Journal of Chromatography A, 1274 (2013) 1927

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Quantitative multi-residue method for determination antibiotics in chicken meat

using turbulent ow chromatography coupled to liquid
chromatographytandem mass spectrometry
Katerina Bousova a, , Hamide Senyuva b , Klaus Mittendorf a
a
b

Food Safety Response Centre, Thermo Fisher Scientic, Im Steingrund 4-6, 63303 Dreieich, Germany
FoodLife International, 06531 Ankara, Turkey

a r t i c l e

i n f o

Article history:
Received 8 August 2012
Received in revised form 30 October 2012
Accepted 25 November 2012
Available online 19 December 2012
Keywords:
Antibiotics
On-line clean-up
LCMS/MS
Chicken meat
Food safety
Validation

a b s t r a c t
A multi-class method for identication and quantication of 36 antibiotics from seven different chemical classes (aminoglycosides, macrolides, lincosamides, sulfonamides, tetracyclines, quinolones and
trimethoprim) has been developed by using liquid chromatographymass spectrometry. The method
was optimised for detection of antibiotics in chicken meat. Sample preparation including extraction with
a mixture of acetonitrile:2% trichloroacetic acid (45:55, v/v), centrifugation and ltration was followed by
on-line clean-up using turbulent ow chromatography. Using this automated on-line technique enabled
a larger number of samples to be analysed per day than with a traditional clean-up technique (e.g. solid
phase extraction). The optimised method was validated according to the European Commission Directive
2002/657/EC. In-house validation was performed by fortifying the blank matrix at three levels 0.5, 1.0
and 1.5 MRL (maximum residue limit), or respectively, at concentrations as low as possible for substances
without an MRL. Precision in terms of repeatability standard deviation ranged from 3 to 28% and recovery
values were between 80 and 120% in most cases. All calculated validation parameters including CC and
CC were in the compliance with the legislative requirements.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Veterinary drugs are widely used in the animal husbandry to
treat and prevent diseases. Despite obvious benets, extensive use
of these drugs can lead to residues in animal products such as
meat, milk and eggs. Consumption of animal products contaminated with veterinary drug residues can cause allergic reactions
in sensitive humans, and can negatively affect the human immune
system. The most serious concern about excessive use of veterinary
dugs is the impact on the effectiveness of the human medicines
as bacteria have started to become resistant to the most common
antibiotics. In order to combat this problem and protect the human
consumers, the use of veterinary drugs is tightly regulated. The EU
Council Directive 96/23/EC [1] requires monitoring of certain veterinary drugs in live animals and in animal products. To ensure that
testing is carried out to the highest analytical standards unambiguous guidelines stipulate the rules for the analytical methods to be
used for testing food samples of animal origin for the presence of

Corresponding author. Tel.: +49 61034081113; fax: +49 61034081122.

E-mail addresses: katerina.bousova@thermosher.com
(K. Bousova), hamide.senyuva@foodlifeint.com (H. Senyuva),
klaus.mittendorf@thermosher.com (K. Mittendorf).
0021-9673/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2012.11.067

residues and contaminants [2]. For the most frequently used veterinary drugs for different types of food products maximum residue
limits (MRLs) have been set [3]. Analytical methods employed for
determination of veterinary drugs must be capable of determining
the residues below their MRLs.
To full the regulatory requirements it is necessary to employ
sensitive, selective and reliable analytical methods. For fast
screening of animal products a screening method should preferably
be used. There are various approaches for residue screening. Very
popular and quite often used are methods based on microbial or
immunological assay [4]. These methods are low-cost and rapid, but
have the disadvantage of either only being suitable for screening
broad classes of drugs or conversely being too specic and suitable
for targeting only at single residue. Instrumental techniques such as
liquid chromatographytandem mass spectrometry (LCMS/MS)
have also been used for screening, providing the added benet of
also being capable of conrming positive results. For example, a
multi-analyte LCMS/MS screening methods for 19 antibiotics in
muscle and kidney [5] and semi-quantitative determination of 39
antibiotics in veal muscle [6] have been reported. High resolution
mass spectrometry (HRMS) is another promising approach for routine screening. This approach has been employed for multi-residue
UHPLCOrbitrapMS screening for veterinary drug residues in milk
[7]. The authors claim that the developed method can be used also

20

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

Table 1
TurboFlowTM loading and eluting conditions.
Step

1.
2.
3.
4.
5.
6.

Step length (s)

60
60
60
720
120
120

Loading pumpa

Valve interface
module (VIM)

Eluting pumpb

Flow (mL/min)

%A

%B

%C

%D

Tee

Loop

Flow (mL/min)

%A

%B

1.5
0.2
1.5
1.5
1.5
1.5

100

50

50

100

50

100

50
100

Out
In
In
In
In
Out

0.3
0.6
0.3
0.3
0.3
0.3

100
100
100
5
5
100

95
95

a
Loading pump: A = 1 mM heptauorobutyric acid + 0.5% formic acid in water. B = 0.5% formic acid in ACN:methanol 1:1 (v/v). C = 2% methanol in water.
D = acetone:ACN:isopropanol 20:40:40 (v/v/v).
b
Eluting pump: A = 1 mM heptauorobutyric acid + 0.5% formic acid in water. B = 0.5% formic acid in ACN:methanol 1:1 (v/v).

for conrmatory purposes. However, the preferred instrumentation to be used for the conrmatory methods is still LCMS/MS.
In the last decades most conrmatory methods were developed only for one class of veterinary drugs. Single-class analytical
methods dealing with meat were reported for such groups as
aminoglycosides [8], tetracyclines [9,10], macrolides [11] and sulfonamides [12,13]. Notwithstanding the success of these methods
currently there is an increasing demand for fast and reliable
multi-class multi-residue methods, which if used by food control
laboratories could reduce costs and achieve high sample throughput.
Poultry meat, just like any other type of meat, having a high proteins and lipid content is a very complex matrix. Considering the
large number of target compounds from different chemical classes
with different chemical properties the most challenging part of
the multi-residue method is the sample preparation in terms of
both the extraction and clean-up. There have been used three main
approaches for sample preparation for the determination of veterinary drug residues in meat. Liquid extraction in combination with
a solid-phase extraction (SPE) as a clean-up procedure has been
used for the determination of sulfonamides in meat [12], tetracyclines in a chicken meat [9] and tetracyclines in pig tissues [10].
The second approach consists of liquid extraction in combination
with a defatting step employing hexane, which has been used in a
multi-residue method for poultry meat [14] and for another multiresidue method for veal muscle [6]. The third approach consists
only of liquid extraction and a subsequent dilution step prior direct
injection into the instrument and has been employed in two multiresidue methods for pig muscle [6,15]. The rst two ways of sample
preparation are quite effective and provide clean sample extracts,
which can be subsequently injected into the mass spectrometer.
The drawback is that the sample preparation is lengthy and in the
case where SPE is used there is the added cost of SPE cartridges. The
last approach appears to be simple and fast, but because of injecting
dirty extracts there is a high risk of MS source contamination.
The aim of the method reported here was to overcome the
disadvantages mentioned above and develop a conrmative and
quantitative multi-class method for the determination of a range of
antibiotics from different classes in chicken meat. For the clean-up
of meat extracts turbulent ow chromatography was used, which
has the added benet of being suitable for automation. This technique has been previously used in the multi-class methods for
the determination of veterinary drug residues in other matrices
such as honey [16] and milk [17]. For animal tissues turbulent ow
chromatography was used only once in one-class method for two
quinolones [18]. The reported method was rst developed and inhouse validated for milk [19]. In this study an adaptation of the
method is reported for more complex matrices, such as chicken
meat. The method was validated for determination of 36 residues
from seven different chemical classes of antibiotics according to
Commission Decision 2002/657/EC [2].

2. Experimental
2.1. Samples and quality control materials
Chicken obtained from a local market, which was repeatedly
measured to conrm that no antibiotics were present, was used
for preparation of matrix-matched calibration standards and for
fortied samples. Because of a lack of chicken tissue certied
test materials, to establish method accuracy a FAPAS test material T02174QC of sh muscle containing a certied amount of
ciprooxacin was used, which was obtained from the Food and
Environment Research Agency (York, United Kingdom). Though
sh muscle is not the same as chicken muscle, considering
the content of proteins, water and fat the differences are not
large and thus sh muscle was used to demonstrate method
accuracy.
2.2. Chemicals and reagents
Optima LCMS grade methanol, acetonitrile, water (Fisher Scientic, Langenselbold, Germany) and HPLC grade isopropanol
and acetone (Fisher Scientic, Langenselbold, Germany) were
used as mobile phases, for sample extraction, preparation
of standard mixtures and washing of the injection port.
Trichloroacetic acid for sample extraction was obtained from
Acros Organics (Geel, Belgium). HPLC grade formic acid (Fisher
Scientic, Loughborough, UK) and heptauorobutyric acid (Acros
Organics, Geel, Belgium) were used as additives for mobile
phases. 35% ammonia solution (Fisher Scientic, Loughborough, UK) was used for preparation of stock standard solutions
for quinolones. Puried water was obtained from Barnstead
EASYpure II water system (Thermo Scientic, Barnstead,
NH).
2.3. Standards
Chlortetracycline, cinoxacin, ciprooxacin, clarithromycin,
clindamycin hydrochloride, danooxacin, dioxacin hydrochloride, doxycycline hyclate, enoxacin, enrooxacin, umequine,
josamycin, kanamycin disulfate salt, lincomycin hydrochloride monohydrate, lomeoxacin hydrochloride, marbooxacin,
nalidixic acid, neomycin, noroxacin, ooxacin, oxolinic acid,
oxytetracycline hydrochloride, saraoxacin hydrochloride trihydrate, spiramycin, sulfachlorpyridazine, sulfadimethoxine,
sulfadoxin, sulfamethoxazole, sulfaphenazole (used as internal
standard), sulfaquinoxaline, tetracycline, tilmicosin, trimethoprim and tylosin tartrate were obtained from SigmaAldrich
(Taufkirchen, Germany); oleandomycin phosphate dehydrate and
sulfaclozine sodium were obtained from Dr. Ehrenstorfer GmbH
(Augsburg, Germany) and tylvalosin tartrate from FarmKemi
(Hubei, China).

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

140

Aminoglycosides

120

100

100

100

80
60

REC (%)

120

80
60

60

40

40

20

20

20

50:50

30:70

70:30

0:100

100:1

50:50

30:70

140

70:30

0:100

100:1

140

Macrolides

140

Sulfonamides

100

100

80
60

REC (%)

100
REC (%)

120

80
60

40

20

20

20

100:1

50:50

30:70

ACN:2%TCA

ACN:2%TCA
140

70:30

0:100

100:1

0:100

100:1

0:100

100:1

Tetracyclines

60

40

0:100

70:30

80

40

70:30

30:70

ACN:2%TCA

120

30:70

50:50

ACN:2%TCA

120

50:50

Trimethoprim

80

40

ACN:2%TCA

REC (%)

140

Lincosamides

120
REC (%)

REC (%)

140

21

50:50

30:70

70:30
ACN:2%TCA

Quinolones

120
REC (%)

100
80
60
40
20
0

50:50

30:70

70:30

0:100

100:1

ACN:2%TCA

Fig. 1. Inuence of extraction mixture on recovery. The range shown at each mixture encompasses the recoveries of all the compounds in the group of antibiotics.

2.4. Standard solutions

Stock standard solutions (1000 g mL1 ) were prepared by
dissolving the analytes in methanol (lincosamides, macrolides,
sulfonamides, tetracyclines and trimethoprim), in water (aminoglycosides) and in methanol with 2% 2 M NH4 OH (quinolones). The
working standard solution containing all analytes with variable
concentrations, according to their LOQ and MRL, was prepared by
dilution of stock standard solutions with ACN. Working standard
solution of internal standard (2000 g L1 ) was prepared by dilution of stock standard solution (sulfaphenazole) with ACN. Stock
standard solutions were kept in brown glass to prevent the photodegradation and stored at 20 C and were stable for three months.
The working standard solutions were also kept at 20 C in brown
glass and were stable for two weeks.

performed in the positive ion mode with a spray voltage set at

3500 V. The sheath and auxiliary gas used was nitrogen at 50 and 10
arbitrary units, respectively. The system was operated in the multiple reaction monitoring (MRM) mode with argon as the collision gas
at a pressure of 1.5 mTorr. The ion transfer tube was kept at 370 C,

2.5. Instrumentation
A turbulent ow chromatograph TranscendTM TLX-1 (TLX)
system (Thermo Fisher Scientic, Franklin, MA) was used for development of this method and included a PAL autosampler (CTC
Analytics, Zwingen, Switzerland), two high-pressure mixing quaternary pumps (loading and eluting pump) and a three-valve
switching device unit with six-port valve. The entire system was
controlled by Aria software, version 1.6. The TurboFlowTM column
was a Cyclone P 50 mm 0.5 mm, 60 m particle size, 60 A pore
size (Thermo Fisher Scientic, Franklin, MA) and the analytical column was a Betasil phenyl hexyl 50 mm 2.1 mm, 3 m (Thermo
Fisher Scientic, Runcorn, UK). The analytical column was operated
at room temperature.
The TranscendTM TLX-1 system was coupled to the TSQ Quantum Access MaxTM triple quadrupole mass spectrometer (Thermo
Fisher Scientic, San Jose, CA) equipped with a heated electrospray ionisation probe that was kept at 400 C. Measurements were

Fig. 2. Comparison of peak shape for two aminoglycosides in a fortied chicken

meat sample (100 g kg1 ) by applying different extraction solvent.

22

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

Fig. 3. Example chromatogram of spiked chicken meat sample for each of 36 target antibiotics.

while the cycle time and peak width were 0.6 s and 0.7 Da, respectively. All data were processed using Xcalibur software version 2.1
in EZ set up mode.
2.6. Sample preparation
Chicken meat of around 500 g was homogenised in a laboratory blender for 5 min. Then, homogenised chicken meat (0.5 g) was
weighed into 2 mL polypropylene tube. Working internal standard
solution (50 L) and solvent mixture ACN:2% TCA (45:55, v/v)
(450 L) were added to the sample. The sample was shaken for
5 min on a vortex mixer equipped with a foam tube holder and
then centrifuged at 12,000 rpm for 5 min. The supernatant was ltered through a nylon micro lter (0.45 m pore size) directly into
the LC vial and nally, the sample was injected into the TLX-MS/MS.
2.7. TurboFlow and LC conditions
Connection of the TurboFlowTM column to the analytical column allows concentration, clean-up and analytical separation in
one step. First the sample was applied by the loading pump onto
the TurboFlowTM column. During this step the macromolecules
were removed, while the target analytes were retained on the
TurboFlowTM column based on their different chemical interactions. In the next step the trapped analytes were transferred by
the eluting pump and the eluent in the loop onto the analytical

column and separated conventionally. The loop can be easily connect and disconnect from the system thanks to a special switching
valve, which enables to perform more steps in parallel. Transfer of
the compounds from the TurboFlowTM column onto analytical column was accomplished by using a loop lled with a mixture with
a certain content of organic phase. While the separation on the
analytical column was running, the loop was lled with the fresh
eluent and the TurboFlowTM column was washed and conditioned
to be ready for the injection of the next sample. The TLX and LC
conditions are given in Table 1.
The analytical column was conditioned during loading step
onto the TurboFlowTM column. The separation of the analytes on
the analytical column was carried out by the gradient (Table 1).
The mobile phases were (A) 1 mM HFBA + 0.5% formic acid (FA) in
water and (B) 0.5% FA in ACN/methanol (1:1, v/v). The gradient
elution programme started with 0% B, it remained for 1 min, then
it increased to 95% in 12 min, remained for 2 min and returned
back to the initial compositions in another 2 min. There was an
additional 2 min at the beginning of the run, which is needed for
loading the sample onto the TurboFlowTM column and transfer
onto the analytical column. The nal run time of the method with
automated on-line sample clean-up and analytical separation was
19 min. The injection volume of the sample was 35 L. To prevent
the possibility of carry-over and cross contamination the injection
syringe as well as the injection valve were washed three times
with cleaning solvent 1 (ACN/water 20/80) and cleaning solvent 2

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

23

Table 2
Performance data of the TLX-MS/MS method for analysis of 36 antibiotics in chicken meat.
Analyte

Spiking levels (g kg1 )

RECa (%)

WDb (%)

BDc (%)

L1

L2

L3

L1

L2

L3

L1

L2

L3

L2

50
250

100
500

150
750

119
84

109
71

120
83

19
23

25
18

21
19

25
28

Lincomycin
Clindamycin

50
10

100
20

150
30

104
111

94
115

102
104

4
6

10
3

10
10

15
10

Trimethoprim

25

50

75

99

91

83

16

Josamycin
Spiramycin
Tilmicosin
Tylosin
Clarithromycin
Oleandomycin
Tylvalosin

10
100
37.5
50
10
10
10

20
200
75
100
20
20
20

30
300
112.5
150
30
30
30

102
108
115
86
101
116
91

91
102
105
84
105
93
101

95
92
102
82
98
92
99

9
8
7
9
11
13
15

6
10
7
7
6
10
6

21
21
9
19
12
10
16

8
22
8
13
10
18
12

Sulfadimethoxine
Sulfamethoxazole
Sulfadoxin
Sulfaquinoxaline
Sulfachlorpyridazine
Sulfaclozine

50
50
50
50
50
50

100
100
100
100
100
100

150
150
150
150
150
150

101
113
101
100
109
118

97
108
104
94
102
110

91
96
98
99
94
106

3
7
14
17
8
14

5
10
9
21
8
14

10
5
6
5
13
10

7
9
11
22
12
11

Oxytetracycline
Tetracycline
Chlortetracycline
Doxycycline

50
50
50
50

100
100
100
100

150
150
150
150

115
102
96
117

109
94
85
98

114
94
87
95

27
10
13
7

13
12
19
8

11
10
12
9

15
11
15
10

10
50
100
50
150
50
200
10
10
10
10
10
10
10

20
100
200
100
300
100
400
20
20
20
20
20
20
20

30
150
300
150
450
150
600
30
30
30
30
30
30
30

104
101
116
112
106
109
108
118
99
101
98
101
105
102

105
114
108
108
105
100
94
103
103
92
100
112
99
94

106
103
109
103
102
95
88
99
88
89
94
101
90
91

9
10
5
11
4
4
6
6
17
9
27
11
24
16

12
8
3
7
8
5
7
6
14
20
19
7
22
19

18
8
9
6
10
7
9
8
22
15
16
16
6
12

15
9
7
9
9
8
8
8
12
15
19
10
17
16

Kanamycin
Neomycin

Marbooxacin
Ciprooxacin
Danooxacin
Enrooxacin
Dioxacin
Oxolinic acid
Flumequine
Nalidixic acid
Enoxacin
Ooxacin
Lomeoxacin
Noroxacin
Saraoxacin
Cinoxacin
a
b
c

Recovery.
Within-day precision.
Between-day precision.

(acetone/ACN/isopropanol 20/40/40) before and ve times after

injection.

groups (classical reversed phase retention together with selectivity

for polar compounds).

3. Results and discussion

3.1.2. Extraction method

For extraction of antibiotics from animal tissues the commonly
used organic solvents are ACN, methanol, ethanol or an aqueous solution of TCA. These compounds cause protein precipitation,
which is necessary to release and extract bound analytes. Recently
a mixture of ACN and 2% TCA (1:1, v/v) was reported as a suitable
solvent for extraction of chicken meat [14]. The target analytes
were not all the same. Therefore the ratio between ACN and 2%
TCA in the extraction solvent was studied by performing experiments with extraction of chicken meat spiked at 100 g kg1 . The
ratios 50:50, 30:70, 70:30, 0:100 and 100:1 between ACN and 2%
TCA were subsequently tested. The results (Fig. 1) showed that
the best recoveries were achieved for different classes with different ratios. For group of aminoglycosides was the best mixture of
0:100 ACN:2% TCA, but the mixtures with 30:70 and 50:50 ACN:2%
TCA were also acceptable. The same statement applies for group of
tetracyclines. For trimethoprim, macrolides and quinolones were
acceptable all ve studied mixtures, because the satised recoveries were reached with all of them. The only one mixture (0:100
ACN:2% TCA) was not suitable for sulfonamides. The two mixtures (50:50 and 30:70 ACN: 2% TCA) were acceptable, because of

3.1. Method development

3.1.1. LCMS/MS
For the group of very polar aminoglycosides application of ionpair reagents was necessary. It is well known, that the presence
of ion-pair agent in the mobile phase can cause ion suppression
[8,20,21]. During optimisation of the chromatographic parameters
two different ion-pair reagents were tested. Mobile phase with only
addition of FA, mobile phase with addition of FA and TFA and nally
mobile phase with addition of FA and HFBA were compared. Chromatographic separation was improved much more by applying
HFBA in comparison to TFA. By applying TFA strong ion suppression effect was observed, while by applying HFBA minimal or no.
After obtaining unsatisfactory results with TFA, HFBA was chosen
as an optimal ion-pairing agent to be added to mobile phase for
the analytical separation of the target compounds. A Betasil phenyl
hexyl 50 mm 2.1 mm, 3 m analytical column provided a good
separation of target compounds thanks to its stationary phase composition with a combination of straight-chain C6 groups and phenyl

24

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

lincomycin, for group of lincosamides. Due to results for this group

mixture 50:50 ACN:2% TCA was chosen as the optimal, but because
of poor shape of aminoglycoside peaks, one more experiment
with mixture 45:55 ACN:2% TCA was performed. The recoveries
of all groups were satisfactory with this mixture and peak shape
of aminoglycosides was improved (Fig. 2). Therefore the mixture
45:55 ACN:2% TCA was used in the nal optimised method.
3.1.3. TurboFlow method
For the development of an effective TurboFlowTM method it is
necessary to optimise certain parameters. Firstly, it is necessary to
select the most appropriate TurboFlowTM column, then optimise
loading of the sample onto the clean-up column, optimise transfer
of target compounds onto the analytical column and then establish
equilibration conditions.
The optimisation of TurboFlow method was comprehensively
described in previously reported turbulent ow LCMS/MS method
[19].
The application of nal method is shown in Fig. 3.
3.2. Method validation
The method was validated in-house according to the criteria
specied in EU Commission Decision 2002/675/EC for a quantitative method. The validation parameters were determined by

spiking blank chicken meat with a working solution at levels of 0.5,

1 and 1.5 times the MRL. For compounds without any MRL, blank
samples were spiked at levels indicated in Table 2. The measured
parameters were specicity, linear range, repeatability, accuracy,
limit of detection (LOD), limit of quantication (LOQ), decision limit
(CC) and detection capability (CC).
3.2.1. Detection and quantication
Mass spectrometric analysis was carried out using a TSQ Quantum Access Max triple quadrupole system. Data acquisition for
quantication and conrmation was performed in the multiple
reaction monitoring mode (MRM). Although two transitions were
followed for the identication, only one of these was used for quantication. All MRM ions (precursor, qualier and quantier ion)
were individually tuned for each target analyte by direct injection
of the individual working standard solution (10 mg mL1 ) (Table 3).
3.2.2. Matrix effects
Matrix effects were evaluated by comparing the calibration
curves for each analyte created from the calibration points (n = 9)
prepared in solvent and in matrix-matched standards. The graphs
prepared for the representatives of each chemical class are shown
in Fig. 4. No matrix effect was observed for group of quinolones
and for most of sulfonamides, when the difference between slope
of calibration curve in solvent and in matrix was minor. Quite

Table 3
MRM transitions for 36 analytes from and sulfaphenazole as internal standard (IS).
Analyte

Classa

Precursor ion (m/z)

Quantier ion

CEb (V)

Qualier ion

CE (V)

TLc

Kanamycin
Neomycin

485.2
615.3

163.1
161.0

25
29

324.1
163.1

15
31

90
101

Lincomycin
Clindamycin

407.1
425.1

126.1
126.1

28
28

359.1
377.1

17
18

97
86

291.1

230.1

23

261.0

24

93

828.4
843.3
869.6
916.5
748.5
688.4
1042.6

173.9
173.9
696.4
174.0
158.1
544.3
109.0

30
32
40
35
28
14
41

109.1
142.0
174.0
772.4
590.3
158.0
173.9

34
32
41
26
17
25
37

18
146
132
141
108
106
133

Sulfadimethoxine
Sulfamethoxazole
Sulfadoxin
Sulfaquinoxaline
Sulfachlorpyridazine
Sulfaclozine
Sulfaphenazole (IS)

311.0
254.0
311.0
301.0
284.9
284.9
315.0

156.0
156.0
156.0
156.0
156.0
92.1
158.1

21
15
18
17
15
29
28

108.1
92.1
108.1
92.1
92.1
108.1
160.1

27
27
26
28
26
26
22

88
96
88
92
90
87
94

Oxytetracycline
Tetracycline
Chlortetracycline
Doxycycline

461.1
445.1
479.0
445.1

426.1
410.1
444.0
428.1

18
18
22
18

426.1
427.1
462.1
321.0

18
11
16
31

93
99
98
82

363.1
332.0
358.1
360.1
400.1
262.0
262.0
233.0
321.0
362.1
352.1
320.0
386.0
263.0

72.3
314.1
340.1
316.1
382.1
244.0
244.0
215.0
303.0
318.1
265.0
302.0
368.1
245.0

22
18
24
19
21
18
19
15
19
18
23
22
23
16

320.0
288.1
314.1
342.1
356.1
216.0
202.0
187.0
257.1
261.0
308.1
276.1
342.1
217.0

14
22
16
22
19
29
33
25
17
27
15
16
18
22

97
89
99
96
98
84
84
77
93
91
100
94
94
90

Trimethoprim
Josamycin
Spiramycin
Tilmicosin
Tylosin
Clarithromycin
Oleandomycin
Tylvalosin

Marbooxacin
Ciprooxacin
Danooxacin
Enrooxacin
Dioxacin
Oxolinic acid
Flumequine
Nalidixic acid
Enoxacin
Ooxacin
Lomeoxacin
Noroxacin
Saraoxacin
Cinoxacin
a
b
c

Class: (1) aminoglycosides, (2) lincosamides, (3) trimethoprim, (4) macrolides, (5) sulfonamides, (6) tetracyclines, (7) quinolones.
CE = collision energy.
TL = tube lens.

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

y = 0.011x - 0.0235
R = 0.9924

Aa/AIS

0.80
y = 0.0075x + 0.0465
R = 0.9902

0.40
0

20

40

60

80

100

120

140

y = 1E-04x - 0.0012
R = 0.9908

0.01
0.00

160

20

40

60

80

c [g/kg]
Matrix
Oxytetracycline

0.00

y = 0.0013x - 0.0166
R = 0.9906
0

20

40

60

80

Solvent

Solvent

20

40

60

20

40

60

80

80

100

120

Solv

120

140

160

180

200

Matrix

y = 0.0018x + 0.0093
R = 0.9938

0.20

y = 0.0019x - 0.0021
R = 0.9902

0.10
0.00

20

40

60

80

100 120 140 160 180 200 220

Matrix

Solvent

Matrix

y = 0.0025x - 0.0007
R = 0.991

0.08

100 120 140 160 180 200 220

100

c [g/kg]

Marbofloxacine

0.12
0.10

y = 0.0009x - 0.0043
R = 0.9909
0

80

Sulfamethoxazole

0.40

y = 0.0115x - 0.0137
R = 0.9927

0.40

Matrix

Aa/AIS

Aa/AIS

0.00

60

Solv

c [g/kg]

Sulfaclozine

0.10

40

0.50

0.80

0.00

100 120 140 160 180 200 220

y = 0.0014x - 0.0097
R = 0.9921

0.20

20

0.30

c [g/kg]

0.30

Matrix

1.20

0.20
0.10

0.40

c [g/kg]

y = 0.0153x + 0.105
R = 0.9975

1.60

Aa/AIS

Aa/AIS

0.30

y = 0.0095x - 0.0587
R = 0.9902

0.80

0.00

100 120 140 160 180 200 220

Solv
Trim
ethoprim

2.00

y = 0.0019x + 0.011
R = 0.9937

0.40

1.20

c [g/kg]

Solvent
0.50

1.60

0.02
0.01

y = 0.0107x + 0.0829
R = 0.9913

2.00

Aa/AIS

Aa/AIS

1.20

0.02

Lincomycin

2.40

y = 0.0001x - 0.0003
R = 0.994

0.03

1.60

0.00

Kanamycin

0.03

Aa/AIS

Tilmicosin

2.00

25

0.06
0.04

y = 0.0023x - 0.0042
R = 0.99

0.02
0.00

10

15

c [g/kg]

20

25

30

35

40

45

c [g/kg]

Fig. 4. Matrix-matched calibration plots for eight representative compounds (1-kanamycin, 2-lincomycin, 3-trimethoprim, 4-tilmicosin, 5-sulfamethoxazole and sulfaclozine,
6-oxytetracycline and 7-marbooxacine) in solvent and chicken meat.

considerable matrix effects were observed for sulfadoxin (signal

suppression) and sulfaclozine (signal enhancement). The signal
suppression occurred for the groups of tetracyclines, aminoglycosides, lincosamides and trimethoprim as calibration curves in
matrix were found to have a slope below the calibration curves in
solvent. Conversely calibration curves in matrix for all macrolides
had higher slopes than the calibration curves in solvent, indicating
a signicant enhancement for all macrolides except oleandomycin
(no matrix effect). Consequently, matrix-matched calibration was
used for quantication purposes to compensate the problems with
suppression or enhancement effects.

3.2.3. Specicity
Using MRM, the specicity was conrmed based on the presence of the transition ions (quantier and qualier) at the correct
retention times corresponding to those of the respective antibiotics.
The measured peak area ratios of qualier/quantier are within the
range (relative intensity of base peak permitted range; >50%
20%, >20%50% 25%, >1020% 30%, 10% 50%) dened in
EU Commission Decision 2002/657/EC when compared to the standards. The calculated ion ratios for solvent and matrix are shown
in Table 4.

3.2.4. Linearity & calibration curve

The linearity of calibration curves was assessed over the range
depending on the target compound. In all cases, the correlation
coefcients of linear functions were >0.99. The calibration curves
were created from 9 matrix-matched calibration standards which
were injected in each batch in duplicate. The matrix-matched calibration standards were prepared by spiking the blank material with
the same spiking solution as for samples for validation with variable
volumes (0400 L).

3.2.5. Precision
Precision (repeatability) of the method was determined using
independently spiked blank samples at three different levels. In
one day, the set of samples at three levels was measured as six
replicates. To determine between-day precision, two other sets at
one level were measured with six repetitions over the next two
days. The results for repeatability ranged from 3 to 28% (Table 2).

3.2.6. Accuracy
Method accuracy was determined using independently spiked
blank samples at three different levels. Accuracy was evaluated
by comparing found values with standard additions of spikes.
Recovery values ranged between 71 and 120% (Table 2). Additionally accuracy was established for ciprooxacin by analysing a test
material (FAPAS T02174QC) which was sh muscle. All measured
concentrations of ciprooxacin were within the acceptable range
(Table 5).

3.2.7. LOD and LOQ

The LOD and LOQ were estimated following the IUPAC approach
which consists of rst analysing the blank sample to establish noise
levels and then estimating LODs and LOQs for signal/noise, 3 and
10 respectively. The values for chicken meat are shown in Table 4
and, in all cases, they are below the level of MRL for analytes, where
it was established.

3.2.8. CC and CC
Both CC and CC were established by the calibration curve
procedure according to ISO 118434. The blank material fortied at
and below the maximum residue limit (for analytes with MRL) or
at and above the lowest possible level (for analytes without MRL)
in equidistant steps was used. The calculated values are shown in
Table 4.

26

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

Table 4
Maximum residue limit (MRL), limit of detection and quantication (LOD and LOQ), limit of decision and capability (CC and CC), ion ratios (qualier/quantier) in solvent
and matrix for 36 antibiotics in chicken meat.
Analyte

MRL (g kg1 )

Kanamycin
Neomycin

100
500

Lincomycin
Clindamycin

LOQ (g kg1 )

CC (g kg1 )

CC (g kg1 )

Ion ratio (solvent)

Ion ratio (matrix)

10.0
40.0

25.0
120.0

121.3
602.2

142.5
704.4

0.53
0.95

0.50
0.94

100

3.0
0.3

10.0
1.0

109.7
1.7

119.5
2.2

0.09
0.05

0.09
0.04

Trimethoprim

50

1.0

3.0

57.1

64.2

0.76

0.70

Josamycin
Spiramycin
Tilmicosin
Tylosin
Clarithromycin
Oleandomycin
Tylvalosin

200
75
100

0.3
0.3
0.3
1.0
0.3
0.3
0.3

1.0
1.0
1.0
3.0
1.0
1.0
1.0

3.2
223.3
79.8
107.2
4.2
3.3
4.8

4.1
246.5
84.6
114.4
5.4
4.2
6.2

0.90
0.17
0.88
0.23
0.62
0.65
0.55

0.91
0.21
0.88
0.23
0.61
0.71
0.50

Sulfadimethoxine
Sulfamethoxazole
Sulfadoxin
Sulfaquinoxaline
Sulfachlorpyridazine
Sulfaclozine

100a
100a
100a
100a
100a
100a

0.3
1.5
0.3
0.3
10.0
3.0

1.0
5.0
1.0
1.0
25.0
10.0

109.8
118.5
107.9
111.0
111.0
116.0

119.6
137.1
115.8
121.9
121.9
132.1

0.60
0.31
0.46
0.24
0.44
0.20

0.56
0.30
0.58
0.27
0.46
0.29

Oxytetracycline
Tetracycline
Chlortetracycline
Doxycycline

100
100
100
100

3.0
3.0
5.0
1.0

10.0
10.0
15.0
3.0

112.4
114.8
111.9
110.0

124.9
129.5
123.8
120.0

0.13
0.80
0.48
0.03

0.10
0.84
0.42
0.05

Marbooxacin
Ciprooxacin
Danooxacin
Enrooxacin
Dioxacin
Oxolinic acid
Flumequine
Nalidixic acid
Enoxacin
Ooxacin
Lomeoxacin
Noroxacin
Saraoxacin
Cinoxacin

100b
200
100b
300
100
400

1.5
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
1.0

5.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
3.0

6.6
104.5
216.6
107.8
334.4
109.0
437.8
1.7
3.5
3.3
4.0
6.1
4.1
4.2

8.4
108.9
233.2
115.7
368.8
118.1
475.6
2.2
4.5
4.2
5.1
7.9
5.3
5.4

0.73
0.13
0.06
0.58
0.58
0.06
0.44
0.30
0.02
0.70
0.58
0.05
0.18
0.28

0.61
0.14
0.04
0.62
0.69
0.08
0.42
0.32
0.03
0.70
0.64
0.09
0.25
0.30

a
b

LOD (g kg1 )

Expressed in form of sum-MRLs of all sulfonamides.

Expressed in form of sum-MRLs of enrooxacine and its metabolite (ciprooxacine).

3.3. Discussion
Turbulent ow chromatography has previously been employed
for on-line clean-up and extraction of samples from food safety
area in wide range of methods such as determination of quinolones
in honey [16], determination of pesticides in oranges and hazelnuts [22], determination of antibiotics in milk [17] and in edible
tissues [18], but just for two quinolones. In this paper we report
the use of turbulent ow technology in multi-residue method
for chicken meat. In comparison with matrices such as honey,
milk or oranges, where a minimal short sample preparation is
needed, here it is necessary perform slightly longer sample preparation prior to injection into the TLX-MS/MS system. The sample
preparation is nevertheless short combining liquid extraction with
on-line clean-up in comparison with a very complicated method
published by others [6] for analysis of similar compounds in
veal muscle. This method was developed as a semi-quantitative

Table 5
Results of measurement the quality control test material FAPAS sh muscle
T02174QC ciprooxacin certied value = 113 50 g kg1 .
Sample

c (g kg1 )

Measurement 1
Measurement 2
Measurement 3

90
103
107

screening with method detection limits for the analytes ranging from 1 to 41 g kg1 . These limits are comparable to LOQs
of the reported method; they ranged from 1 to 25 g kg1 with
the exception for neomycin (120 g kg1 ). These values are also
similar to those reported by Chiaochan et al. [14] for a method
for 24 antibiotics in chicken meat (LOQs ranged from 0.3 to
60 g kg1 ).
The total time needed for measurement of one sample was
34 min, taking around 15 min for sample preparation and 19 min
for instrumental analysis including the sample clean-up. It was a
slightly faster approach than the 25 min for measurement of one
sample as reported by another author [14] and much faster than
method of Martos et al. [6], who needed more than 2 h for one
sample including measurements.
The validated method was applied to a small survey with 20
samples of various chicken meat and chicken meat products from
a local market. Four samples of sausages, ve samples of salami,
ve samples of ham, three samples of raw chicken meat and two
samples of modied chicken meat were analysed. All samples were
measured in parallel and matrix-matched calibration and internal
standardisation were used for the evaluation, whereas retention
times and specic ion ratios were also used for identication. None
of the 36 analytes were detected in any of the samples, except
enrooxacin which was found in one sample of chicken ham at
19.9 g kg1 and met all identication requirements. However, this
value did not exceed the MRL of 100 g kg1 .

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927

4. Conclusion
The method reported here applies a less commonly employed
approach for sample clean-up using turbulent ow chromatography. This brings the benet of automation of the sample
preparation, thus increasing the cost effectiveness and decreasing
the time involved in manual sample handling. With this method
it is possible to determine and quantify 36 antibiotic residues
from seven different classes of drugs in chicken meat. The method
was successfully in-house validated according to the EU Commission Decision 2002/657/EC, and therefore can be recommended for
enforcement of the legislative limits.
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