With better activity of ani-anaerobic bacteria and anti-protozoan, Nitroimidazoles are used in anaerobic infections and protozoal diseases of livestock. Vivo and in vitro toxicological experiments have confirmed that nitroimidazoles can lead to deformity, mutation and cancer. Ministry of Agriculture in China has forbidden the use of metronidazole and dimetridazole as feed additives on food animals and the use of ronidazole on food animals for any purpose.
In this study, the simultaneous detection of 5 nitroimidazoles in fish was eatablished by optimizing preparation methods.
Material and Reagents
HPLC purity of methanol, acetone, n-hexane, ethyl acetate, ammonium acetate; analytical pure sodium chloride; experimental water (ultra-pure water);
Standard solution mix of 5 nitroimidazoles: hydroxyl-methylimidazole, dimetridazole, metronidazole, ronidazole, hydroxy dimetridazole, dissolved in methanol;
Cleanert S C18: 1000 mg/6 mL.
Transfer 20 g comminuted fish sample and 10 g diatomite into a 250 mL conical flask with cover, mix well. Add into 5mL saturated sodium chloride aqueous solution and 70 mL methanol : acetone =3:1(V/V) in proper order and homogenize at high speed for 3 min. Transfer extract into a centrifuge tube, centrifuge at 10000 rpm for 5 min. Then put supernatant into a 250 mL concentrate bottle, extract the residue with methanol: acetone =3:1(V/V) for twice. Put the two extract together, rotary evaporate under 40°C until only aqueous phase left and transfer it into a separatory funnel. Add 50 mL saturated sodium chloride and 25 mL ethyl acetate, shake for 3min, stratify and collect ethyl acetate phase. Repeatedly extract the aqueous phase with ethyl acetate for twice and mix the two ethyl acetate phase. Dehydrate the ethyl acetate phase with 3g anhydrous sodium sulfate, collect and rotary evaporate under 40°C to near dry. Add 5 mL methanol into the residue and filter through 0.45 μm nylon filter for sample preparation.
Activate and equilibrate Cleanert® C18 with 5 mL methanol. Apply all the extract onto the cartridge and collect the eluent. Then elute the cartridge with 10 mL methanol, collect the extract and merge the two eluents. Rotary evaporate under 45°C to dry and reconstitute into 1 mL of mobile phase for further analysis.
Column: Unisol C18, 3 µm, 100 Å, 3.0 × 50 mm;
Mobile phase: A: 5 mmol ammonium acetate; B: methanol;
Flow rate: 0.3 mL/min;
Column temperature: 30 °C;
Injection: 5 mL;
Ion source: ESI+;
Electrospray voltage:5500 V;
Nebulizer pressure: 50 psi;
Curtain gas pressure: 25 psi;
Auxiliary gas pressure: 50 psi;
Ion source temperature: 500 °C;
Scan mode: MRM.
Table 1 Gradien
Table 2 MS Parameters
* underlined were quantitative ions
Results and Discussion
Table 3 shows that the recoveries of spiked samples ranged from 90% to 110% and the CVs were under 10% using Cleanert S C18 by LC-MS/MS to determinate five Nitroimidazoles in fish. Typical method performance results were well within acceptable criteria. Figure 1 shows a good separation was obtained by using Unisol C18 column to detect five Nitroimidazoles simultaneously.
Table 3 Recoveries of five Nitroimidazoles in spiked fish samples (1.0 µg/kg)
Figure 1 Chromatogram of 0.1 µg/mL standard sample mix of five Nitroimidazoles
In this study, the sample preparation methods of five Nitroimidazoles in fish were established, and the samples were detected by LC-MS / MS. The result shows that the overall recoveries ranged from 90% to 110% and the CVs were lower than 10% for 1.0 mg/kg spiking fish samples, which meets the testing requirements. This study indicates that the method was suitable for detection of five Nitroimidazoles in fish.
Cleanert® S C18
1000 mg/6 mL
Qdaura® Automated SPE Workstation
4 channels, 24 positions
3 µm, 100 Å, 3.0 × 50 mm
Maximal 15 samples
1.5 mL vials
Screw neck vials, 12 × 32 mm
Caps and Septa
Screw neck cap, center hole; red silicone/ white PTEE septa, slitted
Syringe Filter (Nylon)
Monofilm, 13 mm, 0.22 μm
Disposable Needle-Free injection systems
2 mL, 100/pk