P6. - EuroResidue

Validation of a Method for the Determination of Residues
of ß-Lactam
ß Lactam Antibiotics in Milk by
y LC-MS/MS
LC MS/MS
D A
D.
A. Bohm
Bohm, S
S. Mönch and J.
J Mankertz
Introduction
The ß
ß-lactam
lactam antibiotic groups penicillins and cephalosporins are prevalently used in the therapy of infectious diseases of animals. Maximum residue
limits (MRL) in different food matrices were established by European Regulation 470/2009/EC [1] and subsequent modifications to ensure human food
safety.
y With regard
g
to this aim,, methods for the determination of antibiotics should be developed
p in the range
g of the MRL. The p
presented method is able
to quantify and confirm penicillins and cephalosporins in cow milk between 0.5
0 5 and 2.5
2 5 x MRL.
MRL Applying a factor-comprehensive
factor comprehensive in-house
in house validation
concept,
p , merelyy eight
g runs on different concentration levels need to be analysed
y
in order to g
gain extensive knowledge
g about the reliability,
y, robustness
and performance of the method [2].
[2] The validation was carried out in accordance with Commission Decision 2002/657/EC [3].
[3] The relevant validation
parameters like the critical concentrations ((CCα
p
(CC and CCβ),
CCβ)
β), the repeatability,
p
y, the within-laboratoryy reproducibility
p
y and the recoveryy were calculated.
E p i
Experimental
t l
R
Results
l
T bl 2
Table
2: V
Validation
lid ti parameters
t
off ß-lactam
ßl t
antibiotics
tibi ti iin milk
ilk att MRL
Sample and Spiking
- weigh in 2 g of cow milk
- add spike solution for validation and internal standards
- wait for 10 min
Extraction
- add 6 ml of acetonitrile
- vortex sample for approximately 1 min
- centrifuge at 3800 g for 5 min at 5 °C
C
- collect
ll the
h supernatant
p
- evaporate to dryness at 40 °C
C under nitrogen
- redissolve
di l iin 5 mll off buffer
b ff solution
l ti
(McIlvaine pH=4
pH 4.00 + 00.11 M Na2EDTA)
Clean-up
Clean
up by solid-phase
solid phase extraction
- condition
di i OASIS HLB cartridge
idg (6
( ml,l, 200 mg)
g))
with 6 ml of methanol and 6 ml of water
- apply
l th
the buffer
b ff solution
l ti onto
t th
the cartridge
t id
- wash with 6 ml of mix water/methanol 95/5 (v/v)
- dry under air stream for 10 min
- elute with 6 ml of methanol under vacuum
- evaporate the eluate to dryness at 40 °C
C under nitrogen
S b
Substances MRL MRL
[ /k ]
[µg/kg] CCα CCα
[ /k ]
[µg/kg] CCβ CCβ
[ /k ]
[µg/kg] RSDr* [%]
[%] RSDwR** [%]
[%] Recovery Recovery
[%]
[%] Penicillins Penicillins
A
Amoxicillin i illi
Ampicillin ll
Penicillin G Cloxacillin
Cloxacillin Dicloxacillin
Dicloxacillin Nafcillin
Nafcillin O illi
Oxacillin Penicillin V (no MRL) Cephalosporins p
p
Cefalexin
Cefalexin Cefquinome
Cefquinome C fti f
Ceftiofur Desfuroylceftiofur Cefapirin p
Desacetylcefapirin
Desacetylcefapirin Cefoperazone
Cefoperazone Cefalonium
Cefalonium Cefazolin f l
4
4 4 4 30
30 30
30 30
30 30
30 10 100
100 20
20 100
100 100 60 60
60 50
50 20
20 50 4 33
4.33 4.81 4.44 33.8
33.8 36 3
36.3 35 5
35.5 35 3
35.3 11.8 121
121 24 7
24.7 121
121 126 69.2 73.4
73.4 58 5
58.5 22 7
22.7 56.8 4 89
4.89 6.42 5.18 40.6
40.6 48 7
48.7 45 6
45.6 46 0
46.0 15.3 160
160 33 1
33.1 160
160 183 85.2 101
101 74 0
74.0 27 4
27.4 68.7 39
3.9 7.0 5.8 5.1
5.1 78
7.8 74
7.4 59
5.9 7.0 66
6.6 88
8.8 61
6.1 10.1 6.5 9.2
9.2 80
8.0 62
6.2 6.4 42
4.2 10.0 5.8 6.2
6.2 10 1
10.1 90
9.0 86
8.6 8.8 10 1
10.1 11 1
11.1 99
9.9 12.6 7.4 11.5
11.5 84
8.4 67
6.7 6.7 99
99 100 98 104
104 102
102 109
109 109
109 102 100
100 99
99 98
98 99 103 99
99 104
104 96
96 99 * ) RSDr = relative repeatability;
p
y; **)) RSDwR = relative within-laboratory
y reproducibility
p
y
Reconstitution
- redissolve
di l iin 00.55 mll off mix
i water/acetonitrile
t / t it il 90/10 ((v/v)
/)
- vortex and centrifuge at 3800 g for 5 min at 5 °C
C
- transfer the supernatant into insert of LC vial
Measurement
- measure with LC
LC-MS/MS
MS/MS (ESI+) in MRM mode
LC-MS/MS measurement:
LC p
parameters:
t
1290 ((Agilent
(Agil t Technologies)
T h l gi )
Column “Aqua”
Aqua from Phenomenex (150 mm x 2 mm,
mm 3 µm) with adequate
guard; mobile phase: A = water (0.1
(0 1 % formic acid) and B = acetonitrile (0.1
(0 1 %
formic acid);
); g
gradient p
program:
g
0 min = 10 % B,, 1 min = 10 % B,, 12 min = 60 %
B 15 min = 60 % B,
B,
B 16 min = 10 % B,
B 25 min = 10 % B; flow: 0.3
0 3 ml/min; oven
t
temperature
t
= 30 °C;
°C injection
i j ti volume
l
= 10 µl;l sampler
l temperature
t
t
= 10 °C.
°C
MS/MS parameters: QTRAP 6500 (SCIEX)
Ionisation mode = ESI+; scan type = MRM-scheduled;
MRM scheduled; resolution Q1 and Q3 =
unit;; g
gas 1 = 70 p
psi;; g
gas 2 = 70 p
psi;; curtain g
gas = 30 p
psi;; collision g
gas = high;
g ;
ion spray voltage = 5500 V; source = 400 °C
C.
Validation
In accordance with Commission Decision 2002/657/EC the validation of the
samples was accomplished with concentrations of 0.5,
0 5 1.0,
1 0 1.5,
1 5 2.0
2 0 and 2.5
25x
MRL or with
ith 5,
5 10,
10 15,
15 20 and
d 25 µg/kg
/k for
f Pen
P V.
V The
Th selection
l ti off substances
b t
was made in agreement with the German Residue Control Plan. The study was
performed applying a specific design on the basis of an in-house
in house concept with
f t l
factor-level
l combinations
bi ti
[[2]] - 6 factors
f t
on two
t
l
levels
l ((see Table
T bl 1).
1)) The
Th
calculation of the validation data was carried out with the help of the InterVal
software (QuoData,
(QuoData Dresden,
Dresden Germany).
Germany)
Table1: Factor-level combinations ((runs)) for milk with 6 factors on 2 levels
Fig 1: LC-MS/MS
Fig.
LC MS/MS chromatograms of ß-lactam
ß lactam antibiotics in milk at MRL
C clusio s
Conclusions
Co
The validation of the method was successfully accomplished with an optimal
number
b off 40 samples
l
att the
th necessary concentrations.
t ti
Th calculated
The
l l t d
relevant validation parameters like the decision limit CCα,
CCα the detection
capability CCβ,
CCβ the repeatability (3.9
(3 9 - 10.1%),
10 1%) the within-laboratory
within laboratory
reproducibility
p
y ((4.2 - 12.6%)) and the recovery
y ((96 - 109%)) are satisfying
y g
and in the ranges required by Commission Decision 2002/657/EC [3] .
Run Storage time Lot of SPE Status of
Duration before
of sample
cartridge
LC column measurement
LC-MS/MS
MRM mode
Operator
1
2d
days
B
old
ld
di tl
directly
scheduled-advanced
h d l d d
d
G
2
2 days
y
B
new
directly
y
scheduled
M
3
2 days
A
old
after 12 h
scheduled-advanced
M
4
2d
days
A
new
after
ft 12 h
scheduled
h d l d
G
5
1 dayy
B
old
after 12 h
scheduled
G
6
1 day
B
new
after 12 h
scheduled-advanced
scheduled
advanced
M
[ ] Commission Regulation
[1]
g
470/2009/EC,, Off. J. Eur. Commun. L152 ((2009)) 11.
[2] S. Uhlig, P. Gowik and W. Radeck, Anal. Chim. Acta, 483 (2003) 351 – 362.
[3] Commission Decision 2002/657/EC,
2002/657/EC Off.
Off J.
J Eur.
Eur Commun.
Commun L221 (2002) 8
8.
7
1 da
day
A
old
directl
directly
sched led
scheduled
M
8
1 day
A
new
directly
scheduled-advanced
G
Acknowledgement
g
References:
Special
p
thanks to Ms. M. Jüsgen
g for p
proofreading.
g
Detlef
D
tl f A
A. B
Bohm,
h ,S
Sabine
bi Mö
Mönch
h and
d Joachim
J
hi Mankertz
M k t
NRL for Residues of Pharmacologically
g
y Active Substances • Federal Office of Consumer Protection and Food Safety
y
Mauerstr 39
Mauerstr.
39-42
42 • 10117 Berlin • Germany • NRL
[email protected]
TAM@bvl bund de
EuroResidue VIII • Egmond aan Zee • 23 - 25 May 2016 • Poster No
No. 6

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