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B. Delépine and J.P. Morétain AFSSA – Fougères, France

Antibiotic residues in milk : LC/MS-MS confirmation (identification and quantification) of positive results obtained with microbial screening tests. B. Delépine and J.P. Morétain AFSSA – Fougères, France. Microbial screening tests in France :. For milk payment to the producers

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B. Delépine and J.P. Morétain AFSSA – Fougères, France

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  1. Antibiotic residues in milk :LC/MS-MS confirmation (identification and quantification) of positive results obtained with microbial screening tests B. Delépine and J.P. Morétain AFSSA – Fougères, France EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  2. Microbial screening tests in France : • For milk payment to the producers • Milk channels – Interprofessional control labs. • For monitoring plans • Ministry of Agriculture – Approved official labs. EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  3. Milk payment scheme : • Delvotest MCS (cow milk) or Eclipse 100 ZEU Inmunotech (goat, ewe milk) • with Bacillus stearothermophilus • Agar diffusion tests when positive • with B. stearothermophilus and B. subtilis EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  4. Monitoring plans (Dir. 96/23/EC) : • Acidification test • with Streptococcus thermophilus • Agar diffusion tests when positive • with B. stearo., B. subtilis and B. megaterium EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  5. These qualitative methods are not sufficient to fulfil the requirements of the Regulation 2377/90Could LC/MS-MS be useful ? EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  6. Equipment : A.B. API 2000 EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  7. Chromatographic conditions : • 2 columns : • Waters Symmetry C18 – 5µm – 150x3.9 mm • Merck Lichrospher RP18 – 5 µm – 125x4 mm • 2 mobile phases : • Formic acid 0.2% in water / MeOH • PFPA 0.1% in water / MeOH and (or) ACN • Flow rate : 0.6 ml/min – Injection volume : 100 µl EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  8. Spectrometric conditions : • 2 interfaces : • APCI positive mode • ESI positive mode • Nitrogen as collision gas • All parameters optimised with standard solutions EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  9. Sample preparation : • 2 fractions of 2 ml per sample • 2 extraction procedures : • Procedure A : TCA 5% • Procedure B : ACN - dryness - NH4 acetate 0.2 M • Filtration on a 0.45 µm membrane EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  10. Strategy for the different antibiotics EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  11. Example : Some penicillins : chromato. cloxacillin ampicillin amoxycillin penicillin G EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  12. Example : Some penicillins : data EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  13. Example : Some penicillins : validation (calibration curve procedure) EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  14. Results ( with 61 samples tested) : EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  15. Comments : • ßlactam AB are widely represented • extensive use in dairy cows • high detection level with B. stearothermophilus • 30 % non confirmed samples • some samples were not studied for all AB families • possible false positive results with microbial tests • some AB are not taken into account EuroResidue V - Noordwijkerhout - 10-12 May, 2004

  16. Conclusion : Development needs : • Improvement of the method • With a more efficient equipment • Addition of missing AB and metabolites EuroResidue V - Noordwijkerhout - 10-12 May, 2004

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