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

EuroResidue V - Noordwijkerhout May, Equipment : A.B. API 2000

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

EuroResidue V - Noordwijkerhout May, 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 May, 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

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

EuroResidue V - Noordwijkerhout May, Example : Some penicillins : data Rt (min)Parent ions Product ions MRLLOD estimation Amoxicillin Ampicillin Penicillin G Cloxacillin

EuroResidue V - Noordwijkerhout May, Example : Some penicillins : validation (calibration curve procedure) Penicillin GCloxacillin 2 : 2.14 – : 13.5 – : 4.15 – : 27.8 – : 7.81 – : 59.5 – 63.4 y = x y = x – 3.25  =  = CC  = 4.73CC  = 33.71

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

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