Lincomycin is mainly obtained by fermentation of streptomyces. It is often used in the 50S subunit of the bacterial ribosome, inhibiting bacterial protein synthesis by extending the inhibitory peptide. Then it removes the bacterial surface protein A and fluffy coat and makes it easily devoured and killed. Lincomycin can enhance the immune regulation of the immune system, improve nuclear leukocyte phagocytosis and bactericidal function and change the bacterial surface activity, inhibit of bacterial toxins. It can be used in the treatment of gram positive bacteria and mycoplasma infection and it has a strong effect in many treatments especially has strong effect on Staphylococcus aureus, Streptococcus and it also has inhibitory effect on anaerobic bacteria. Used as a feed additive, it can promote the growth of broilers and pigs. Nowadays, people are concerned about the animal derived food safety problems and therefore China and EU stipulate the maximum residue levels of lincomycin is 1 μ g/kg in animal derived food.
After weighing 5.0 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. The supernate was transferred to another 50 mL centrifuge tube and added 10 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 before adding 2 g NaCl and 10.0 mL n-hexane in it. Then oscillated the sample for 10min and centrifuged it for 10 min at 4200 r/min speed again. Then 10 mL liquid was absorbed from the middle acetonitrile layer into a centrifuge tube for concentration until nearly dry in 55 °C water bath and nitrogen.
As the last step, the dried residues were dissolved in 7 mL phosphate buffer in twice in order to be purified.
The Cleanert PEP-2 (500mg / 6mL) cartridge was activated using 10.0 mL methanol, 10.0 mL water, 5.0 mL 0.2% NaCl solution 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 10.0 mL water and 5.0 mL methanol: water (2:3. v/v) in turn, then put away the waste and dried it using vacuum pump. The sample was eluted using 10.0 mL methanol into a pipe then further dried it at 45 oC in nitrogen. As the last step, the residues were dissolved by 1 mL 10mmolacetic ammonia solution (contained 15%formic acid) and Filter (0.22 mm) in order to detection.
Column: Venusil ASB C18, 2.1 × 100 mm, 5 μm, 150 Å;
Mobile phase: A: 0.3% formic acid and water mixture, B: 0.3% formic acid and acetonitrile mixture;
Column Temperature: 35 °C;
Injection Volume: 10 μL;
Ion source: ESI
Scan mode: positive
Electrospray voltage: 5500 V
Atomizer pressure: 45 psi
Curtain Gas pressure: 15 psi
Aux Gas Pressure: 50 psi
Ion source temperature: 550 °C
Detection mode: MRM
Table 1 Gradient elution conditions of HPLC chromatography
Table 2 Mass spectrum parameters of lincomycin
Results and Discussion
The standards of Lincomycin were added in samples at 1.0 μg/kg followed by detection using SPE-HPLC-MS/MS. As showed in table 3, the spiked recoveries were in the range of 84%-105%, and the RSDs under 10% showed good reproducibility. As showed in fig.1 and fig.2, the peak shape of Lincomycin 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 Recoveries and retention time for Lincomycin (n = 3)
Figure 1 HPLC-MS/MS chromatogram of Lincomycin (adding standards 1.0 μg/kg)
This study developed a LC-MS/MS method for the detection of Lincomycin. Combined with solid phase extraction, it also achieved quantification of Lincomycin residues in pork. With this method, 1.0 μg/kg spiked samples could be directly analyzed, and the spiked recoveries were in the range of 84%-105%. 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 Lincomycin residues in animal derived food.