GB/T 22988-2008 PDF in English
GB/T 22988-2008 (GB/T22988-2008, GBT 22988-2008, GBT22988-2008)
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Determination of spiramycin, pirlimycin, oleandomycin, tilmicosin, erythromycin and tylosin residues in milk and milk powder -- LC-MS-MS method
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Standards related to (historical): GB/T 22988-2008
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GB/T 22988-2008: PDF in English (GBT 22988-2008) GB/T 22988-2008
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.050
X 04
Determination of spiramycin, pirlimycin, oleandomycin,
tilmicosin, erythromycin and tylosin residues in milk and
milk powder - LC-MS-MS method
ISSUED ON: DECEMBER 31, 2008
IMPLEMENTED ON: MAY 01, 2009
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine of PRC;
National Standardization Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Principle ... 5
4 Reagents and materials ... 5
5 Instruments and equipment ... 6
6 Preparation and storage of specimens ... 6
7 Determination steps ... 6
8 Result calculation ... 10
9 Precision ... 10
Appendix A (Informative) Multiple reaction monitoring (MRM) chromatograms of
standard substances ... 12
Appendix B (Informative) Recovery ... 13
Determination of spiramycin, pirlimycin, oleandomycin,
tilmicosin, erythromycin and tylosin residues in milk and
milk powder - LC-MS-MS method
1 Scope
This standard specifies the HPLC-tandem mass spectrometry method for the
determination of spiramycin, pirlimycin, oleandomycin, tilmicascin, erythromycin,
tylosin residues in milk and milk powder.
This standard is applicable to the determination and confirmation of spiramycin,
pirlimycin, oleandomycin, tilmicascin, erythromycin, tylosin residues in milk and milk
powder.
The method detection limit of this standard is 1 μg/kg for milk and 8 μg/kg for milk
powder.
2 Normative references
The clauses in the following documents become the clauses of this standard through
reference in this standard. For any dated referenced document, all subsequent
amendments (excluding corrections) or revisions are not applicable to this standard.
However, parties that reach an agreement based on this standard are encouraged to study
whether the latest versions of these documents can be used. For any undated referenced
document, the latest version applies to this standard.
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 Measurement methods and results - Accuracy (trueness and precision)
- Part 2: Determine the standard methods of measurement repeatability and
reproducibility of the basic method (GB/T 6379.2-2004, ISO 5725-2:1994, IDT)
GB/T 6682 Water for analytical laboratory use - Specification and test methods
(GB/T 6682-2008, ISO 3696:1987, MOD)
3 Principle
The analytes in the specimen are extracted with acetonitrile, cleaned up with solid phase
extraction column, determined by high performance liquid chromatography-tandem
mass spectrometry, quantified by external standard method.
4 Reagents and materials
Unless otherwise specified, all reagents used are analytical grade; the water is grade 1
water as specified in GB/T 6682.
4.1 Acetonitrile, HPLC grade.
4.2 Methanol, HPLC grade.
4.3 Acetic acid, HPLC grade.
4.4 Ammonium formate, HPLC grade.
4.5 Disodium hydrogen phosphate.
4.6 Sodium hydroxide.
4.7 Methanol-water solution (3 + 7): 300 mL of methanol mixed with 700 mL of water.
4.8 5 mol/L sodium hydroxide solution: Weigh 20 g of sodium hydroxide; dissolve it in
water; dilute it to 100 mL.
4.9 0.1 mol/L phosphate buffer solution: Dissolve 6 g of disodium hydrogen phosphate
(4.5) in 450 mL of water; use sodium hydroxide (4.8) solution to adjust pH to 8; add
water to 500 mL; prepare it before use.
4.10 0.1 mol/L ammonium formate solution: Add water to dissolve 0.63 g of ammonium
formate (4.4) to 1000 mL.
4.11 Standard products: Spiramycin (CAS: 8025-81-8), pirlimycin (CAS: 79548-73-5),
oleandomycin (CAS: 7060-74-4), tilmicosin (CAS: 108050-54-0), erythromycin (CAS:
114-07-8), tylosin (CAS: 1401-69-0), purity greater than or equal to 98%.
4.12 Standard stock solution: Accurately weigh appropriate amount of standard
products; use methanol to prepare 100 μg/mL standard stock solution.
4.13 Mixed standard intermediate working solution: Take 1 mL of each standard stock
solution (4.12) into a 100 mL volumetric flask; use methanol to dilute to the mark;
prepare a mixed standard working solution which has a concentration of 1 μg/mL.
10 mL of acetonitrile to repeat extraction once. Combine the supernatant in the same
chicken heart bottle.
7.1.2 Milk powder
Weigh 0.5 g, accurate to 0.01 g, of the milk powder sample. Add 4 mL of water to a 50
mL stoppered centrifuge tube and mix well. Add 20 mL of acetonitrile. Oscillate for
extraction for 2 min. Centrifuge at 3000 r/min for 10 min. Remove the supernatant and
filter it into a chicken heart bottle. Use 10 mL of acetonitrile to repeat the extraction
once. Combine the supernatant in the same chicken heart bottle.
7.2 Purification
Rotate and evaporate the extract at 45 °C to about 4 mL. Add 2 mL of phosphate buffer
(4.9). Mix well. Transfer to the conditioned HLB solid phase extraction column (4.15).
Wash the chicken heart bottle twice with 2 mL of phosphate buffer. Transfer the washing
liquid to the column. Drip it at a flow rate of less than 2 mL/min. Then, wash with 3
mL of water and 2 mL of methanol-water solution (4.7) in turn. Drain the column.
Finally elute with 6 mL of acetonitrile and collect in a 10 mL graduated glass tube (the
flow rate in this process is less than 2 mL/min). The eluent is blown to about 1 mL with
nitrogen in a 45 °C water bath; use ammonium formate solution (4.10) to make its
volume reach to 2 mL. After vortex mixing, filter it through a 0.45 µm filter membrane
for HPLC-MS-MS analysis.
7.3 Preparation of blank matrix solution
Take 4 g of milk negative sample and 0.5 g of milk powder negative sample, accurate
to 0.01 g. Operate according to 7.1 and 7.2.
7.4 Determination conditions
7.4.1 Liquid chromatography reference conditions
Liquid chromatography reference conditions are as follows:
a) Chromatographic column: Phenyl column, 5 μm, 150 mm × 2.1 mm (inner
diameter) or equivalent;
b) Chromatographic column temperature: 30 °C;
c) Injection volume: 15 μL;
d) Mobile phase gradient and flow rate are shown in Table 1.
can be found in Figure A.1 in Appendix A.
7.5 Parallel test
According to the above steps, parallel test determination is carried out on the same
specimen.
7.6 Recovery test
Pipette an appropriate amount of mixed standard working solution. Use blank matrix
solution to dilute it to the standard calibration solution of required concentration. Add
standard solution to negative sample and operate according to 7.1 and 7.2. After
determination, calculate the recovery of sample addition. The recovery range is shown
in Table B.1 in Appendix B.
7.7 Blank test
Follow the above steps except that the sample is not weighed.
8 Result calculation
The residual amount of analyte in the specimen is calculated using the data processing
system or according to formula (1):
Where:
Xi - The residual amount of the measured component in the specimen, in micrograms
per kilogram (μg/kg);
ci - The concentration of the measured component solution obtained from the
standard working curve, in nanograms per milliliter (ng/mL);
Vi - The final volume of the sample solution, in milliliters (mL);
mi - The amount of the final sample represented by the sample solution, in grams
(g).
The blank value shall be deducted from the calculation result.
9 Precision
9.1 General provisions
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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