Yan, CQ; Li, SP; Zhang, S; Xie, S; Wei, KY; Huang, XH
Objective: Cefalonium is a first generation semi-synthetic cephalosporin with broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The cefalonium is used intramammarily during the dry period of cattle at a recommended dose of 250 mg per quarter to treat existing sub-clinical infections and to prevent new infections. The bactericidal activity of cefalonium is a result of its inhibitory action on bacterial cell wall synthesis due to binding to one or more penicillin binding proteins located under the cell wall susceptible bacteria. The resulting high internal osmotic pressure leads to rupture of the cytoplasmic membrane. The objective of this study was to establish a withdrawal period for cefalonium in milk, following intramammary infusion treatment.
Method: Twenty healthy dairy cows in the dry period were injected with a cefalonium injection into each breast area. The average dry period was 50 days, and they were mixed with the samples of 4 milk areas collected per cow at 6, 12, 24, 36, 48, 60, 72, 96 and 120 h after the start of milking. Residues of cefalonium were detectable in milk for up to 6, 12, 24, 36, 48, 60, 72, 96 and 120 h post-treatment, respectively. Phenomenex Luna C18 (150 mm×2 mm, 5 μm) was used. Acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase. The gradient elution procedure was used. The flow rate was 0.25 mL·min -1, the column temperature was 35°C, and the injection volume was 5.0 μL. Phenomenex Luna C18 (150 mm×2.1 mm,3.5 μm) was used. Acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase. The gradient elution procedure was used. The flow rate was 0.25 mL/min, the column temperature was 35°C, and the injection volume was 5.0 μL. MS conditions of detection method: ESI ion source, positive ion scan, multiple reaction monitoring, ion spray voltage: 4kV, the pressure of GS1: 55 psi, the pressure of CUR: 15 psi, the pressure of GS2: 35 psi, TEM: 600℃, the quantitative ion is: cefalonium m/z→459.4/337.3. The sample was thawed naturally, 5g of milk was accurately pipetted into a 50 mL centrifuge tube, 20 mL of acetonitrile was added, vortexed and shaken for 2min, and centrifuged at 10 000 r/min for 10 min. The supernatant was extracted once with 15 mL of 75% acetonitrile aqueous solution, and the extracts were combined twice, and 10 mL of acetonitrile saturated n-hexane was added, shaken for 1 min, the n-hexane was discarded, and the extract was transferred to a 100 mL heart bottle. Acetonitrile was removed by rotary evaporation at 40 ° C using a rotary concentrator. To the above sample solution, 20 mL of a sodium dihydrogen phosphate buffer solution was added, followed by adjustment to pH = 8.5 with a sodium hydroxide solution. The sample was passed through the HLB solid phase extraction column, then the heart bottle was washed with 5 mL of sodium dihydrogen phosphate buffer solution and passed through the column, and the column was washed with 2 mL of water. It was eluted with 2 mL of acetonitrile, dried at 40 °C with a nitrogen concentrator, dissolved in 2 mL of water, shaken, passed through a 0.22μm microporous membrane, LC-MS/MS detection analysis.
Result: Cefalonium showed a good linearity in the concentration range of 2-200 μg·kg -1, with a correlation coefficient of 0.991-0.997, a detection limit of 0.5 μg·kg -1, and a limit of quantification of 2 μg·kg -1. The relative recovery rate of this method at the four levels of addition of 2, 20, 40 and 200 μg·kg -1 was 82.21%-89.08%. The coefficient of intra-assay coefficient of variation was 1.85%-10.41%, and the inter-assay coefficient of variation was 3.41%-8.97%. The experimental method has high sensitivity and simple operation, and can be used for the residue depletion study of cefalonium in milk. Milk samples from these cows after calving were analyzed for antibiotic residues by a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) procedure. Primiparous dairy cows that used cefalonium intramammary infusion (dry cow) were below the limit of quantitation at 96 hours after calving. Dairy cows were below the limit of quantitation at 36 hours after calving.
Conclusion: After the cefalonium intramammary infusion (dry cow) was administered at the recommended dose of 4 milk areas (250 mg/milk area), the withdrawal period in milk was 24 h.