GB/T 20756-2006Method for the determination of chloramphenicol, thiamphenicol，and florfenicol residues in edible animal muscles, liver and aquatic products - LC-MS-MS method (English PDF)

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

7 Test Procedures