Tylosin, also known as Tai, tylosin, is a macrolide antibiotic which is obtained from Streptomyces fradiae culturemedium in USA in 1959. It has the specific effects on the mycoplasma and it not only has the very strong antibacterial effects on a variety of G+ bacteria, but also has an inhibitory effect on the part of the G- bacterium, campylobacter, spiral and coccidiosis. It exists in the form of tartaric acid salt, phosphate, chloride, sulfate and lactate and it is soluble in water. It is widely used in the prevention and control of animal husbandry and feed additive. In 2006 the EU Member States fully ban the use of antibiotics as growth promoters. The EU provide the standard that the highest residue of tylosin is 100 μg/kg in the meat. In 2002 China's Ministry of agriculture announced "the highest on veterinary drug residues in animal food notice", it said that the maximum residue levels of tylosin is 200 μg/kg in muscle, fat, liver, kidney of chicken, pigs, cattle.
After weighing 5.0 g (accurate to 0.01g) homogenized pork, the sample was put into a 50.0 mL centrifuge tube and then added in 20.0 mL acetonitrile. Then homogenized it for 1 min, and oscillated it for 10min before centrifuging for 5 min at 4200 r/min speed. The supernate was transferred to another 50 mL centrifuge tube and added 20 mL acetonitrile to the residues that still in the first tube, then took the same extraction programs again. After that, two tubes of supernate were combined in a 50 mL centrifuge tube and diluted with acetonitrile to 50 mL. Then 10 mL liquid was transferred out and added in 30 mL n-hexane. Put away the n-hexane after oscillating for 2 min and centrifugation for 5 min at 4200 r/min speed. The under layer was concentrated until nearly dry in 50 °C water bath and nitrogen. As the last step, the dried residues were dissolved in 10 mL phosphate buffer in twice in order to be purified.
The Cleanert® PEP-2 (500mg / 6mL) column was activated and equilibrated using 5.0 mL methanol, 5.0 mL water and 5.0 mL phosphate buffer solution in sequence. After that, the liquid sample to purify was loaded on the column (the flow rate was controlled at 1 mL/min). The SPE column was washed using 5.0 mL water and 5.0 mL methanol : water (2:8. v/v) in turn, then put away the waste and dried it using vacuum pump for 30 min. 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 water contained 10% methanol and filtered (0.22 μm) in order to detection.
Column: Venusil® MP C18, 2.1 × 50 mm, 5 μm, 100 Å;
Mobile phase: A: water contained 0.1% formic acid, B: acetonitrile contained 0.1% formic acid;
Column Temperature: 30 °C;
Injection Volume: 10 μL;
Ion source: ESI
Scan mode: positive
Electrospray voltage: 5500 V
Atomizer pressure: 50 psi
Curtain Gas pressure: 15 psi
Aux Gas Pressure: 45 psi
Ion source temperature: 550 °C
Detection mode: multiple reaction monitoring (MRM)
Table 1 Gradient elution conditions of HPLC chromatography
Table 2 Mass spectrum parameters of Tylosin
Results and Discussion
The standards of Tylosin were added in samples at 4.0 μg/kg and 20.0 μg/kg followed by detection using SPE-HPLC-MS/MS. As showed in table 3, the spiked recoveries were in the range of 80%-100%, and the RSDs under 10% showed good reproducibility. As showed in fig.1 and figure 2, the peak shape of Tylosin was satisfactory and its retention time was stable after purification and separation by Cleanert® PEP-2 SPE column and Venusil® MP C18 column.
Table 3 Recoveries and retention time for Lincomycin in pork (n = 3)
Figure 1 Chromatogram of 4.0 μg/kg spiked pork Figure 2 Chromatogram of 20.0 μg/kg spiked pork
This study developed a LC-MS/MS method for the detection of Tylosin. Combined with solid phase extraction, it also achieved quantification of Tylosin residues in pork. With this method, 4.0 μg/kg and 20.0 μg/kg spiked samples could be directly analyzed, and the spiked recoveries were in the range of 80%-100. 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 Tylosin residues in animal derived food.