Comprehensive fast multiresidue screening of
150 veterinary drugs in milk
by UPLC coupled to ToF mass spectrometry
COGNARD Emmanuelle, ORTELLI Didier and EDDER Patrick
Official Food Control Authority and Veterinary Affairs of Geneva
Swiss Centre for
Applied Toxicology
CP 76, 22 Quai Ernest-Ansermet, 1211 Genève 4 Plainpalais, Switzerland
E-mails: [email protected] and [email protected]
www.scaht.org
Introduction
In food analysis, techniques involving mass spectrometry (MS) as a detection tool have evolved substantially these last years. Nowadays, the use of ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time-of-flight mass
spectrometry (TOF-MS) allows the screening of veterinary drugs residues in different matrices.
An easy sample preparation based on protein precipitation associated with ultrafiltration was hyphenated to fast chromatography to perform a comprehensive screening of veterinary compounds in raw milk. The method enabled the screening for 150 drugs and
metabolites including avermectines, benzimidazoles, β-antagonists, β-lactams, corticoids, macrolides, nitroimidazoles, quinolones, sulfonamides, tetracyclines and some other veterinary medicinal products. Identification of contaminants is based on accurate mass
measurement. UPLC-TOF showed very good performances for quantification and allowed the determination of majority of compounds below maximum residue levels (MRL).
UPLC-TOF
Matrix effect
The matrix effect on response was evaluated by using a post-column infusion system. Several blank milk and blank water
extracts from different origins were injected. A syringe pump system was used for continuous post-column infusion of analyte
95
standard solution at 100 ng/mL and 10 µL/min flow rate between the analytical column and the MS source. Data were recorded
Gradient
and signal intensity with or without matrix was compared. Chromatograms for 8 representative compounds are reported below.
% B 50
Significant signal
suppression or enhancement
No matrix effect
5
0
6
Time
9 min
7
Tetracycline
100
Levamisol
100
Sulfamethazine
Ipronidazole
mean matrix effect -26%
mean matrix effect -7%
water
Materials
7.16 7.39
6.66
LC-MS :
Column UPLC :
Precolumn :
Mobile phase :
Flow :
Injection :
Column temperature :
Sample temperature :
Source :
Acquity UPLC (Waters®) – TOF LCT Premier XE (Waters®)
Acquity BEH C18 1.7 µm, 100 x 2.1 mm (Waters®)
VanGuard Acquity BEH C18 1.7 µm, 5 x 2.1 mm (Waters®)
A : H2O + 0.1 % formic acid – B : MeCN + 0.1 % formic acid
400 µL/min – Total run time = 9 minutes
5 µL
40°C
10°C
Temperature = 120°C – Capillary voltage = 3 kV
ESI+, V mode (resolution ~ 7'000 FWHM)
without DRE * for screening – with DRE * for confirmation
Cone :
Voltage = 40 V – N2 flow = 20 L/Hr
Desolvation :
Temperature = 380°C – N2 flow = 750 L/Hr
Tune (sensitivity + resolution): 0.5 ng/µL leucine-enkephalin solution infused at 5 µL/min
Lockspray :
Leucine enkephalin reference solution infused
Daily calibration :
With sodium formate solution
Acquisition :
Mass range of 50-1150 m/z – Scan time 200 ms
Data processing :
150 veterinary coumpounds – Mass window 20 mDa
milk + water
water
milk + water
50 %
5.09
4.65
4.26
7.41
3.51
6.08
5.555.95
50 %
2.83
3.86
3.73
5.52
4.78
5.20
6.85
7.047.25
3.05
3.64
3.37
3.26
4.57
4.334.48
3.463.60
2.11
milk
1.58
milk
0.76
min
0
1
2
3
4
5
6
7
1
2
3
Cefquinom
100
4
5
6
7
Penicillin G
min
0
1
2
3
4
5
6
1
7
2
3
4
5
6
7
Enrofloxacine
Febantel
mean matrix effect +1300%
matrix effect from +18% to -95%
100
milk
6.78
milk + water
milk + water
water
50 %
1.58
50 %
6.75
5.83
1.76
4.52
3.55
1.771.89
0.96
0
1.39
1.51
water
4.46
4.19
4.64
4.83
2.95
2.28 2.79
2.46
2
3
4
5
6
1
7
2
3
2.36
milk
min
1
1.96
4
5
6
7
min
0
1
2
3
4
5
6
1
7
2
3
4
5
6
7
* DRE = dynamic range enhancement – The use of DRE mode extends the dynamic range and reduce shifts of exact masses caused by peak saturation
but can reduce drastically sensitivity. During confirmation analyses, DRE mode was activated to ensure a wider dynamic range and more accurate
quantitation.
Matrix effect varies from case to case but was mainly compound-dependent. Febantel and Erythromycin showed significant
change in signal response according to milk sample. A great number of quinolones were subjected to signal enhancement
explaining high accuracy during validation process.
Sample preparation : generic milk preparation
Example of a real positive milk sample
The complete sample preparation was carried out using single-use disposable in order to resolve problems of contamination
from sample to sample.
Milk samples (at least 50 mL) were mixed to a homogeneous mixture before considering a test portion of 750 µL.
100
100
0.59
A
B
Extract ion
for Tetracyclin
2.35
0.74
STEP 1
Protein precipitation
with ACN containing IS
500 µL
STEP 2
STEP 3
Ultrafiltration
with cut-off of 3 kDa
Evaporation of ACN fraction
Total ion
chromatogram
0.89
50 %
15 min
50°C
50 rpm
300 mbar
Centrifugation
17'000 g
Filter
3 kDa
50 %
2.23
1.08
1.17
Agitation
1 min
+
Centrifugation
17'000 g
2.35
1.94
1.98
1.60
7.36
2.92
1.53
N2
2.21
5.80
1.70
6.63
4.824.90
3.05
2.432.53
1.43
6.03
2
3.18
3.43
3.54
3.84 3.98
5.07
5.27
3.32
zoom
2.5
2.21
4.42
4.36
3.69
Centrifugation
17'000 g
0
min
1
2
3
4
5
6
0
min
1
7
2
3
4
5
6
7
750 µL ACN
5 min
750 µL Milk
60 min
2 ml microtube
5 min
microcon-3
avoid dryness
100
C
445.1581
Positive sample
mass spectra
40
446.1596
30
50 %
447.1540
0
100
Cefalexin
100
1.46
50 %
Oxytetracyclin
X
20
X
Isotope model
X
50 %
10
100
2.01
100
2.12
444
445
446
447
448
0
0
Compound name: Tetracyclin (M018)
Correlation coefficient: r = 0.999728, r^2 = 0.999456
Calibration curve: 0.614886 * x + 10.6588
Response type: Area
Curve type: Linear, Origin: Exclude, Weighting: Null
10
20
30
40
µg/L
50
2.22
Conclusion
50 %
50 %
50 %
m/z
447.1668
0
Enrofloxacin
X
m/z
446.1643
Metronidazol-hydroxy
Quantification by
standard addition :
Tetracyclin 17 µg/L
445.1611
100
Extract ion chromatograms for a milk spiked at 10 µg/L
D
This poster presents the potential of UPLC hyphenated to TOF-MS to carry out mutliclass veterinary drugs residues screening
0
0
1
2
0
min
2
3
min
0
2
3
min
2
3
min
in milk. This technique combines the good efficiency and resolution of separation with the high sensitivity and selectivity of
accurate mass spectra detection. Joined to an easy and rapid sample preparation, these advantages allow to perform very fast
Ipronidazol
Sulfamethoxazol
100
100
2.80
2.96
Erythromycin
100
Mebendazol
100
3.17
and generic analysis with a simultaneous identification of few hundred contaminants, a good preliminary quantification and a
detection of possible not compliant samples. This procedure is easily applied in routine for official control of veterinary drugs
3.30
residues in milk since it allows the comprehensive screening of 150 veterinary drugs residues in raw milk. Around 50 samples
can be analysed per day including calibration and stabilisation of the analytical system.
50 %
50 %
50 %
50 %
The major drawback of this generic sample preparation is inevitable matrix effects which can reduce or enhance substantially
the response signal. Rather than trying to relieve these matrix effects by a more complex sample preparation, it was decided to
0
0
2
min
3
0
2
3
Penicillin G
100
min 0
Oxacillin
100
3.39
3.78
3
4
min
3
100
4.09
accommodate and to react case by case for the lower number positive samples. Confirmation quantitative analysis are carry
out either by standard addition quantification, by using labelled standards or even by using a dedicated method with specific
sample preparation for the corresponding compounds.
Febantel
Nafcillin
100
4
min
4.86
According to high sensitivity and selectivity of TOF-MS detection, achieved limits of detection were between 0.5 and 25 µg/L
and largely below MRL for the majority of compounds. Except some problems with avermectines, the method allowed
50 %
50 %
50 %
screening and quantitation for benzimidazoles, β-agonists, β-lactams, corticoides, macrolides, nitroimidazoles, quinolones,
50 %
sulfonamides, tetracyclines and some others veterinary drugs.
0
0
3
4
min
0
3
4
min 0
4
5
min
4
5
min
References
Above, examples of extract ion chromatograms for 12 compounds of the main families of veterinary drugs which can potentially
be observed in a raw milk sample. These results were obtained by spiking at 10 µg/L a blank milk with different standard
(1) Ortelli D., Cognard E., Jan P. and Edder P., J. Chrom. B, 2009, 877 (23) : 2363-2374.
solutions of antibiotics or other veterinary products. Spiked samples were analysed by UPLC-TOF after the previously
(2) Stolker A.A.M., Rutgers P., Oosterink E., Lasaroms J.J.P., Peters R.J.B., van Rhijn J.A., Nielen M.W.F., Anal. Bioanal.
described sample preparation (protein precipitation + ultrafiltration).
Chem., 2008, 391(6) : 2309-2322.
Département de l'économie, des affaires régionales et de la santé
Service de la consommation et des affaires vétérinaires