Antibiotic residues in milk : LCMSMS confirmation identification and quantification of positive resu

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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

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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.

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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

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Monitoring plans (Dir. 96/23/EC)

• Acidification test
• with Streptococcus thermophilus
• Agar diffusion tests when positive
• with B. stearo., B. subtilis and B. megaterium

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These qualitative methods are not sufficient to
fulfil the requirements of the Regulation
2377/90Could LC/MS-MS be useful ?
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Equipment A.B. API 2000
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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

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Spectrometric conditions

• 2 interfaces
• APCI positive mode
• ESI positive mode
• Nitrogen as collision gas
• All parameters optimised with standard solutions

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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

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Strategy for the different antibiotics
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Example Some penicillins chromato.
cloxacillin
ampicillin
amoxycillin
penicillin G
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Example Some penicillins data
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Example Some penicillins validation
(calibration curve procedure)
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Results ( with 61 samples tested)
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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

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Conclusion Development needs

• Improvement of the method
• With a more efficient equipment
• Addition of missing AB and metabolites