-hexane liquid-liquid partitioning. Take liquid chromatography-
tandem mass spectrometer for detection.
4 Reagents
Unless otherwise specified, the used reagents are of analytically pure; and the water is the first-
grade water specified in GB/T 6682.
4.1 Methanol: chromatographically pure.
4.2 Ethyl acetate
4.3 n-hexane.
4.4 Ammonium hydroxide: 25% ~ 28%.
4.5 Anhydrous sodium sulfate: burn at 650 °C for 4 h; and store in a desiccator for later-use.
4.6 Standard substances of chloramphenicol, thiamphenicol and florfenicol: with purity ≥99.5%.
4.7 Internal standard solution of Chloramphenicol D5: 100μg/mL.
4.8 Standard stock solution: 100μg/mL. Accurately weigh appropriate amounts of standard
substances of chloramphenicol, thiamphenicol and florfenicol, respectively; and use methanol
to make a standard stock solution of 100μg/mL. The solution can be stored at -18 °C and can
be used for 1 year.
4.9 Mixed standard stock solution: 1μg/mL. Accurately pipette 1 mL of chloramphenicol,
thiamphenicol and florfenicol standard stock solution (4.8) respectively into a 100 mL
volumetric flask; and dilute to the mark with methanol. The solution can be stored at -18°C and
can be used for 6 months.
4.10 Intermediate concentration mixed standard solution: 20ng/mL. Accurately pipette 1 mL of
the mixed standard stock solution (4.9) in a 50 mL volumetric flask and dilute with water to the
mark. The solution can be stored at 4°C and can be used for 3 months.
4.11 Internal standard stock solution: 1μg/mL. Accurately pipette 100μL of chloramphenicol-
D5 (d5-chloramphenicol) standard solution (4.7) into a 10 mL volumetric flask; and dilute to
the mark with methanol. The solution is stored at -18°C and can be used for 6 months.
4.12 Intermediate concentration internal standard solution: 20ng/mL. Accurately pipette 1 mL
of internal standard stock solution (4.11) into a 50 mL volumetric flask; and dilute to the mark
with water. The solution is stored the at 4°C and can be used for 3 months.
4.13 Matrix mixed standard working solution: according to the sensitivity of each standard and
the linear range of the instrument, pipette a certain amount of intermediate concentration mixed
standard solution (4.10) and intermediate concentration internal standard solution (4.12); and
use the blank sample extract to prepare a series of matrix mixed standard working solutions;
and the internal standard concentration is 0.3ng/mL. It shall be prepared in the same day.
4.14 Filter membrane: 0.2μm.
5 Instruments
5.1 Liquid chromatography-tandem mass spectrometer: equipped with an electrospray ion
source.
5.2 Analytical balance: with sensitivity of 0.1 mg and 0.01 g.
5.3 Centrifuge: 4000 r/min.
5.4 High-speed desktop centrifuge: 13000 r/min.
5.5 Tissue masher.
5.6 Homogenizer.
5.7 Rotary evaporator.
5.8 Ultrasound.
5.9 Liquid mixer.
5.10 Polypropylene centrifuge tubes: 50mL, 1.5mL, with stoppers.
5.11 Heart-shaped bottle: 25mL.
5.12 Colorimetric tube: 50mL, with stopper.
6 Preparation and Storage of Specimen
6.1 Preparation of specimen
Take a sample of about 500g; grind it with a meat tissue grinder; put it into a clean container as
a specimen; seal it; and mark it.
6.2 Storage of specimen
Store the specimen in a refrigerator at -18°C.
7 Test Procedures
7.1 Extraction
Weigh 5g of specimen, accurate to 0.01g. Place it in a 50 mL polypropylene centrifuge tube;
add 75.0μL of intermediate concentration internal standard solution (4.12); add 15 mL of ethyl
acetate; add 0.45 mL of ammonium hydroxide (4.4); and add 5 g of anhydrous sodium sulfate;
and extract homogeneously for 30 s; centrifuge at 4000 r/min for 5 min; and transfer the
supernatant to a 50 mL colorimetric tube. Take another 50 mL centrifuge tube; add 15 mL of
ethyl acetate and 0.45 mL ammonium hydroxide; wash the homogeneous knife head for 10 s;
transfer the washing liquid into the first centrifuge tube; stir the residue with a glass rod; and
place it in a liquid mixer (5.9) to vortex for 1 min; ultrasonically extract for 5 min; centrifuge
at 4000 r/min for 5 min; and put the supernatant into a 50 mL colorimetric tube. Add 15 mL of
ethyl acetate to the residue; repeat the above operation; combine all supernatants into a 50 mL
colorimetric tube; and make constant volume to 50 mL with ethyl acetate. After shaking well,
pipette 10 mL of ethyl acetate extract into a 25 mL heart-shaped bottle; and concentrate to
dryness at 45°C with rotation.
7.2 Purification
Dissolve the residue in the heart-shaped bottle with 3 mL of water; sonicate for 5 min; add 3
mL of n-hexane and vortex mix for 30 s; let stand to separate layers; discard the upper layer of
n-hexane. Then add 3 mL of n-hexane and vortex mix for 30 s; after standing and separating
layers, pipette 1 mL of the aqueous phase into a 1.5 mL polypropylene centrifuge tube;
centrifuge at 1 3000 r/min for 5 min; pass through a 0.2μm filter membrane; and use it for liquid
chromatography-tandem mass spectrometry.
7.3 Chromatographic determination
7.3.1 Liquid chromatography conditions
a) Chromatographic column: Discovery C18 chromatographic column; 5μm, 150 mm × 2.1
mm (inner diameter) or equivalent;
b) Column temperature: 40°C;
c) Mobile phase: methanol + water (40 + 60);
d) Flow rate: 0.30 mL/min;
e) Injection volume: 20μL.
7.3.2 Mass spectrometry conditions
a) Ion source: electrospray ion source;
...