Introduction
Tilmicosin is a relatively new special macrolide antibiotic for livestock with tylosin for semi synthetic precursor. The British Elanco animal health care products company was the first one to develop the medicine successfully in 1980s. It is mainly used for infectious diseases of cattle, dairy cattle, goats, sheep, pigs, chicken and other animal caused by sensitive bacteria, especially for respiratory disease of livestock such as porcine Actinobacilluspleuropneumonia, cattle Hemolytic and Pasteurella multocida disease and avian mycoplasmosis. Besides, it has a good antibacterial effects on dairy cow mastitis mainly strains.
Experimental
Sample extraction
After weighing 5.00 g (accurate to 0.01 g) homogenized drug, the sample was put into a 50.0 mL centrifuge tube that had been added in 15.0 mL acetonitrile. Then homogenized it with homogenizer for 1 min, and oscillated it for 10min before centrifuging for 5 min at 4200 r/min speed. After that 2 g NaCl and 10.0 mL n-hexane were added into the separated supernate, then oscillated it for 10 min and centrifuged it for 5 min at 4200 r/min speed again. As the last step, transferred the middle acetonitrile layer 12.0 mL on another centrifuge tube carefully, and dried it in the nitrogen.
Purification
The Cleanert PEP-2 (500mg / 6mL)column was activated using 10.0 mL methanol, 10.0 mL water, 5.0 mL 2% NaCl solution and 5.0 mL ammonium dihydrogen phosphate buffer solution in sequence. After that, dissolved the residual liquid sample using 7.0 mL ammonium dihydrogen phosphate buffer in twice (the flow rate in the range of 2-3 mL/min) and put away the filtrate. The SPE column was washed using 10.0 mL water and 5.0 mL methanol: water (2:3. v/v) in turn, then put away the waste and dried it in negative pressure. The sample was eluted using 10.0 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 0.1% acetic ammonia solution and Filter (0.22 μm) in order to detection.
Instrumentation
Column: Venusil ASB C18, 2.1 × 100 mm, 5 μm, 150 Å;
Mobile phase: A: 10 mM ammonium acetate solution, B: acetonitrile;
Column Temperature: 35 °C;
Injection Volume: 5 μL;
Ion source: ESI
Scan mode: positive
Electrospray voltage: 5500 V
Atomizer pressure: 60 psi
Curtain Gas pressure: 13 psi
Aux Gas Pressure: 65 psi
Ion source temperature: 600 °C
Detection mode: MRM
Table 1 Gradient elution conditions of HPLC chromatography
Time (min) | Flow (mL/min) | A% | B% |
0.00 | 0.35 | 95 | 5 |
1.50 | 0.35 | 95 | 5 |
2.00 | 0.35 | 60 | 40 |
4.00 | 0.35 | 60 | 40 |
4.01 | 0.35 | 10 | 90 |
5.00 | 0.35 | 10 | 90 |
5.01 | 0.35 | 95 | 5 |
7.00 | 0.35 | 95 | 5 |
Table 2 Mass spectrum parameters of Tilmicosin
Compound | Q1 | Q3 | CE/V |
Tilmicosin | 869.8 | 696.7 | 58 |
174.5 | 60 |
Results and Discussion
The standards of Tilmicosin were spiked in samples at 1.0 μg/kg and 20.0 μg/kg respectively, followed by detection using SPE-HPLC-MS/MS.As showed in table 3, the spiked recoveries were in the range of 90%-100%, and the RSDs under10% showed good reproducibility. As showed in fig.1 and fig.2, the peak shape of Tilmicosin was satisfactory and its retention time was stable after purification and separation by Cleanert PEP-2 SPE column and Venusil ASB C18 column.
Table 3 Spiked recoveries and retention time for Tilmicosin (n = 3)
Compound | Concentration(μg/kg) | Recovery (%) | RSD (%) | RT (min) |
Tilmicosin | 1.0 | 92.52 | 3.88 | 2.50 |
20.0 | 91.91 | 4.21 |
Fig.1 the HPLC-MS/MS chromatogram of Tilmicosin(1.0 μg/kg)
Fig.2 the HPLC-MS/MS chromatogram of Tilmicosin(20.0 μg/kg)
Conclusion
This study developed a LC-MS/MS method for the detection of Tilmicosin. Combined with solid phase extraction, it also achieved quantification of Tilmicosin residues in livestock meat. With this method,1.0 μg/kg and 20.0 μg/kg spiked samples could be directly analyzed, and the spiked recoveries were in the range of 90%-100%. Solid phase extraction method showed good stability and the columns showed excellent reproducibility, which pointed out that this method could be used for the quantification of Tilmicosin residues in livestock meat.
