Determination of chloramphenicol and its related compounds residues in animal tissues

Summary

There are many reported methods for the determination of chloramphenicol, and among the screening methods, GC-ECD and ELISA are more commonly used. source: Food Safety Monitoring Technology (Chemical Industry Press)

Operation method

Gas Chromatography-Mass Spectrometry

Materials and Instruments

Animal tissues
Acetic acid buffer solution Glucuronidase Protease Potassium hydroxide solution Trichloroacetic acid Methyl tert-butyl ether-cyclohexane
Centrifuge tubes Incubator Ultrasonic water bath Silanized pear-shaped vials Chromatographic columns

Move

1. Sample extraction

Take 10 g of fully homogenized tissue samples in 100 mL glass centrifuge tubes, add 20 mL of 0.1 mol/L acetic acid buffer solution, 20 mg of glucuronidase and protease, homogenize for 1 min on a homogenizer, and place in an incubator at 60 for 1 min. After cooling, add 15 mL of 200 g/L trichloroacetic acid (using a wide-range paper, adjusted to pH 1.5), and sonicate for 10 min on a sonicating water bath. The extract was centrifuged at 10/15 ℃ at 4000 g for 15 min, and the temperature could be lowered to 8 ℃ if the suspension still existed. The supernatant was decanted and the pH was adjusted to 5.8-6.2 with 20% potassium hydroxide solution. If precipitation occurred during the neutralization process, it should be filtered or centrifuged again. Drench the electrode with 200 g/L trichloroacetic acid and incorporate into the solution and make the total volume close to 500 mL. Pipette 20 mL of the above solution into an ISOLUTE HM-N column and equilibrate for 20 min. Prepare two 20 mL solutions by adding 30 mL of methyl tertiary-butyl ether-cyclohexane (8 + 2) to each in a 100 mL silanized pear-shaped flask under reduced pressure of 250 mbar at 50 ℃. Rotary evaporation was performed to dryness.

2. Purification

Place ISOLUTE-SiFen on an SPE apparatus, pre-add 4 mL of ethyl acetate, 4 mL of ethyl acetate-toluene (20 + 80) in that order, and evacuate to dryness. The residue was dissolved with 2 mL ethyl acetate-toluene (20 + 80), sonicated and shaken, and transferred to the column. The column was then drenched with 4 mL ethyl acetate-toluene (20 + 80) and the drench was discarded. The chloramphenicol fraction was eluted with 4 mL ethyl acetate-toluene (65 + 35) and collected.

2. Derivatization

The solution was collected and blown dry at 40 °C under a slow stream of nitrogen. Transfer into the reaction vial with 200 uL of ethyl acetate, add 20 uL of internal standard solution m-CAP and again blow dry at 40 °C under a slow stream of nitrogen. Add 50 uL of BSTFA-TMCS (99 + 1) solution, tightly capped with a screw cap lined with polytetrafluoroethylene (PTFE), and react at 60 ℃ for l h. After cooling, blow-dry slowly under a stream of nitrogen. Add 25uL of iso-octane to dissolve the residue. For GC-MS determination.

3. Instrumental conditions

Chromatographic column: 5% phenyl, 95% methyl siloxane, 30 m×0.25 mm×0.25 um;

Column temperature: programmed temperature increase, 60 ℃ (l min), to 40 ℃/min, to l80 ℃, to 8 ℃/min, to 280 ℃, to 5 ℃/min, to 300 ℃;

Inlet mode: pulse, no shunt;

Injector temperature: 270 ℃;

Carrier gas: helium;

Interface temperature: 300 ℃;

Ion source temperature: l85 °C;

NCI ionization mode;

Reaction gas: methane at a pressure of 533 Pa (4000 mTorr);

Collision gas: argon at a pressure of 0.27 Pa (2mTorr);

Electron energy: 200 eV;

Collision current: 200 mA;

CAP: m/z 466 (70), 468 (70), 376 (30), 378 (30);

Internal standard: m/z 47l (70).

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