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The Quantification of Fluoroquinolones Residues in Pork Using LC-MS/MS Method

Application Introduction

Fluoroquinolones, also called the pyridine acid class, are a series of new synthetic sterilization antimicrobial drugs in recent years.

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Introduction

Fluoroquinolones, also called the pyridine acid class, are a series of new synthetic sterilization antimicrobial drugs in recent years. Because of its broad antimicrobial spectrum, strong antibacterial activity, and no cross with other antibacterial drugs and small side effects, it is widely used in the prevention of various infectious diseases of animal and human. Besides the side effects of fluoroquinolone residues can cause harm to human directly, the more serious is that long-term consumption of animal source foods containing low concentration of FQs drugs may easily induce human disease to become bacterial resistant and may have the potential teratogenic and carcinogenic risk. China and EU stipulate the maximum residue levels for the series of quinolones is 10~90 μg/kg in animal tissues.

Experimental

Sample extraction

Weigh 5.0 g (accurate to 0.01 g) homogenized sample, then put it into 50.0 mL centrifuge tube, and 20 mL 0.1 mol/L buffer solution was added. Mixed it for 1 min at 1000 r/min speed and ultrasonic extraction for 10 min then centrifuged for 10 min at 8000 r/min speed. Repeat the same process for three times and finally combined the supernatant for detection.

Purification

After activating the Cleanert PEP-2 cartridge using 6 mL methanol and 6 mL water in sequence, the purifying liquid was loaded on the column, and flowed through the column at the speed of 2 to 3 mL/min. Then put away the filtrate. The SPE column was washed using 3mL methanol and put away the waste before drying it in negative pressure. At last, the sample was eluted using 6 mL methanol into a pipe then further dried it at 45 °C in nitrogen. As the last step, the residues were dissolved by 1 mL 0.2 % formic acid solution and filtered (0.22 μm) in order to detection.

Instrumentation

Column: Unisol C18, 2.1 × 50 mm, 5 μm, 100 Å;

Mobile phase: A: 0.1 % formic acid and water, B: acetonitrile;

Column Temperature: 30 °C ;

Injection Volume: 10 μL;

Gradient elution is shown in Table 1

Table 1 Gradient elution conditions of HPLC chromatography

Time (min)

Flow (mL/min)

A%

B%

0.00

0.3

85

15

6.00

0.3

80

20

7.00

0.3

10

90

7.10

0.3

85

15

12.00

0.3

85

15

Table 2 Mass spectrum parameters of fluoroquinolones

Compound

Q1

Q3

CE/V

Norfloxacin

320.3

302.3

25

276.3

25

Ciprofloxacin

332.2

314.3

23

288.3

23

Pefloxacin

334.3

290.3

29

233.2

29

Lomefloxacin

352.3

265.2

27

308.3

27

Enrofloxacin


360.3

316.4

27

342.3

27

Ofloxacin

362.2

318.3

27

261.2

27

Results and Discussion

The standards of different drugs were added in samples at 2.0 μg/kg, 20 μg/kg and 100 μg/kg followed by detection using SPE-HPLC-MS/MS.As showed in table 3, the spiked recoveries were in the range of 70%-120%. As showed in Fig.1to Fig.3, the peak shape of six different drugs was satisfactory and its retention time was stable after purification and separation by Cleanert PEP-2 SPE cartridge and Unisol C18 column.

Fig.1 the HPLC-MS/MS chromatogram of fluoroquinolones(adding standards 2.0 μg/kg)

Fig.2the HPLC-MS/MS chromatogram of fluoroquinolones(adding standards 20 μg/kg)

Fig.3the HPLC-MS/MS chromatogram of fluoroquinolones(adding standards 100 μg/kg)

Table 3 Recoveries and retention time for fluoroquinolones (n = 3)

Compound

Adding standard(mg/kg)

Recovery (%)

RSD (%)

RT/min

Norfloxacin

2.0

78.77

0.15

3.26

20

84.95

0.12

100

100.34

0.03

Ciprofloxacin

2.0

87.98

0.08

3.79

20

71.63

0.11

100

79.66

0.01

Pefloxacin

2.0

86.21

0.08

3.57

20

83.65

0.15

100

92.67

0.18

Lomefloxacin

2.0

101.39

0.07

4.34

20

92.97

0.12

100

108.73

0.04

Enrofloxacin

2.0

94.27

0.03

5.02

20

88.12

0.16

100

93.05

0.05

Ofloxacin

2.0

102.73

0.08

3.23

20

103.91

0.15

100

114.35

0.12

Conclusion

This study developed a LC-MS/MS method for the detection of fluoroquinolones. Combined with solid phase extraction, it also achieved quantification of fluoroquinolones residues in pork. With this method, 2.0 μg/kg, 20 μg/kg and 100 μg/kg spiked samples could be directly analyzed, and the spiked recoveries were in the range of 70%-120%. Solid phase extraction method showed good stability and the columns showed excellent reproducibility, which pointed out that the method could be used for the quantification of fluoroquinolones residues in animal derived food.


  • Unisol Technology and Column Products

    Bonna-Agela's unique sol-gel process technology generates an uniform surface that produces an universal column applicable to several kinds of HPLC applications.

  • Cleanert PEP-2

    Cleanert PEP-2 is made of polydivinylbenzene on which the surface is functionalized with vinyl pyrrolidone and urea.

  • Qdaura Automated SPE System

    The Qdaura Automated SPE system is specifically designed for high throughput sample preparation. The system automates routine SPE procedures from conditioning, sample loading, washing, dry and elution. The Qdaura utilizes positive pressure to ensure more reproducible flow rates, more reproducible recoveries, less sample to sample contamination, and higher precision.

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