Phenicols, a kind of amide alcohol broad-spectrum antibiotic, includes chloramphenicol and its derivatives. Chloramphenicol, thiamphenicol and florfennicol all belong to phenicols. Chloramphenicol can lead to aplastic anemia with its side effect, and the side effect is irreversible and can’t be reduced by the use of dose and frequency. The United States, European Union, Japan and many other countries have prohibited the existence of chloramphenicol in animal-derived food. China also classified it as a forbidden drug and prohibited the existence of it in animal food in 2002. Thiamphenicol is less toxic to the blood system than chloramphenicol, but it can damage the formation of body's immune system, red blood cells and platelet. The European Community and the United States have banned the thiamphenicol on food animals except China and Japan. Florfenicol can impact animals’ embryos though it is not the cause of aplastic anemia.
In this paper, the method of simultaneous determination of chloramphenicol, florfenicol and thiamphenicol in fish was established by the reappearance of the national standard.
Instruments, reagents and materials
AB SCIEX API 4000+ Liquid Chromatography Tandem Mass Spectrometer
HPLC purity of acetonitrile, n-hexane and acetone; experimental water (ultra-pure water); analytical pure n-propanol; mixed standard solution: chloramphenicol, thiamphenicol, florfenicol, methanol solution; Cleanert® Silica: 200 mg/3 mL.
Firstly, transfer 5 g homogenized fish sample and 30 mL acetonitrile into a 50 mL centrifuge tube. Then vortex 1 min, and sonicate for 5 min then centrifuge at 8000 rpm for 5 min. Secondly, all supernatant in the centrifuge tube was put into a liquid funnel. Add 15 mL acetonitrile saturated n-hexane solution into the same liquid funnel and shake for 5min, stratified it. Thirdly, the acetonitrile layer was transferred into a heart-shaped bottle. Another 30mL acetonitrile was added into the residue to extract another acetonitrile layer according to the above process. Fourthly, the two acetonitrile layers and 5mL n-propyl alcohol were mixed in the heart-shaped bottle by shaking. Rotary evaporate under 40℃ to near dry and dry with N2 flow. Add 5mL acetone-n-hexane (1 + 9) into the mixture to dissolve, shake and wait for sample preparation.
The Cleanert® Silica was activated and equilibrated with 5mL acetone / n-hexane (V / V, 1 + 9). Load the extraction onto the cartridge. Elute the cartridge with 5mL acetone hexane (V/V, 6+4). Collect elution and rotary evaporate under 40℃ to near dry. Reconstitute into 1 mL of water for further analysis.
Above steps was operated on Qdaura® Automated SPE Workstation.
HPLC column: Unisol C18, 3 µm, 100 Å，3.0 × 50 mm
Mobile phase: 0.1% formic acid aqueous (A), 0.1% Formic acid acetonitrile (B)
Column temperature: 30℃
Injection volume: 5µL
Table 1. Gradient
Mass spectrometer conditions
Ion source：ESI-；ESI voltage：-4500 V; atomizer pressure：50 psi; air curtain pressure：10 psi; Auxiliary gas pressure: 60 psi; ion source temperature: 550℃; collection method: multiple-reaction monitoring (MRM).
Table 2. MS Parameters
Notes: The quantitive ions are underlined.
Results and discussion
Table 3 shows that the recovery of spike sample was 70%-150% by LC/MS/MS using Cleanert Silica and the method can meet the demand of fish testing. It can also be seen from Figure 1 that the peak shape is sharp and the retention time of phenicols is stable using Unisol C18 column.
Table 3. The Recoveries of the 3 kinds of Phenicols in Fish
Figure 1. Chromatogram of 0.02 µg/mL Standard Solution of Phenicols Mix
In this study, the sample preparation methods of 3 kinds of phenicols in fish were established, and the samples were detected by LC-MS / MS. The result shows that the overall recoveries ranged from 70% to 115% of 4.0µg/kg spiking fish samples, which meets the testing requirements. This study indicated that the method was suitable for simultaneous detection of phenicols in fish.
200 mg/3 mL
3 µm, 100 Å，3.0 × 50 mm
Qdaura® Automated SPE Workstation
4 channel, 24 position