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Antibiotics residues

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Antibiotics residues
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
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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.
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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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
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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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
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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These qualitative methods are not
sufficient to fulfil the requirements of
the Regulation 2377/90
Could LC/MS-MS be useful ?
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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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
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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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
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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
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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Strategy for the different antibiotics
Antibiotics Extraction
Column
Elution
Source/T°
ß lactams
Proc. B
Lichrospher
FA/MeOH
ESI+/300°C
Tetracyclins
Aminoglyc.
Quinolones
Proc. A
Proc. A
Proc. A
Symmetry
Symmetry
Symmetry
PFPA/ACN
PFPA/MeOH
PFPA/ACN
APCI+/450°C
APCI+/500°C
APCI+/500°C
Symmetry
PFPA/MeOH/
APCI+/450°C
ACN
Macrolides
and Sulfa.
Proc. B
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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penicillin G
ampicillin
amoxycillin
cloxacillin
Example : Some penicillins : chromato.
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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Example : Some penicillins : data
Rt (min)
Parent Product MRL
LOD
ions
ions
estimation
366 114-349
4
3.0
Amoxicillin
4.70
Ampicillin
9.05
350
106-160
4
1.0
Penicillin G
12.3
335
160-176
4
0.6
Cloxacillin
13.6
436
160-277
30
0.8
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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Example : Some penicillins : validation
(calibration curve procedure)
Penicillin G
Cloxacillin
2 : 2.14 – 2.34
15 : 13.5 – 13.7
4 : 4.15 – 3.58
30 : 27.8 – 27.7
8 : 7.81 – 8.21
y = 0.972 x + 01675
60 : 59.5 – 63.4
y = 1.072 x – 3.25
 = 0.3070
 =1.7172
CC = 4.73
CC = 33.71
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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Results ( with 61 samples tested) :
Antibiotics
Nb of + samples Concent. range Conc. > MRL
Penicillin G
29
1 – 700,000
25
Cefalonium
6
7 – 2000
5
Cloxacillin
5
28 – 135
3
Oxacillin
2
4 and 2500
1
Cephapirin
1
570
1
Ampicillin
1
184
1
Spiramycin
1
10
0
Neomycin
1
400
0
Tetracycline
1
15
0
EuroResidue V - Noordwijkerhout - 10-12 May, 2004
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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
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