1. EuroResidue V - Noordwijkerhout - 10-12 May, 20041 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

2. EuroResidue V - Noordwijkerhout - 10-12 May, 20042 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.

3. EuroResidue V - Noordwijkerhout - 10-12 May, 20043 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

4. EuroResidue V - Noordwijkerhout - 10-12 May, 20044 Monitoring plans (Dir. 96/23/EC) : Acidification test –with Streptococcus thermophilus Agar diffusion tests when positive –with B. stearo., B. subtilis and B. megaterium

5. EuroResidue V - Noordwijkerhout - 10-12 May, 20045 These qualitative methods are not sufficient to fulfil the requirements of the Regulation 2377/90 Could LC/MS-MS be useful ?

6. EuroResidue V - Noordwijkerhout - 10-12 May, 20046 Equipment : A.B. API 2000

7. EuroResidue V - Noordwijkerhout - 10-12 May, 20047 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

8. EuroResidue V - Noordwijkerhout - 10-12 May, 20048 Spectrometric conditions : 2 interfaces : –APCI positive mode –ESI positive mode Nitrogen as collision gas All parameters optimised with standard solutions

9. EuroResidue V - Noordwijkerhout - 10-12 May, 20049 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

10. EuroResidue V - Noordwijkerhout - 10-12 May, 200410 Strategy for the different antibiotics AntibioticsExtractionColumnElutionSource/T° ß lactamsProc. B Lichrospher FA/MeOHESI+/300°C TetracyclinsProc. ASymmetryPFPA/ACNAPCI+/450°C Aminoglyc.Proc. ASymmetryPFPA/MeOHAPCI+/500°C QuinolonesProc. ASymmetryPFPA/ACNAPCI+/500°C Macrolides and Sulfa. Proc. BSymmetry PFPA/MeOH/ ACN APCI+/450°C

11. EuroResidue V - Noordwijkerhout - 10-12 May, 200411 Example : Some penicillins : chromato. amoxycillin ampicillin cloxacillin penicillin G

12. EuroResidue V - Noordwijkerhout - 10-12 May, 200412 Example : Some penicillins : data Rt (min)Parent ions Product ions MRLLOD estimation Amoxicillin4.70366114-34943.0 Ampicillin9.05350106-16041.0 Penicillin G12.3335160-17640.6 Cloxacillin13.6436160-277300.8

13. EuroResidue V - Noordwijkerhout - 10-12 May, 200413 Example : Some penicillins : validation (calibration curve procedure) Penicillin GCloxacillin 2 : 2.14 – 2.3415 : 13.5 – 13.7 4 : 4.15 – 3.5830 : 27.8 – 27.7 8 : 7.81 – 8.2160 : 59.5 – 63.4 y = 0.972 x + 01675y = 1.072 x – 3.25  = 0.3070  =1.7172 CC  = 4.73CC  = 33.71

14. EuroResidue V - Noordwijkerhout - 10-12 May, 200414 Results ( with 61 samples tested) : AntibioticsNb of + samplesConcent. rangeConc. > MRL Penicillin G291 – 700,00025 Cefalonium67 – 20005 Cloxacillin528 – 1353 Oxacillin24 and 25001 Cephapirin15701 Ampicillin11841 Spiramycin1100 Neomycin14000 Tetracycline1150

15. EuroResidue V - Noordwijkerhout - 10-12 May, 200415 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

16. EuroResidue V - Noordwijkerhout - 10-12 May, 200416 Conclusion : Development needs : Improvement of the method –With a more efficient equipment Addition of missing AB and metabolites