Journal of Chromatography A, 1274 (2013) 1927

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

a r t i c l e i n f o a b s t r a c t

Article history: A multi-class method for identication and quantication of 36 antibiotics from seven different chem-
Received 8 August 2012 ical classes (aminoglycosides, macrolides, lincosamides, sulfonamides, tetracyclines, quinolones and
Received in revised form 30 October 2012 trimethoprim) has been developed by using liquid chromatographymass spectrometry. The method
Accepted 25 November 2012
was optimised for detection of antibiotics in chicken meat. Sample preparation including extraction with
Available online 19 December 2012
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
Keywords:
a larger number of samples to be analysed per day than with a traditional clean-up technique (e.g. solid
Antibiotics
On-line clean-up
phase extraction). The optimised method was validated according to the European Commission Directive
LCMS/MS 2002/657/EC. In-house validation was performed by fortifying the blank matrix at three levels 0.5, 1.0
Chicken meat and 1.5 MRL (maximum residue limit), or respectively, at concentrations as low as possible for substances
Food safety without an MRL. Precision in terms of repeatability standard deviation ranged from 3 to 28% and recovery
Validation 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 contami-
nated 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 vet-
erinary drugs in live animals and in animal products. To ensure that
testing is carried out to the highest analytical standards unambigu-
ous 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).
residues and contaminants [2]. For the most frequently used veteri-
nary 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 rou-
tine 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

0021-9673/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2012.11.067
20 K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927
Table 1
TurboFlowTM loading and eluting conditions.
Step Step length (s) Loading pumpa
Flow (mL/min) %A %B %C
1. 60 1.5 100
2. 60 0.2 100
3. 60 1.5 50
4. 720 1.5
5. 120 1.5 50 50
6. 120 1.5 100
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 instrumenta-
tion to be used for the conrmatory methods is still LCMS/MS.
In the last decades most conrmatory methods were devel-
oped 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 sul-
fonamides [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 through-
put.
Poultry meat, just like any other type of meat, having a high pro-
teins 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 veteri-
nary 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], tetracy-
clines 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 multi-
residue 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 multi-
residue 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 tech-
nique 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 in-
house 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].
Valve interface Eluting pumpb
module (VIM)
%D Tee Loop Flow (mL/min) %A %B
Out 0.3 100
T In 0.6 100
50 In 0.3 100
100 In 0.3 5 95
In 0.3 5 95
Out 0.3 100
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 mate-
rial 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 Sci-
entic, 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, Loughbor-
ough, 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 hydrochlo-
ride, doxycycline hyclate, enoxacin, enrooxacin, umequine,
josamycin, kanamycin disulfate salt, lincomycin hydrochlo-
ride monohydrate, lomeoxacin hydrochloride, marbooxacin,
nalidixic acid, neomycin, noroxacin, ooxacin, oxolinic acid,
oxytetracycline hydrochloride, saraoxacin hydrochloride tri-
hydrate, spiramycin, sulfachlorpyridazine, sulfadimethoxine,
sulfadoxin, sulfamethoxazole, sulfaphenazole (used as internal
standard), sulfaquinoxaline, tetracycline, tilmicosin, trimetho-
prim 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 21

140 140 140

Aminoglycosides Lincosamides Trimethoprim
120 120 120

100 100 100

REC (%)

REC (%)

REC (%)
80 80 80

60 60 60

40 40 40

20 20 20

0 0 0
50:50 30:70 70:30 0:100 100:1 50:50 30:70 70:30 0:100 100:1 50:50 30:70 70:30 0:100 100:1
ACN:2%TCA ACN:2%TCA ACN:2%TCA

140 140 140

Macrolides Sulfonamides Tetracyclines
120 120 120

100 100 100

REC (%)
REC (%)
REC (%)

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0
50:50 30:70 70:30 0:100 100:1 50:50 30:70 70:30 0:100 100:1 50:50 30:70 70:30 0:100 100:1
ACN:2%TCA ACN:2%TCA ACN:2%TCA

140
Quinolones
120

100
REC (%)

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 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
Stock standard solutions (1000 g mL1 ) were prepared by arbitrary units, respectively. The system was operated in the multi-
dissolving the analytes in methanol (lincosamides, macrolides, ple reaction monitoring (MRM) mode with argon as the collision gas
sulfonamides, tetracyclines and trimethoprim), in water (amino- at a pressure of 1.5 mTorr. The ion transfer tube was kept at 370 C,
glycosides) 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 dilu-
tion of stock standard solution (sulfaphenazole) with ACN. Stock
standard solutions were kept in brown glass to prevent the photo-
degradation 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.

2.5. Instrumentation

A turbulent ow chromatograph TranscendTM TLX-1 (TLX)

system (Thermo Fisher Scientic, Franklin, MA) was used for devel-
opment of this method and included a PAL autosampler (CTC
Analytics, Zwingen, Switzerland), two high-pressure mixing qua-
ternary 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 col-
umn 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 Quan-
tum Access MaxTM triple quadrupole mass spectrometer (Thermo
Fisher Scientic, San Jose, CA) equipped with a heated electro- Fig. 2. Comparison of peak shape for two aminoglycosides in a fortied chicken
spray ionisation probe that was kept at 400 C. Measurements were 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, respec-
tively. 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 labora-
tory 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 l-
tered 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 col-
umn 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 interac-
tions. 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 con-
nect 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 col-
umn 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

Kanamycin 50 100 150 119 109 120 19 25 21 25

Neomycin 250 500 750 84 71 83 23 18 19 28

Lincomycin 50 100 150 104 94 102 4 10 10 15

Clindamycin 10 20 30 111 115 104 6 3 10 10

Trimethoprim 25 50 75 99 91 83 9 7 9 16

Josamycin 10 20 30 102 91 95 9 6 21 8
Spiramycin 100 200 300 108 102 92 8 10 21 22
Tilmicosin 37.5 75 112.5 115 105 102 7 7 9 8
Tylosin 50 100 150 86 84 82 9 7 19 13
Clarithromycin 10 20 30 101 105 98 11 6 12 10
Oleandomycin 10 20 30 116 93 92 13 10 10 18
Tylvalosin 10 20 30 91 101 99 15 6 16 12

Sulfadimethoxine 50 100 150 101 97 91 3 5 10 7

Sulfamethoxazole 50 100 150 113 108 96 7 10 5 9
Sulfadoxin 50 100 150 101 104 98 14 9 6 11
Sulfaquinoxaline 50 100 150 100 94 99 17 21 5 22
Sulfachlorpyridazine 50 100 150 109 102 94 8 8 13 12
Sulfaclozine 50 100 150 118 110 106 14 14 10 11

Oxytetracycline 50 100 150 115 109 114 27 13 11 15

Tetracycline 50 100 150 102 94 94 10 12 10 11
Chlortetracycline 50 100 150 96 85 87 13 19 12 15
Doxycycline 50 100 150 117 98 95 7 8 9 10

Marbooxacin 10 20 30 104 105 106 9 12 18 15

Ciprooxacin 50 100 150 101 114 103 10 8 8 9
Danooxacin 100 200 300 116 108 109 5 3 9 7
Enrooxacin 50 100 150 112 108 103 11 7 6 9
Dioxacin 150 300 450 106 105 102 4 8 10 9
Oxolinic acid 50 100 150 109 100 95 4 5 7 8
Flumequine 200 400 600 108 94 88 6 7 9 8
Nalidixic acid 10 20 30 118 103 99 6 6 8 8
Enoxacin 10 20 30 99 103 88 17 14 22 12
Ooxacin 10 20 30 101 92 89 9 20 15 15
Lomeoxacin 10 20 30 98 100 94 27 19 16 19
Noroxacin 10 20 30 101 112 101 11 7 16 10
Saraoxacin 10 20 30 105 99 90 24 22 6 17
Cinoxacin 10 20 30 102 94 91 16 19 12 16
a
Recovery.
b
Within-day precision.
c
Between-day precision.

(acetone/ACN/isopropanol 20/40/40) before and ve times after groups (classical reversed phase retention together with selectivity
injection. for polar compounds).

3. Results and discussion 3.1.2. Extraction method

3.1. Method development
3.1.1. LCMS/MS
For the group of very polar aminoglycosides application of ion-
pair 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. Chro-
matographic separation was improved much more by applying
HFBA in comparison to TFA. By applying TFA strong ion suppres-
sion 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 com-
position with a combination of straight-chain C6 groups and phenyl
For extraction of antibiotics from animal tissues the commonly
used organic solvents are ACN, methanol, ethanol or an aque-
ous 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 experi-
ments 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 dif-
ferent 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 recover-
ies were reached with all of them. The only one mixture (0:100
ACN:2% TCA) was not suitable for sulfonamides. The two mix-
tures (50:50 and 30:70 ACN: 2% TCA) were acceptable, because of
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 quan-
titative 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 Quan-
tum 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 quan-
tication. 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 485.2 163.1 25 324.1 15 90

1
Neomycin 615.3 161.0 29 163.1 31 101

Lincomycin 407.1 126.1 28 359.1 17 97

2
Clindamycin 425.1 126.1 28 377.1 18 86

Trimethoprim 3 291.1 230.1 23 261.0 24 93

Josamycin 828.4 173.9 30 109.1 34 18

Spiramycin 843.3 173.9 32 142.0 32 146
Tilmicosin 869.6 696.4 40 174.0 41 132
Tylosin 4 916.5 174.0 35 772.4 26 141
Clarithromycin 748.5 158.1 28 590.3 17 108
Oleandomycin 688.4 544.3 14 158.0 25 106
Tylvalosin 1042.6 109.0 41 173.9 37 133

Sulfadimethoxine 311.0 156.0 21 108.1 27 88

Sulfamethoxazole 254.0 156.0 15 92.1 27 96
Sulfadoxin 311.0 156.0 18 108.1 26 88
Sulfaquinoxaline 5 301.0 156.0 17 92.1 28 92
Sulfachlorpyridazine 284.9 156.0 15 92.1 26 90
Sulfaclozine 284.9 92.1 29 108.1 26 87
Sulfaphenazole (IS) 315.0 158.1 28 160.1 22 94

Oxytetracycline 461.1 426.1 18 426.1 18 93

Tetracycline 445.1 410.1 18 427.1 11 99
6
Chlortetracycline 479.0 444.0 22 462.1 16 98
Doxycycline 445.1 428.1 18 321.0 31 82

Marbooxacin 363.1 72.3 22 320.0 14 97

Ciprooxacin 332.0 314.1 18 288.1 22 89
Danooxacin 358.1 340.1 24 314.1 16 99
Enrooxacin 360.1 316.1 19 342.1 22 96
Dioxacin 400.1 382.1 21 356.1 19 98
Oxolinic acid 262.0 244.0 18 216.0 29 84
Flumequine 262.0 244.0 19 202.0 33 84
7
Nalidixic acid 233.0 215.0 15 187.0 25 77
Enoxacin 321.0 303.0 19 257.1 17 93
Ooxacin 362.1 318.1 18 261.0 27 91
Lomeoxacin 352.1 265.0 23 308.1 15 100
Noroxacin 320.0 302.0 22 276.1 16 94
Saraoxacin 386.0 368.1 23 342.1 18 94
Cinoxacin 263.0 245.0 16 217.0 22 90
a
Class: (1) aminoglycosides, (2) lincosamides, (3) trimethoprim, (4) macrolides, (5) sulfonamides, (6) tetracyclines, (7) quinolones.
b
CE = collision energy.
c
TL = tube lens.
 K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927 25

Tilmicosin Kanamycin Lincomycin

2.00 0.03 2.40

1.60 0.03 y = 0.0001x - 0.0003 2.00 y = 0.0107x + 0.0829

y = 0.011x - 0.0235 R = 0.994 R = 0.9913
0.02 1.60
1.20 R = 0.9924
0.02 1.20
Aa/AIS

Aa/AIS

Aa/AIS
0.80
0.01 0.80 y = 0.0095x - 0.0587
y = 1E-04x - 0.0012 R = 0.9902
0.40 y = 0.0075x + 0.0465
0.01 R = 0.9908 0.40
R = 0.9902
0.00 0.00 0.00
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 180 200 220 0 20 40 60 80 100 120 140 160 180 200
c [g/kg] c [g/kg] c [g/kg]

Solvent Matrix Solv Matrix Solv Matrix

Oxytetracycline Trim ethoprim Sulfamethoxazole
0.50 2.00 0.50

0.40 y = 0.0019x + 0.011 1.60 y = 0.0153x + 0.105 0.40

R = 0.9975 y = 0.0018x + 0.0093
R = 0.9937 R = 0.9938
0.30 1.20 0.30
Aa/AIS

Aa/AIS

Aa/AIS
0.20 0.80 0.20 y = 0.0019x - 0.0021
y = 0.0115x - 0.0137 R = 0.9902
0.10 y = 0.0013x - 0.0166 0.40 0.10
R = 0.9927
R = 0.9906
0.00 0.00 0.00
0 20 40 60 80 100 120 140 160 180 200 220 0 20 40 60 80 100 120 0 20 40 60 80 100 120 140 160 180 200 220
c [g/kg] c [g/kg] c [g/kg]

Solvent Solvent Matrix Solv Matrix Solvent Matrix

Sulfaclozine Marbofloxacine
0.30 0.12
0.10 y = 0.0025x - 0.0007
y = 0.0014x - 0.0097
R = 0.991
0.20 R = 0.9921 0.08
0.06
Aa/AIS

Aa/AIS

0.10 y = 0.0009x - 0.0043 0.04 y = 0.0023x - 0.0042

R = 0.9909 R = 0.99
0.02

0.00 0.00
0 20 40 60 80 100 120 140 160 180 200 220 0 5 10 15 20 25 30 35 40 45
c [g/kg] 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 3.2.5. Precision
suppression) and sulfaclozine (signal enhancement). The signal Precision (repeatability) of the method was determined using
suppression occurred for the groups of tetracyclines, aminogly- independently spiked blank samples at three different levels. In
cosides, lincosamides and trimethoprim as calibration curves in one day, the set of samples at three levels was measured as six
matrix were found to have a slope below the calibration curves in replicates. To determine between-day precision, two other sets at
solvent. Conversely calibration curves in matrix for all macrolides one level were measured with six repetitions over the next two
had higher slopes than the calibration curves in solvent, indicating days. The results for repeatability ranged from 3 to 28% (Table 2).
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 3.2.6. Accuracy
suppression or enhancement effects. 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). Addition-
3.2.3. Specicity ally accuracy was established for ciprooxacin by analysing a test
Using MRM, the specicity was conrmed based on the pres- material (FAPAS T02174QC) which was sh muscle. All measured
ence of the transition ions (quantier and qualier) at the correct concentrations of ciprooxacin were within the acceptable range
retention times corresponding to those of the respective antibiotics. (Table 5).
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 3.2.7. LOD and LOQ
EU Commission Decision 2002/657/EC when compared to the stan- The LOD and LOQ were estimated following the IUPAC approach
dards. The calculated ion ratios for solvent and matrix are shown which consists of rst analysing the blank sample to establish noise
in Table 4. 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.
3.2.4. Linearity & calibration curve
The linearity of calibration curves was assessed over the range
depending on the target compound. In all cases, the correlation 3.2.8. CC and CC
coefcients of linear functions were >0.99. The calibration curves Both CC and CC were established by the calibration curve
were created from 9 matrix-matched calibration standards which procedure according to ISO 118434. The blank material fortied at
were injected in each batch in duplicate. The matrix-matched cali- and below the maximum residue limit (for analytes with MRL) or
bration standards were prepared by spiking the blank material with at and above the lowest possible level (for analytes without MRL)
the same spiking solution as for samples for validation with variable in equidistant steps was used. The calculated values are shown in
volumes (0400 L). 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 ) LOD (g kg1 ) LOQ (g kg1 ) CC (g kg1 ) CC (g kg1 ) Ion ratio (solvent) Ion ratio (matrix)

Kanamycin 100 10.0 25.0 121.3 142.5 0.53 0.50

Neomycin 500 40.0 120.0 602.2 704.4 0.95 0.94

Lincomycin 100 3.0 10.0 109.7 119.5 0.09 0.09

Clindamycin 0.3 1.0 1.7 2.2 0.05 0.04

Trimethoprim 50 1.0 3.0 57.1 64.2 0.76 0.70

Josamycin 0.3 1.0 3.2 4.1 0.90 0.91

Spiramycin 200 0.3 1.0 223.3 246.5 0.17 0.21
Tilmicosin 75 0.3 1.0 79.8 84.6 0.88 0.88
Tylosin 100 1.0 3.0 107.2 114.4 0.23 0.23
Clarithromycin 0.3 1.0 4.2 5.4 0.62 0.61
Oleandomycin 0.3 1.0 3.3 4.2 0.65 0.71
Tylvalosin 0.3 1.0 4.8 6.2 0.55 0.50

Sulfadimethoxine 100a 0.3 1.0 109.8 119.6 0.60 0.56

Sulfamethoxazole 100a 1.5 5.0 118.5 137.1 0.31 0.30
Sulfadoxin 100a 0.3 1.0 107.9 115.8 0.46 0.58
Sulfaquinoxaline 100a 0.3 1.0 111.0 121.9 0.24 0.27
Sulfachlorpyridazine 100a 10.0 25.0 111.0 121.9 0.44 0.46
Sulfaclozine 100a 3.0 10.0 116.0 132.1 0.20 0.29

Oxytetracycline 100 3.0 10.0 112.4 124.9 0.13 0.10

Tetracycline 100 3.0 10.0 114.8 129.5 0.80 0.84
Chlortetracycline 100 5.0 15.0 111.9 123.8 0.48 0.42
Doxycycline 100 1.0 3.0 110.0 120.0 0.03 0.05

Marbooxacin 1.5 5.0 6.6 8.4 0.73 0.61

Ciprooxacin 100b 0.3 1.0 104.5 108.9 0.13 0.14
Danooxacin 200 0.3 1.0 216.6 233.2 0.06 0.04
Enrooxacin 100b 0.3 1.0 107.8 115.7 0.58 0.62
Dioxacin 300 0.3 1.0 334.4 368.8 0.58 0.69
Oxolinic acid 100 0.3 1.0 109.0 118.1 0.06 0.08
Flumequine 400 0.3 1.0 437.8 475.6 0.44 0.42
Nalidixic acid 0.3 1.0 1.7 2.2 0.30 0.32
Enoxacin 0.3 1.0 3.5 4.5 0.02 0.03
Ooxacin 0.3 1.0 3.3 4.2 0.70 0.70
Lomeoxacin 0.3 1.0 4.0 5.1 0.58 0.64
Noroxacin 0.3 1.0 6.1 7.9 0.05 0.09
Saraoxacin 0.3 1.0 4.1 5.3 0.18 0.25
Cinoxacin 1.0 3.0 4.2 5.4 0.28 0.30
a
Expressed in form of sum-MRLs of all sulfonamides.
b
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 hazel-
nuts [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 prepa-
ration 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 90
Measurement 2 103
Measurement 3 107
screening with method detection limits for the analytes rang-
ing 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 chromatog-
raphy. 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 Commis-
sion Decision 2002/657/EC, and therefore can be recommended for
enforcement of the legislative limits.
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