RESIDUE DEPLETION STUDY OF AMOXICILLIN
IN THE EDIBLE TISSUES OF CHICKENS FOLLOWING CONTINUOUS DOSING OF
SURAMOX 50% OSP IN DRINKING WATER AND DETERMINATION OF THE
WITHDRAWAL PERIOD
X. REY-GROBELLET1, B. HEAL2, P.BIRCKEL2 & L.MAYNARD1
1Virbac,
13ème rue LID, 06516 Carros, France ; 2Avogadro, 31470 Fontenilles, France .
OBJECTIVE
The aim of the studies was the determination of the
withdrawal period for SURAMOX® 50% OSP when
administered in the drinking water (continuous dosing) at the
recommended therapeutic dosage regimen to poultry, the
target species. SURAMOX® 50% OSP is an amoxicillin-based
oral soluble powder.
MATERIALS AND METHODS
Animals and treatments
After 7-day acclimatization period and random allocation to 5
groups of 6 birds, 24-day old chicken were treated with
SURAMOX® 50% OSP at the dosage regimen of 20 mg
amoxicillin per kg body weight per day, and for 5 consecutive
days. Water intakes were measured to compute actual dose
rate. Animals were slaughtered 6, 12, 24, 48 and 72 hours
after the last day of treatment, and samples of muscle, liver,
kidneys and skin plus fat (in natural proportion) were collected
and frozen at ca -80°C for further analysis of amoxicillin
residues.
Residues determination
Amoxicillin (the marker residue (1)) was determined using GLP
HPLC-MS/MS method, fully validated according to the criterion
set in the Volume 8 (2). After liquid solid extraction with
H2O/methanol (25/75) using Oasis HLB 1cc 30 mg cartridges,
dried residues were solubilised in water/hexane (25/10) and
further injected onto the HPLC system. Analytical conditions
were as follows:
Column :
Hypersil® BDS C18, 100 x 3.0 mm, 3 µm
Flow rate:
0.2 mL.min-1
MSMS conditions:
Ionization mode: Ionspray in the positive ion mode
Mobile phase and gradient
Time (min)
0.0
5.0
12.0
13.0
13.1
20.0
H2O/Acetonitrile (70/30)
80%
80%
0%
0%
80%
80%
H2O/Acetonitrile (70/30)
20%
20%
100%
100%
20%
20%
Withdrawal period determination
The withdrawal period (WT) was determined in compliance
with the European Guideline in force (3).
RESULTS
Analytical method parameters
Muscle
Specificity
Selectivity
Linearity
LOD
LLOQ
Accuracy %
Stability
MRL
Kidney
Skin + fat
Yes
Against ceftiofur, clavulanic acid, enrofloxacin,
ciprofloxacin, oxolinic acid, oxytetracycline and
chlortetracycline
10 - 1000
20 - 1000 20 - 1000
10 - 1000
2.5
6.2
6.8
4.3
10
20
20
10
-3.2 - 3.7
-3.8 - 3.0 1.5 - 9.2
-3.4 - 0.0
138
131
125
141
50
This method complies with the criterion set in the Volume 8
and in particular, the LOQ ≤ ½ LMR.
(2)
,
Administered doses
Average daily administered doses ranged from 18.7 ± 0.9 to
20.0 ± 1.3 mg.kg-1.
Residues determination
Amoxicillin residues levels were all below the MRL, whatever
the bird, the tissue and the slaughter time.
Slaughter
time (h)
6
12
24
48
72
MRL
LOQ
LOD
Muscle
Liver
Kidney
Skin + fat
BLD (1.25)
ND
BLD (1.25)
BLD (1.25)
BLD (1.25)
50
10
2.5
BLD (3.1)
BLD (3.1)
BLD (3.1)
BLD (3.1)
BLD (3.1)
50
20
6.2
BLD (3.4)
BLD (3.4)
BLD (3.4)
BLD (3.4)
BLD (3.4)
50
20
6.8
BLD (2.15)
ND
BLD (2.15)
BLD (2.15)
BLD (2.15)
50
10
4.3
ND: not determined; LOD: limit of detection; LOQ: limit of
quantification; BLD: below the limit of detection. Values in brackets:
value set as ½ LOD, as stated in (3).
Withdrawal period determination
Because all residues (whatever the tissue, the sampling time
and the bird) were below the MRL and the LOD, the use of the
statistical method (EMEA software WT14), recommended by
the European guideline in force (3) for the withdrawal period
determination, was not possible. The alternative approach was
thus retained, in compliance with this guideline. Because all
residues were below the LOD from 6 hours after dosing, this
target value was retained, together with a safety span. A
withdrawal period of 1 day was thus determined.
CONCLUSIONS
Two in vivo GLP residues depletion studies (with slaughter
times of 6, 24, 48, 72 Hours, and 12 hours respectively) were
performed in the target species, using the amoxicillin-based
SURAMOX® 50% OSP product. After treatment at the
recommended therapeutic scheme and slaughtering at
different times, amoxicillin residues were determined in edible
tissues using a GLP HPLC-MS/MS method fully validated
according to the Volume 8 (2). From 6 hours after the last
treatment, all residues were found below the LOD. Using a
safety span, a withdrawal period of 1 day was proposed. This
withdrawal period perfectly complies with the recent CVMP
opinion about penicillins residues markers (1) and the guideline
in force in the EC (3), perfectly ensures the consumer safety,
and was accepted by different European Agencies
Liver
Linearity, LOD, LOQ; MRL: µg.kg-1; Stability: long term stability at ca -80°C
(1)
: Summary report: Penicillins – Rev. 1
: Volume 8: Notice to applicants and guideline:
establishment of maximum residue limits (MRLs) for
residues of veterinary medicinal products in foodstuffs of
animal origin – October 2005.
(3)
: EMEA/CVMP/036/95/-Final: Note for guidance: approach
toward harmonization of withdrawal periods.
(2)
11th EAVPT International Congress
Leipzig, 12-16 July 2009