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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 residues and contaminants [2]. For the most frequently used veteri-
nary drugs for different types of food products maximum residue
Veterinary drugs are widely used in the animal husbandry to limits (MRLs) have been set [3]. Analytical methods employed for
treat and prevent diseases. Despite obvious benets, extensive use determination of veterinary drugs must be capable of determining
of these drugs can lead to residues in animal products such as the residues below their MRLs.
meat, milk and eggs. Consumption of animal products contami- To full the regulatory requirements it is necessary to employ
nated with veterinary drug residues can cause allergic reactions sensitive, selective and reliable analytical methods. For fast
in sensitive humans, and can negatively affect the human immune screening of animal products a screening method should preferably
system. The most serious concern about excessive use of veterinary be used. There are various approaches for residue screening. Very
dugs is the impact on the effectiveness of the human medicines popular and quite often used are methods based on microbial or
as bacteria have started to become resistant to the most common immunological assay [4]. These methods are low-cost and rapid, but
antibiotics. In order to combat this problem and protect the human have the disadvantage of either only being suitable for screening
consumers, the use of veterinary drugs is tightly regulated. The EU broad classes of drugs or conversely being too specic and suitable
Council Directive 96/23/EC [1] requires monitoring of certain vet- for targeting only at single residue. Instrumental techniques such as
erinary drugs in live animals and in animal products. To ensure that liquid chromatographytandem mass spectrometry (LCMS/MS)
testing is carried out to the highest analytical standards unambigu- have also been used for screening, providing the added benet of
ous guidelines stipulate the rules for the analytical methods to be also being capable of conrming positive results. For example, a
used for testing food samples of animal origin for the presence of 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-
Corresponding author. Tel.: +49 61034081113; fax: +49 61034081122.
tine screening. This approach has been employed for multi-residue
E-mail addresses: katerina.bousova@thermosher.com
(K. Bousova), hamide.senyuva@foodlifeint.com (H. Senyuva), UHPLCOrbitrapMS screening for veterinary drug residues in milk
klaus.mittendorf@thermosher.com (K. Mittendorf). [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 Valve interface Eluting pumpb
module (VIM)

Flow (mL/min) %A %B %C %D Tee Loop Flow (mL/min) %A %B

1. 60 1.5 100 Out 0.3 100


2. 60 0.2 100 T In 0.6 100
3. 60 1.5 50 50 In 0.3 100
4. 720 1.5 100 In 0.3 5 95
5. 120 1.5 50 50 In 0.3 5 95
6. 120 1.5 100 Out 0.3 100
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- 2. Experimental


tion to be used for the conrmatory methods is still LCMS/MS.
In the last decades most conrmatory methods were devel- 2.1. Samples and quality control materials
oped only for one class of veterinary drugs. Single-class analytical
methods dealing with meat were reported for such groups as Chicken obtained from a local market, which was repeatedly
aminoglycosides [8], tetracyclines [9,10], macrolides [11] and sul- measured to conrm that no antibiotics were present, was used
fonamides [12,13]. Notwithstanding the success of these methods for preparation of matrix-matched calibration standards and for
currently there is an increasing demand for fast and reliable fortied samples. Because of a lack of chicken tissue certied
multi-class multi-residue methods, which if used by food control test materials, to establish method accuracy a FAPAS test mate-
laboratories could reduce costs and achieve high sample through- rial T02174QC of sh muscle containing a certied amount of
put. ciprooxacin was used, which was obtained from the Food and
Poultry meat, just like any other type of meat, having a high pro- Environment Research Agency (York, United Kingdom). Though
teins and lipid content is a very complex matrix. Considering the sh muscle is not the same as chicken muscle, considering
large number of target compounds from different chemical classes the content of proteins, water and fat the differences are not
with different chemical properties the most challenging part of large and thus sh muscle was used to demonstrate method
the multi-residue method is the sample preparation in terms of accuracy.
both the extraction and clean-up. There have been used three main
approaches for sample preparation for the determination of veteri- 2.2. Chemicals and reagents
nary drug residues in meat. Liquid extraction in combination with
a solid-phase extraction (SPE) as a clean-up procedure has been Optima LCMS grade methanol, acetonitrile, water (Fisher Sci-
used for the determination of sulfonamides in meat [12], tetracy- entic, Langenselbold, Germany) and HPLC grade isopropanol
clines in a chicken meat [9] and tetracyclines in pig tissues [10]. and acetone (Fisher Scientic, Langenselbold, Germany) were
The second approach consists of liquid extraction in combination used as mobile phases, for sample extraction, preparation
with a defatting step employing hexane, which has been used in a of standard mixtures and washing of the injection port.
multi-residue method for poultry meat [14] and for another multi- Trichloroacetic acid for sample extraction was obtained from
residue method for veal muscle [6]. The third approach consists Acros Organics (Geel, Belgium). HPLC grade formic acid (Fisher
only of liquid extraction and a subsequent dilution step prior direct Scientic, Loughborough, UK) and heptauorobutyric acid (Acros
injection into the instrument and has been employed in two multi- Organics, Geel, Belgium) were used as additives for mobile
residue methods for pig muscle [6,15]. The rst two ways of sample phases. 35% ammonia solution (Fisher Scientic, Loughbor-
preparation are quite effective and provide clean sample extracts, ough, UK) was used for preparation of stock standard solutions
which can be subsequently injected into the mass spectrometer. for quinolones. Puried water was obtained from Barnstead
The drawback is that the sample preparation is lengthy and in the EASYpure II water system (Thermo Scientic, Barnstead,
case where SPE is used there is the added cost of SPE cartridges. The NH).
last approach appears to be simple and fast, but because of injecting
dirty extracts there is a high risk of MS source contamination. 2.3. Standards
The aim of the method reported here was to overcome the
disadvantages mentioned above and develop a conrmative and Chlortetracycline, cinoxacin, ciprooxacin, clarithromycin,
quantitative multi-class method for the determination of a range of clindamycin hydrochloride, danooxacin, dioxacin hydrochlo-
antibiotics from different classes in chicken meat. For the clean-up ride, doxycycline hyclate, enoxacin, enrooxacin, umequine,
of meat extracts turbulent ow chromatography was used, which josamycin, kanamycin disulfate salt, lincomycin hydrochlo-
has the added benet of being suitable for automation. This tech- ride monohydrate, lomeoxacin hydrochloride, marbooxacin,
nique has been previously used in the multi-class methods for nalidixic acid, neomycin, noroxacin, ooxacin, oxolinic acid,
the determination of veterinary drug residues in other matrices oxytetracycline hydrochloride, saraoxacin hydrochloride tri-
such as honey [16] and milk [17]. For animal tissues turbulent ow hydrate, spiramycin, sulfachlorpyridazine, sulfadimethoxine,
chromatography was used only once in one-class method for two sulfadoxin, sulfamethoxazole, sulfaphenazole (used as internal
quinolones [18]. The reported method was rst developed and in- standard), sulfaquinoxaline, tetracycline, tilmicosin, trimetho-
house validated for milk [19]. In this study an adaptation of the prim and tylosin tartrate were obtained from SigmaAldrich
method is reported for more complex matrices, such as chicken (Taufkirchen, Germany); oleandomycin phosphate dehydrate and
meat. The method was validated for determination of 36 residues sulfaclozine sodium were obtained from Dr. Ehrenstorfer GmbH
from seven different chemical classes of antibiotics according to (Augsburg, Germany) and tylvalosin tartrate from FarmKemi
Commission Decision 2002/657/EC [2]. (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- column and separated conventionally. The loop can be easily con-
tively. All data were processed using Xcalibur software version 2.1 nect and disconnect from the system thanks to a special switching
in EZ set up mode. valve, which enables to perform more steps in parallel. Transfer of
the compounds from the TurboFlowTM column onto analytical col-
2.6. Sample preparation umn was accomplished by using a loop lled with a mixture with
a certain content of organic phase. While the separation on the
Chicken meat of around 500 g was homogenised in a labora- analytical column was running, the loop was lled with the fresh
tory blender for 5 min. Then, homogenised chicken meat (0.5 g) was eluent and the TurboFlowTM column was washed and conditioned
weighed into 2 mL polypropylene tube. Working internal standard to be ready for the injection of the next sample. The TLX and LC
solution (50 L) and solvent mixture ACN:2% TCA (45:55, v/v) conditions are given in Table 1.
(450 L) were added to the sample. The sample was shaken for The analytical column was conditioned during loading step
5 min on a vortex mixer equipped with a foam tube holder and onto the TurboFlowTM column. The separation of the analytes on
then centrifuged at 12,000 rpm for 5 min. The supernatant was l- the analytical column was carried out by the gradient (Table 1).
tered through a nylon micro lter (0.45 m pore size) directly into The mobile phases were (A) 1 mM HFBA + 0.5% formic acid (FA) in
the LC vial and nally, the sample was injected into the TLX-MS/MS. 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
2.7. TurboFlow and LC conditions 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
Connection of the TurboFlowTM column to the analytical col- additional 2 min at the beginning of the run, which is needed for
umn allows concentration, clean-up and analytical separation in loading the sample onto the TurboFlowTM column and transfer
one step. First the sample was applied by the loading pump onto onto the analytical column. The nal run time of the method with
the TurboFlowTM column. During this step the macromolecules automated on-line sample clean-up and analytical separation was
were removed, while the target analytes were retained on the 19 min. The injection volume of the sample was 35 L. To prevent
TurboFlowTM column based on their different chemical interac- the possibility of carry-over and cross contamination the injection
tions. In the next step the trapped analytes were transferred by syringe as well as the injection valve were washed three times
the eluting pump and the eluent in the loop onto the analytical 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


For extraction of antibiotics from animal tissues the commonly
3.1. Method development used organic solvents are ACN, methanol, ethanol or an aque-
ous solution of TCA. These compounds cause protein precipitation,
3.1.1. LCMS/MS which is necessary to release and extract bound analytes. Recently
For the group of very polar aminoglycosides application of ion- a mixture of ACN and 2% TCA (1:1, v/v) was reported as a suitable
pair reagents was necessary. It is well known, that the presence solvent for extraction of chicken meat [14]. The target analytes
of ion-pair agent in the mobile phase can cause ion suppression were not all the same. Therefore the ratio between ACN and 2%
[8,20,21]. During optimisation of the chromatographic parameters TCA in the extraction solvent was studied by performing experi-
two different ion-pair reagents were tested. Mobile phase with only ments with extraction of chicken meat spiked at 100 g kg1 . The
addition of FA, mobile phase with addition of FA and TFA and nally ratios 50:50, 30:70, 70:30, 0:100 and 100:1 between ACN and 2%
mobile phase with addition of FA and HFBA were compared. Chro- TCA were subsequently tested. The results (Fig. 1) showed that
matographic separation was improved much more by applying the best recoveries were achieved for different classes with dif-
HFBA in comparison to TFA. By applying TFA strong ion suppres- ferent ratios. For group of aminoglycosides was the best mixture of
sion effect was observed, while by applying HFBA minimal or no. 0:100 ACN:2% TCA, but the mixtures with 30:70 and 50:50 ACN:2%
After obtaining unsatisfactory results with TFA, HFBA was chosen TCA were also acceptable. The same statement applies for group of
as an optimal ion-pairing agent to be added to mobile phase for tetracyclines. For trimethoprim, macrolides and quinolones were
the analytical separation of the target compounds. A Betasil phenyl acceptable all ve studied mixtures, because the satised recover-
hexyl 50 mm 2.1 mm, 3 m analytical column provided a good ies were reached with all of them. The only one mixture (0:100
separation of target compounds thanks to its stationary phase com- ACN:2% TCA) was not suitable for sulfonamides. The two mix-
position with a combination of straight-chain C6 groups and phenyl 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 spiking blank chicken meat with a working solution at levels of 0.5,
mixture 50:50 ACN:2% TCA was chosen as the optimal, but because 1 and 1.5 times the MRL. For compounds without any MRL, blank
of poor shape of aminoglycoside peaks, one more experiment samples were spiked at levels indicated in Table 2. The measured
with mixture 45:55 ACN:2% TCA was performed. The recoveries parameters were specicity, linear range, repeatability, accuracy,
of all groups were satisfactory with this mixture and peak shape limit of detection (LOD), limit of quantication (LOQ), decision limit
of aminoglycosides was improved (Fig. 2). Therefore the mixture (CC) and detection capability (CC).
45:55 ACN:2% TCA was used in the nal optimised method.
3.2.1. Detection and quantication
3.1.3. TurboFlow method Mass spectrometric analysis was carried out using a TSQ Quan-
For the development of an effective TurboFlowTM method it is tum Access Max triple quadrupole system. Data acquisition for
necessary to optimise certain parameters. Firstly, it is necessary to quantication and conrmation was performed in the multiple
select the most appropriate TurboFlowTM column, then optimise reaction monitoring mode (MRM). Although two transitions were
loading of the sample onto the clean-up column, optimise transfer followed for the identication, only one of these was used for quan-
of target compounds onto the analytical column and then establish tication. All MRM ions (precursor, qualier and quantier ion)
equilibration conditions. were individually tuned for each target analyte by direct injection
The optimisation of TurboFlow method was comprehensively of the individual working standard solution (10 mg mL1 ) (Table 3).
described in previously reported turbulent ow LCMS/MS method
[19]. 3.2.2. Matrix effects
The application of nal method is shown in Fig. 3. Matrix effects were evaluated by comparing the calibration
curves for each analyte created from the calibration points (n = 9)
3.2. Method validation prepared in solvent and in matrix-matched standards. The graphs
prepared for the representatives of each chemical class are shown
The method was validated in-house according to the criteria in Fig. 4. No matrix effect was observed for group of quinolones
specied in EU Commission Decision 2002/675/EC for a quan- and for most of sulfonamides, when the difference between slope
titative method. The validation parameters were determined by 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 screening with method detection limits for the analytes rang-
ing from 1 to 41 g kg1 . These limits are comparable to LOQs
Turbulent ow chromatography has previously been employed of the reported method; they ranged from 1 to 25 g kg1 with
for on-line clean-up and extraction of samples from food safety the exception for neomycin (120 g kg1 ). These values are also
area in wide range of methods such as determination of quinolones similar to those reported by Chiaochan et al. [14] for a method
in honey [16], determination of pesticides in oranges and hazel- for 24 antibiotics in chicken meat (LOQs ranged from 0.3 to
nuts [22], determination of antibiotics in milk [17] and in edible 60 g kg1 ).
tissues [18], but just for two quinolones. In this paper we report The total time needed for measurement of one sample was
the use of turbulent ow technology in multi-residue method 34 min, taking around 15 min for sample preparation and 19 min
for chicken meat. In comparison with matrices such as honey, for instrumental analysis including the sample clean-up. It was a
milk or oranges, where a minimal short sample preparation is slightly faster approach than the 25 min for measurement of one
needed, here it is necessary perform slightly longer sample prepa- sample as reported by another author [14] and much faster than
ration prior to injection into the TLX-MS/MS system. The sample method of Martos et al. [6], who needed more than 2 h for one
preparation is nevertheless short combining liquid extraction with sample including measurements.
on-line clean-up in comparison with a very complicated method The validated method was applied to a small survey with 20
published by others [6] for analysis of similar compounds in samples of various chicken meat and chicken meat products from
veal muscle. This method was developed as a semi-quantitative 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
Table 5 measured in parallel and matrix-matched calibration and internal
Results of measurement the quality control test material FAPAS sh muscle standardisation were used for the evaluation, whereas retention
T02174QC ciprooxacin certied value = 113 50 g kg1 .
times and specic ion ratios were also used for identication. None
Sample c (g kg1 ) of the 36 analytes were detected in any of the samples, except
Measurement 1 90 enrooxacin which was found in one sample of chicken ham at
Measurement 2 103 19.9 g kg1 and met all identication requirements. However, this
Measurement 3 107 value did not exceed the MRL of 100 g kg1 .
K. Bousova et al. / J. Chromatogr. A 1274 (2013) 1927 27

4. Conclusion [4] C. Chfer-Perics, . Maquieira, R. Puchades, Trends Anal. Chem. 29 (2010)


1038.
[5] K. Granelli, C. Branzell, Anal. Chim. Acta 586 (2007) 289.
The method reported here applies a less commonly employed [6] P.A. Martos, F. Jayasundara, J. Dolbeer, W. Jin, L. Spilsbury, M. Mitchell, C. Varilla,
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[7] R. Romero-Gonzlez, M.M. Aguilera-Luiz, P. Plaza-Bolanos, A. Garrido Frenich,
raphy. This brings the benet of automation of the sample
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preparation, thus increasing the cost effectiveness and decreasing [8] W. Zhu, J. Yang, W. Wei, Y. Liu, S. Zhang, J. Chromatogr. A 1207 (2008) 29.
the time involved in manual sample handling. With this method [9] A.R. Shalaby, N.A. Salama, S.H. Abou-Raya, W.H. Emam, F.M. Mehaya, Food
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it is possible to determine and quantify 36 antibiotic residues
[10] M. Cherlet, M. Schelkens, S. Croubels, P. De Backer, Anal. Chim. Acta 492 (2003)
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