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Journal of Chromatography A, 1274 (2013) 19– 27

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

jou rn al h om epage: www.elsev ier .com/ locat e/chroma

uantitative multi-residue method for determination antibiotics in chicken meatsing turbulent flow chromatography coupled to liquidhromatography–tandem mass spectrometry

aterina Bousovaa,∗, Hamide Senyuvab, Klaus Mittendorfa

Food Safety Response Centre, Thermo Fisher Scientific, Im Steingrund 4-6, 63303 Dreieich, GermanyFoodLife International, 06531 Ankara, Turkey

r t i c l e i n f o

rticle history:eceived 8 August 2012eceived in revised form 30 October 2012ccepted 25 November 2012vailable online 19 December 2012

eywords:ntibiotics

a b s t r a c t

A multi-class method for identification and quantification of 36 antibiotics from seven different chem-ical classes (aminoglycosides, macrolides, lincosamides, sulfonamides, tetracyclines, quinolones andtrimethoprim) has been developed by using liquid chromatography–mass spectrometry. The methodwas optimised for detection of antibiotics in chicken meat. Sample preparation including extraction witha mixture of acetonitrile:2% trichloroacetic acid (45:55, v/v), centrifugation and filtration was followed byon-line clean-up using turbulent flow chromatography. Using this automated on-line technique enableda larger number of samples to be analysed per day than with a traditional clean-up technique (e.g. solid

n-line clean-upC–MS/MShicken meatood safetyalidation

phase extraction). The optimised method was validated according to the European Commission Directive2002/657/EC. In-house validation was performed by fortifying the blank matrix at three levels 0.5, 1.0and 1.5 MRL (maximum residue limit), or respectively, at concentrations as low as possible for substanceswithout an MRL. Precision in terms of repeatability standard deviation ranged from 3 to 28% and recoveryvalues were between 80 and 120% in most cases. All calculated validation parameters including CC� andCC� were in the compliance with the legislative requirements.

. Introduction

Veterinary drugs are widely used in the animal husbandry toreat and prevent diseases. Despite obvious benefits, extensive usef these drugs can lead to residues in animal products such aseat, milk and eggs. Consumption of animal products contami-

ated with veterinary drug residues can cause allergic reactionsn sensitive humans, and can negatively affect the human immuneystem. The most serious concern about excessive use of veterinaryugs is the impact on the effectiveness of the human mediciness bacteria have started to become resistant to the most commonntibiotics. In order to combat this problem and protect the humanonsumers, the use of veterinary drugs is tightly regulated. The EUouncil Directive 96/23/EC [1] requires monitoring of certain vet-rinary drugs in live animals and in animal products. To ensure that

esting is carried out to the highest analytical standards unambigu-us guidelines stipulate the rules for the analytical methods to besed for testing food samples of animal origin for the presence of

∗ Corresponding author. Tel.: +49 61034081113; fax: +49 61034081122.E-mail addresses: katerina.bousova@thermofisher.com

K. Bousova), hamide.senyuva@foodlifeint.com (H. Senyuva),laus.mittendorf@thermofisher.com (K. Mittendorf).

021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2012.11.067

© 2012 Elsevier B.V. All rights reserved.

residues and contaminants [2]. For the most frequently used veteri-nary drugs for different types of food products maximum residuelimits (MRLs) have been set [3]. Analytical methods employed fordetermination of veterinary drugs must be capable of determiningthe residues below their MRLs.

To fulfil the regulatory requirements it is necessary to employsensitive, selective and reliable analytical methods. For fastscreening of animal products a screening method should preferablybe used. There are various approaches for residue screening. Verypopular and quite often used are methods based on microbial orimmunological assay [4]. These methods are low-cost and rapid, buthave the disadvantage of either only being suitable for screeningbroad classes of drugs or conversely being too specific and suitablefor targeting only at single residue. Instrumental techniques such asliquid chromatography–tandem mass spectrometry (LC–MS/MS)have also been used for screening, providing the added benefit ofalso being capable of confirming positive results. For example, amulti-analyte LC–MS/MS screening methods for 19 antibiotics inmuscle and kidney [5] and semi-quantitative determination of 39antibiotics 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-residueUHPLC–Orbitrap–MS 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) 19– 27

Table 1TurboFlowTM loading and eluting conditions.

Step Step length (s) Loading pumpa Valve interfacemodule (VIM)

Eluting pumpb

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 5 955. 120 1.5 50 50 – – – In 0.3 5 956. 120 1.5 – – 100 – – Out 0.3 100 –

ater.

D 0.5% f

ft

omafcmlp

tlwtbanaucTwmroirpwTcl‘

dqaohntscqhmmfC

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

or confirmatory purposes. However, the preferred instrumenta-ion to be used for the confirmatory methods is still LC–MS/MS.

In the last decades most confirmatory methods were devel-ped only for one class of veterinary drugs. Single-class analyticalethods dealing with meat were reported for such groups as

minoglycosides [8], tetracyclines [9,10], macrolides [11] and sul-onamides [12,13]. Notwithstanding the success of these methodsurrently there is an increasing demand for fast and reliableulti-class multi-residue methods, which if used by food control

aboratories could reduce costs and achieve high sample through-ut.

Poultry meat, just like any other type of meat, having a high pro-eins and lipid content is a very complex matrix. Considering thearge number of target compounds from different chemical classes

ith different chemical properties the most challenging part ofhe multi-residue method is the sample preparation in terms ofoth the extraction and clean-up. There have been used three mainpproaches for sample preparation for the determination of veteri-ary drug residues in meat. Liquid extraction in combination with

solid-phase extraction (SPE) as a clean-up procedure has beensed for the determination of sulfonamides in meat [12], tetracy-lines in a chicken meat [9] and tetracyclines in pig tissues [10].he second approach consists of liquid extraction in combinationith a defatting step employing hexane, which has been used in aulti-residue method for poultry meat [14] and for another multi-

esidue method for veal muscle [6]. The third approach consistsnly of liquid extraction and a subsequent dilution step prior directnjection into the instrument and has been employed in two multi-esidue methods for pig muscle [6,15]. The first two ways of samplereparation are quite effective and provide clean sample extracts,hich can be subsequently injected into the mass spectrometer.

he drawback is that the sample preparation is lengthy and in thease where SPE is used there is the added cost of SPE cartridges. Theast approach appears to be simple and fast, but because of injectingdirty’ extracts there is a high risk of MS source contamination.

The aim of the method reported here was to overcome theisadvantages mentioned above and develop a confirmative anduantitative multi-class method for the determination of a range ofntibiotics from different classes in chicken meat. For the clean-upf meat extracts turbulent flow chromatography was used, whichas the added benefit of being suitable for automation. This tech-ique has been previously used in the multi-class methods forhe determination of veterinary drug residues in other matricesuch as honey [16] and milk [17]. For animal tissues turbulent flowhromatography was used only once in one-class method for twouinolones [18]. The reported method was first developed and in-ouse validated for milk [19]. In this study an adaptation of the

ethod is reported for more complex matrices, such as chickeneat. The method was validated for determination of 36 residues

rom seven different chemical classes of antibiotics according toommission Decision 2002/657/EC [2].

B = 0.5% formic acid in ACN:methanol 1:1 (v/v). C = 2% methanol in water.

ormic acid in ACN:methanol 1:1 (v/v).

2. Experimental

2.1. Samples and quality control materials

Chicken obtained from a local market, which was repeatedlymeasured to confirm that no antibiotics were present, was usedfor preparation of matrix-matched calibration standards and forfortified samples. Because of a lack of chicken tissue certifiedtest materials, to establish method accuracy a FAPAS test mate-rial T02174QC of fish muscle containing a certified amount ofciprofloxacin was used, which was obtained from the Food andEnvironment Research Agency (York, United Kingdom). Thoughfish muscle is not the same as chicken muscle, consideringthe content of proteins, water and fat the differences are notlarge and thus fish muscle was used to demonstrate methodaccuracy.

2.2. Chemicals and reagents

Optima LCMS grade methanol, acetonitrile, water (Fisher Sci-entific, Langenselbold, Germany) and HPLC grade isopropanoland acetone (Fisher Scientific, Langenselbold, Germany) wereused as mobile phases, for sample extraction, preparationof standard mixtures and washing of the injection port.Trichloroacetic acid for sample extraction was obtained fromAcros Organics (Geel, Belgium). HPLC grade formic acid (FisherScientific, Loughborough, UK) and heptafluorobutyric acid (AcrosOrganics, Geel, Belgium) were used as additives for mobilephases. 35% ammonia solution (Fisher Scientific, Loughbor-ough, UK) was used for preparation of stock standard solutionsfor quinolones. Purified water was obtained from Barnstead®

EASYpure® II water system (Thermo Scientific, Barnstead,NH).

2.3. Standards

Chlortetracycline, cinoxacin, ciprofloxacin, clarithromycin,clindamycin hydrochloride, danofloxacin, difloxacin hydrochlo-ride, doxycycline hyclate, enoxacin, enrofloxacin, flumequine,josamycin, kanamycin disulfate salt, lincomycin hydrochlo-ride monohydrate, lomefloxacin hydrochloride, marbofloxacin,nalidixic acid, neomycin, norfloxacin, ofloxacin, oxolinic acid,oxytetracycline hydrochloride, sarafloxacin hydrochloride tri-hydrate, spiramycin, sulfachlorpyridazine, sulfadimethoxine,sulfadoxin, sulfamethoxazole, sulfaphenazole (used as internalstandard), sulfaquinoxaline, tetracycline, tilmicosin, trimetho-prim and tylosin tartrate were obtained from Sigma–Aldrich

(Taufkirchen, Germany); oleandomycin phosphate dehydrate andsulfaclozine 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) 19– 27 21

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

RE

C (

%)

ACN:2%TCA

Trimethoprim

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

ACN:2%TCA

Lincosamides

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

ACN:2%TCA

Aminoglycosides

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

ACN:2%TCA

Macrolides

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

Quinolones

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

RE

C (

%)

ACN:2%TCA

Sulfonamides

0

20

40

60

80

100

120

140

50:50 30:70 70:30 0:100 100:1

ACN:2%TC A

Tetracyclines

ixtur

2

dsgwcdstsdTg

2

soAtscwsuFa

tFs

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

ACN:2%TCA

Fig. 1. Influence of extraction mixture on recovery. The range shown at each m

.4. Standard solutions

Stock standard solutions (1000 �g mL−1) were prepared byissolving the analytes in methanol (lincosamides, macrolides,ulfonamides, tetracyclines and trimethoprim), in water (amino-lycosides) and in methanol with 2% 2 M NH4OH (quinolones). Theorking standard solution containing all analytes with variable

oncentrations, according to their LOQ and MRL, was prepared byilution of stock standard solutions with ACN. Working standardolution of internal standard (2000 �g L−1) was prepared by dilu-ion of stock standard solution (sulfaphenazole) with ACN. Stocktandard solutions were kept in brown glass to prevent the photo-egradation and stored at −20 ◦C and were stable for three months.he working standard solutions were also kept at −20 ◦C in brownlass and were stable for two weeks.

.5. Instrumentation

A turbulent flow chromatograph TranscendTM TLX-1 (TLX)ystem (Thermo Fisher Scientific, Franklin, MA) was used for devel-pment of this method and included a PAL autosampler (CTCnalytics, Zwingen, Switzerland), two high-pressure mixing qua-

ernary pumps (loading and eluting pump) and a three-valvewitching device unit with six-port valve. The entire system wasontrolled by Aria software, version 1.6. The TurboFlowTM columnas a Cyclone P 50 mm × 0.5 mm, 60 �m particle size, 60 A pore

ize (Thermo Fisher Scientific, Franklin, MA) and the analytical col-mn was a Betasil phenyl hexyl 50 mm × 2.1 mm, 3 �m (Thermoisher Scientific, Runcorn, UK). The analytical column was operatedt room temperature.

The TranscendTM TLX-1 system was coupled to the TSQ Quan-um Access MaxTM triple quadrupole mass spectrometer (Thermoisher Scientific, San Jose, CA) equipped with a heated electro-pray ionisation probe that was kept at 400 ◦C. Measurements were

e encompasses the recoveries of all the compounds in the group of antibiotics.

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

Fig. 2. Comparison of peak shape for two aminoglycosides in a fortified chickenmeat sample (100 �g kg−1) by applying different extraction solvent.

22 K. Bousova et al. / J. Chromatogr. A 1274 (2013) 19– 27

cken m

wti

2

tws(5ttt

2

uotwTtt

Fig. 3. Example chromatogram of spiked chi

hile the cycle time and peak width were 0.6 s and 0.7 Da, respec-ively. All data were processed using Xcalibur software version 2.1n EZ set up mode.

.6. Sample preparation

Chicken meat of around 500 g was homogenised in a labora-ory blender for 5 min. Then, homogenised chicken meat (0.5 g) waseighed into 2 mL polypropylene tube. Working internal standard

olution (50 �L) and solvent mixture ACN:2% TCA (45:55, v/v)450 �L) were added to the sample. The sample was shaken for

min on a vortex mixer equipped with a foam tube holder andhen centrifuged at 12,000 rpm for 5 min. The supernatant was fil-ered through a nylon micro filter (0.45 �m pore size) directly intohe LC vial and finally, the sample was injected into the TLX-MS/MS.

.7. TurboFlow and LC conditions

Connection of the TurboFlowTM column to the analytical col-mn allows concentration, clean-up and analytical separation inne step. First the sample was applied by the loading pump ontohe TurboFlowTM column. During this step the macromolecules

ere removed, while the target analytes were retained on the

urboFlowTM column based on their different chemical interac-ions. In the next step the trapped analytes were transferred byhe eluting pump and the eluent in the loop onto the analytical

eat sample for each of 36 target antibiotics.

column and separated conventionally. The loop can be easily con-nect and disconnect from the system thanks to a special switchingvalve, which enables to perform more steps in parallel. Transfer ofthe compounds from the TurboFlowTM column onto analytical col-umn was accomplished by using a loop filled with a mixture witha certain content of organic phase. While the separation on theanalytical column was running, the loop was filled with the fresheluent and the TurboFlowTM column was washed and conditionedto be ready for the injection of the next sample. The TLX and LCconditions are given in Table 1.

The analytical column was conditioned during loading steponto the TurboFlowTM column. The separation of the analytes onthe analytical column was carried out by the gradient (Table 1).The mobile phases were (A) 1 mM HFBA + 0.5% formic acid (FA) inwater and (B) 0.5% FA in ACN/methanol (1:1, v/v). The gradientelution programme started with 0% B, it remained for 1 min, thenit increased to 95% in 12 min, remained for 2 min and returnedback to the initial compositions in another 2 min. There was anadditional 2 min at the beginning of the run, which is needed forloading the sample onto the TurboFlowTM column and transferonto the analytical column. The final run time of the method withautomated on-line sample clean-up and analytical separation was

19 min. The injection volume of the sample was 35 �L. To preventthe possibility of carry-over and cross contamination the injectionsyringe as well as the injection valve were washed three timeswith cleaning solvent 1 (ACN/water – 20/80) and cleaning solvent 2

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 19– 27 23

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

Analyte Spiking levels (�g kg−1) RECa (%) WDb (%) BDc (%)

L1 L2 L3 L1 L2 L3 L1 L2 L3 L2

Kanamycin 50 100 150 119 109 120 19 25 21 25Neomycin 250 500 750 84 71 83 23 18 19 28

Lincomycin 50 100 150 104 94 102 4 10 10 15Clindamycin 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 8Spiramycin 100 200 300 108 102 92 8 10 21 22Tilmicosin 37.5 75 112.5 115 105 102 7 7 9 8Tylosin 50 100 150 86 84 82 9 7 19 13Clarithromycin 10 20 30 101 105 98 11 6 12 10Oleandomycin 10 20 30 116 93 92 13 10 10 18Tylvalosin 10 20 30 91 101 99 15 6 16 12

Sulfadimethoxine 50 100 150 101 97 91 3 5 10 7Sulfamethoxazole 50 100 150 113 108 96 7 10 5 9Sulfadoxin 50 100 150 101 104 98 14 9 6 11Sulfaquinoxaline 50 100 150 100 94 99 17 21 5 22Sulfachlorpyridazine 50 100 150 109 102 94 8 8 13 12Sulfaclozine 50 100 150 118 110 106 14 14 10 11

Oxytetracycline 50 100 150 115 109 114 27 13 11 15Tetracycline 50 100 150 102 94 94 10 12 10 11Chlortetracycline 50 100 150 96 85 87 13 19 12 15Doxycycline 50 100 150 117 98 95 7 8 9 10

Marbofloxacin 10 20 30 104 105 106 9 12 18 15Ciprofloxacin 50 100 150 101 114 103 10 8 8 9Danofloxacin 100 200 300 116 108 109 5 3 9 7Enrofloxacin 50 100 150 112 108 103 11 7 6 9Difloxacin 150 300 450 106 105 102 4 8 10 9Oxolinic acid 50 100 150 109 100 95 4 5 7 8Flumequine 200 400 600 108 94 88 6 7 9 8Nalidixic acid 10 20 30 118 103 99 6 6 8 8Enoxacin 10 20 30 99 103 88 17 14 22 12Ofloxacin 10 20 30 101 92 89 9 20 15 15Lomefloxacin 10 20 30 98 100 94 27 19 16 19Norfloxacin 10 20 30 101 112 101 11 7 16 10Sarafloxacin 10 20 30 105 99 90 24 22 6 17Cinoxacin 10 20 30 102 94 91 16 19 12 16

(i

3

3

3

po[tammHsAathsp

a Recovery.b Within-day precision.c Between-day precision.

acetone/ACN/isopropanol – 20/40/40) before and five times afternjection.

. Results and discussion

.1. Method development

.1.1. LC–MS/MSFor the group of very polar aminoglycosides application of ion-

air reagents was necessary. It is well known, that the presencef ion-pair agent in the mobile phase can cause ion suppression8,20,21]. During optimisation of the chromatographic parameterswo different ion-pair reagents were tested. Mobile phase with onlyddition of FA, mobile phase with addition of FA and TFA and finallyobile phase with addition of FA and HFBA were compared. Chro-atographic separation was improved much more by applyingFBA in comparison to TFA. By applying TFA strong ion suppres-

ion effect was observed, while by applying HFBA minimal or no.fter obtaining unsatisfactory results with TFA, HFBA was chosens an optimal ion-pairing agent to be added to mobile phase for

he analytical separation of the target compounds. A Betasil phenylexyl 50 mm × 2.1 mm, 3 �m analytical column provided a goodeparation of target compounds thanks to its stationary phase com-osition with a combination of straight-chain C6 groups and phenyl

groups (classical reversed phase retention together with selectivityfor polar compounds).

3.1.2. Extraction methodFor 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. Recentlya mixture of ACN and 2% TCA (1:1, v/v) was reported as a suitablesolvent for extraction of chicken meat [14]. The target analyteswere 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 kg−1. Theratios 50:50, 30:70, 70:30, 0:100 and 100:1 between ACN and 2%TCA were subsequently tested. The results (Fig. 1) showed thatthe best recoveries were achieved for different classes with dif-ferent ratios. For group of aminoglycosides was the best mixture of0:100 ACN:2% TCA, but the mixtures with 30:70 and 50:50 ACN:2%TCA were also acceptable. The same statement applies for group oftetracyclines. For trimethoprim, macrolides and quinolones were

acceptable all five studied mixtures, because the satisfied recover-ies were reached with all of them. The only one mixture (0:100ACN:2% TCA) was not suitable for sulfonamides. The two mix-tures (50:50 and 30:70 ACN: 2% TCA) were acceptable, because of

2 matog

lmowoo4

3

nsloe

d[

3

st

TM

4 K. Bousova et al. / J. Chro

incomycin, for group of lincosamides. Due to results for this groupixture 50:50 ACN:2% TCA was chosen as the optimal, but because

f poor shape of aminoglycoside peaks, one more experimentith mixture 45:55 ACN:2% TCA was performed. The recoveries

f all groups were satisfactory with this mixture and peak shapef aminoglycosides was improved (Fig. 2). Therefore the mixture5:55 ACN:2% TCA was used in the final optimised method.

.1.3. TurboFlow methodFor the development of an effective TurboFlowTM method it is

ecessary to optimise certain parameters. Firstly, it is necessary toelect the most appropriate TurboFlowTM column, then optimiseoading of the sample onto the clean-up column, optimise transferf target compounds onto the analytical column and then establishquilibration conditions.

The optimisation of TurboFlow method was comprehensivelyescribed in previously reported turbulent flow LC–MS/MS method19].

The application of final method is shown in Fig. 3.

.2. Method validation

The method was validated in-house according to the criteriapecified in EU Commission Decision 2002/675/EC for a quan-itative method. The validation parameters were determined by

able 3RM transitions for 36 analytes from and sulfaphenazole as internal standard (IS).

Analyte Classa Precursor ion (m/z) Quantifi

Kanamycin1

485.2 163.1

Neomycin 615.3 161.0

Lincomycin2

407.1 126.1

Clindamycin 425.1 126.1

Trimethoprim 3 291.1 230.1

Josamycin

4

828.4 173.9

Spiramycin 843.3 173.9

Tilmicosin 869.6 696.4

Tylosin 916.5 174.0

Clarithromycin 748.5 158.1

Oleandomycin 688.4 544.3

Tylvalosin 1042.6 109.0

Sulfadimethoxine

5

311.0 156.0

Sulfamethoxazole 254.0 156.0

Sulfadoxin 311.0 156.0

Sulfaquinoxaline 301.0 156.0

Sulfachlorpyridazine 284.9 156.0

Sulfaclozine 284.9 92.1

Sulfaphenazole (IS) 315.0 158.1

Oxytetracycline

6

461.1 426.1

Tetracycline 445.1 410.1

Chlortetracycline 479.0 444.0

Doxycycline 445.1 428.1

Marbofloxacin

7

363.1 72.3

Ciprofloxacin 332.0 314.1

Danofloxacin 358.1 340.1

Enrofloxacin 360.1 316.1

Difloxacin 400.1 382.1

Oxolinic acid 262.0 244.0

Flumequine 262.0 244.0

Nalidixic acid 233.0 215.0

Enoxacin 321.0 303.0

Ofloxacin 362.1 318.1

Lomefloxacin 352.1 265.0

Norfloxacin 320.0 302.0

Sarafloxacin 386.0 368.1

Cinoxacin 263.0 245.0

a Class: (1) – aminoglycosides, (2) – lincosamides, (3) – trimethoprim, (4) – macrolidesb CE = collision energy.c TL = tube lens.

r. A 1274 (2013) 19– 27

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, blanksamples were spiked at levels indicated in Table 2. The measuredparameters were specificity, linear range, repeatability, accuracy,limit of detection (LOD), limit of quantification (LOQ), decision limit(CC�) and detection capability (CC�).

3.2.1. Detection and quantificationMass spectrometric analysis was carried out using a TSQ Quan-

tum Access Max triple quadrupole system. Data acquisition forquantification and confirmation was performed in the multiplereaction monitoring mode (MRM). Although two transitions werefollowed for the identification, only one of these was used for quan-tification. All MRM ions (precursor, qualifier and quantifier ion)were individually tuned for each target analyte by direct injectionof the individual working standard solution (10 mg mL−1) (Table 3).

3.2.2. Matrix effectsMatrix 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 shownin Fig. 4. No matrix effect was observed for group of quinolonesand for most of sulfonamides, when the difference between slopeof calibration curve in solvent and in matrix was minor. Quite

er ion CEb (V) Qualifier ion CE (V) TLc

25 324.1 15 9029 163.1 31 101

28 359.1 17 9728 377.1 18 86

23 261.0 24 93

30 109.1 34 1832 142.0 32 14640 174.0 41 13235 772.4 26 14128 590.3 17 10814 158.0 25 10641 173.9 37 133

21 108.1 27 8815 92.1 27 9618 108.1 26 8817 92.1 28 9215 92.1 26 9029 108.1 26 8728 160.1 22 94

18 426.1 18 9318 427.1 11 9922 462.1 16 9818 321.0 31 82

22 320.0 14 9718 288.1 22 8924 314.1 16 9919 342.1 22 9621 356.1 19 9818 216.0 29 8419 202.0 33 8415 187.0 25 7719 257.1 17 9318 261.0 27 9123 308.1 15 10022 276.1 16 9423 342.1 18 9416 217.0 22 90

, (5) – sulfonamides, (6) – tetracyclines, (7) – quinolones.

K. Bousova et al. / J. Chromatogr. A 1274 (2013) 19– 27 25

y = 0.0075x + 0.046 5

R² = 0.990 2

y = 0.011x - 0.023 5

R² = 0.992 4

0.00

0.40

0.80

1.20

1.60

2.00

0 20 40 60 80 100 120 140 160

Aa/A

IS

c [µg/ kg]

Til micosin

Solven t Ma trix

y = 0.0001x - 0.000 3

R² = 0.99 4

y = 1E -04x - 0.001 2

R² = 0.990 8

0.00

0.01

0.01

0.02

0.02

0.03

0.03

0 20 40 60 80 100 120 140 160 180 200 220

Aa/A

IS

c [µg/ kg]

Kanamyci n

Solv Ma trix

y = 0.0107x + 0.082 9

R² = 0.991 3

y = 0.0095x - 0.058 7

R² = 0.990 2

0.00

0.40

0.80

1.20

1.60

2.00

2.40

0 20 40 60 80 100 120 140 160 180 200

Aa/A

IS

c [µg/ kg]

Lincomycin

Solv Ma trix

y = 0.0019x + 0.01 1

R² = 0.993 7

y = 0.0013x - 0.016 6

R² = 0.990 60.00

0.10

0.20

0.30

0.40

0.50

0 20 40 60 80 100 120 140 160 180 200 220

Aa/A

IS

c [µg/ kg]

Oxytetra cycl ine

Solven t Sol ven t Ma trix

y = 0.0009x - 0.004 3

R² = 0.990 9

y = 0.0014x - 0.009 7

R² = 0.992 1

0.00

0.10

0.20

0.30

0 20 40 60 80 100 120 140 160 180 200 220

Aa/A

IS

c [µg/ kg]

Sulfaclozin e

y = 0.0153x + 0.10 5

R² = 0.997 5

y = 0.0115x - 0.013 7

R² = 0.992 7

0.00

0.40

0.80

1.20

1.60

2.00

0 20 40 60 80 100 120A

a/A

IS

c [µg/ kg]

Trim ethopri m

Solv Ma trix

y = 0.0018x + 0.009 3

R² = 0.993 8

y = 0.0019x - 0.002 1

R² = 0.990 2

0.00

0.10

0.20

0.30

0.40

0.50

0 20 40 60 80 100 120 140 160 180 200 220

Aa/A

IS

c [µg/ kg]

Sulfamethoxazo le

Solven t Ma trix

y = 0.0025x - 0.000 7

R² = 0.99 1

y = 0.0023x - 0.004 2

R² = 0.99

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0 5 10 15 20 25 30 35 40 45

Aa/A

IS

c [µg/ kg]

Marbofloxacin e

F mycin6

csscmsha(us

3

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3

dcwwbtv

ig. 4. Matrix-matched calibration plots for eight representative compounds (1-kana-oxytetracycline and 7-marbofloxacine) in solvent and chicken meat.

onsiderable matrix effects were observed for sulfadoxin (signaluppression) and sulfaclozine (signal enhancement). The signaluppression occurred for the groups of tetracyclines, aminogly-osides, lincosamides and trimethoprim as calibration curves inatrix were found to have a slope below the calibration curves in

olvent. Conversely calibration curves in matrix for all macrolidesad higher slopes than the calibration curves in solvent, indicating

significant enhancement for all macrolides except oleandomycinno matrix effect). Consequently, matrix-matched calibration wassed for quantification purposes to compensate the problems withuppression or enhancement effects.

.2.3. SpecificityUsing MRM, the specificity was confirmed based on the pres-

nce of the transition ions (quantifier and qualifier) at the correctetention times corresponding to those of the respective antibiotics.he measured peak area ratios of qualifier/quantifier are within theange (relative intensity of base peak – permitted range; >50% –20%, >20%–50% – ±25%, >10–20% – ±30%, ≤10% – ±50%) defined inU Commission Decision 2002/657/EC when compared to the stan-ards. The calculated ion ratios for solvent and matrix are shown

n Table 4.

.2.4. Linearity & calibration curveThe linearity of calibration curves was assessed over the range

epending on the target compound. In all cases, the correlationoefficients of linear functions were >0.99. The calibration curvesere created from 9 matrix-matched calibration standards which

ere injected in each batch in duplicate. The matrix-matched cali-

ration standards were prepared by spiking the blank material withhe same spiking solution as for samples for validation with variableolumes (0–400 �L).

, 2-lincomycin, 3-trimethoprim, 4-tilmicosin, 5-sulfamethoxazole and sulfaclozine,

3.2.5. PrecisionPrecision (repeatability) of the method was determined using

independently spiked blank samples at three different levels. Inone day, the set of samples at three levels was measured as sixreplicates. To determine between-day precision, two other sets atone level were measured with six repetitions over the next twodays. The results for repeatability ranged from 3 to 28% (Table 2).

3.2.6. AccuracyMethod accuracy was determined using independently spiked

blank samples at three different levels. Accuracy was evaluatedby comparing found values with standard additions of spikes.Recovery values ranged between 71 and 120% (Table 2). Addition-ally accuracy was established for ciprofloxacin by analysing a testmaterial (FAPAS T02174QC) which was fish muscle. All measuredconcentrations of ciprofloxacin were within the acceptable range(Table 5).

3.2.7. LOD and LOQThe LOD and LOQ were estimated following the IUPAC approach

which consists of first analysing the blank sample to establish noiselevels and then estimating LODs and LOQs for signal/noise, 3 and10 respectively. The values for chicken meat are shown in Table 4and, in all cases, they are below the level of MRL for analytes, whereit was established.

3.2.8. CC and CCˇBoth CC� and CC� were established by the calibration curve

procedure according to ISO 118434. The blank material fortified at

and below the maximum residue limit (for analytes with MRL) orat and above the lowest possible level (for analytes without MRL)in equidistant steps was used. The calculated values are shown inTable 4.

26 K. Bousova et al. / J. Chromatogr. A 1274 (2013) 19– 27

Table 4Maximum residue limit (MRL), limit of detection and quantification (LOD and LOQ), limit of decision and capability (CC� and CC�), ion ratios (qualifier/quantifier) in solventand matrix for 36 antibiotics in chicken meat.

Analyte MRL (�g kg−1) LOD (�g kg−1) LOQ (�g kg−1) CC� (�g kg−1) CC� (�g kg−1) Ion ratio (solvent) Ion ratio (matrix)

Kanamycin 100 10.0 25.0 121.3 142.5 0.53 0.50Neomycin 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 0.09Clindamycin – 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 0.91Spiramycin 200 0.3 1.0 223.3 246.5 0.17 0.21Tilmicosin 75 0.3 1.0 79.8 84.6 0.88 0.88Tylosin 100 1.0 3.0 107.2 114.4 0.23 0.23Clarithromycin – 0.3 1.0 4.2 5.4 0.62 0.61Oleandomycin – 0.3 1.0 3.3 4.2 0.65 0.71Tylvalosin – 0.3 1.0 4.8 6.2 0.55 0.50

Sulfadimethoxine 100a 0.3 1.0 109.8 119.6 0.60 0.56Sulfamethoxazole 100a 1.5 5.0 118.5 137.1 0.31 0.30Sulfadoxin 100a 0.3 1.0 107.9 115.8 0.46 0.58Sulfaquinoxaline 100a 0.3 1.0 111.0 121.9 0.24 0.27Sulfachlorpyridazine 100a 10.0 25.0 111.0 121.9 0.44 0.46Sulfaclozine 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 0.10Tetracycline 100 3.0 10.0 114.8 129.5 0.80 0.84Chlortetracycline 100 5.0 15.0 111.9 123.8 0.48 0.42Doxycycline 100 1.0 3.0 110.0 120.0 0.03 0.05

Marbofloxacin – 1.5 5.0 6.6 8.4 0.73 0.61Ciprofloxacin 100b 0.3 1.0 104.5 108.9 0.13 0.14Danofloxacin 200 0.3 1.0 216.6 233.2 0.06 0.04Enrofloxacin 100b 0.3 1.0 107.8 115.7 0.58 0.62Difloxacin 300 0.3 1.0 334.4 368.8 0.58 0.69Oxolinic acid 100 0.3 1.0 109.0 118.1 0.06 0.08Flumequine 400 0.3 1.0 437.8 475.6 0.44 0.42Nalidixic acid – 0.3 1.0 1.7 2.2 0.30 0.32Enoxacin – 0.3 1.0 3.5 4.5 0.02 0.03Ofloxacin – 0.3 1.0 3.3 4.2 0.70 0.70Lomefloxacin – 0.3 1.0 4.0 5.1 0.58 0.64Norfloxacin – 0.3 1.0 6.1 7.9 0.05 0.09Sarafloxacin – 0.3 1.0 4.1 5.3 0.18 0.25

cine).

3

fainttfmnrpopv

TRT

Cinoxacin – 1.0 3.0

a Expressed in form of sum-MRLs of all sulfonamides.b Expressed in form of sum-MRLs of enrofloxacine and its metabolite (ciprofloxa

.3. Discussion

Turbulent flow chromatography has previously been employedor on-line clean-up and extraction of samples from food safetyrea in wide range of methods such as determination of quinolonesn honey [16], determination of pesticides in oranges and hazel-uts [22], determination of antibiotics in milk [17] and in edibleissues [18], but just for two quinolones. In this paper we reporthe use of turbulent flow technology in multi-residue methodor chicken meat. In comparison with matrices such as honey,

ilk or oranges, where a minimal short sample preparation iseeded, here it is necessary perform slightly longer sample prepa-ation prior to injection into the TLX-MS/MS system. The samplereparation is nevertheless short combining liquid extraction with

n-line clean-up in comparison with a very complicated methodublished by others [6] for analysis of similar compounds ineal muscle. This method was developed as a semi-quantitative

able 5esults of measurement the quality control test material FAPAS – fish muscle02174QC – ciprofloxacin – certified value = 113 ± 50 �g kg−1.

Sample c (�g kg−1)

Measurement 1 90Measurement 2 103Measurement 3 107

4.2 5.4 0.28 0.30

screening with method detection limits for the analytes rang-ing from 1 to 41 �g kg−1. These limits are comparable to LOQsof the reported method; they ranged from 1 to 25 �g kg−1 withthe exception for neomycin (120 �g kg−1). These values are alsosimilar to those reported by Chiaochan et al. [14] for a methodfor 24 antibiotics in chicken meat (LOQs ranged from 0.3 to60 �g kg−1).

The total time needed for measurement of one sample was34 min, taking around 15 min for sample preparation and 19 minfor instrumental analysis including the sample clean-up. It was aslightly faster approach than the 25 min for measurement of onesample as reported by another author [14] and much faster thanmethod of Martos et al. [6], who needed more than 2 h for onesample including measurements.

The validated method was applied to a small survey with 20samples of various chicken meat and chicken meat products froma local market. Four samples of sausages, five samples of salami,five samples of ham, three samples of raw chicken meat and twosamples of modified chicken meat were analysed. All samples weremeasured in parallel and matrix-matched calibration and internalstandardisation were used for the evaluation, whereas retentiontimes and specific ion ratios were also used for identification. None

of the 36 analytes were detected in any of the samples, exceptenrofloxacin which was found in one sample of chicken ham at19.9 �g kg−1 and met all identification requirements. However, thisvalue did not exceed the MRL of 100 �g kg−1.

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K. Bousova et al. / J. Chro

. Conclusion

The method reported here applies a less commonly employedpproach for sample clean-up using turbulent flow chromatog-aphy. This brings the benefit of automation of the samplereparation, thus increasing the cost effectiveness and decreasinghe time involved in manual sample handling. With this methodt is possible to determine and quantify 36 antibiotic residuesrom seven different classes of drugs in chicken meat. The methodas successfully in-house validated according to the EU Commis-

ion Decision 2002/657/EC, and therefore can be recommended fornforcement of the legislative limits.

eferences

[1] Council Directive 96/23/EC of 29 April 1996 on measures to monitor cer-tain substances and residues thereof in live animals and animal products andrepealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and91/664/EEC, Off. J. Eur. Commun. L125 (1996) 10.

[2] Commission Decision 2002/657/EC of 12th August implementing Council

Directive 96/23/EC concerning the performance of analytical methods and theinterpretation of the results, Off. J. Eur. Commun. L221 (2002) 8.

[3] Commission Regulation 37/2010 of 22nd December 2009 on pharmacologicallyactive substances and their classification regarding maximum residue limits infoodstuffs of animal origin, Off. J. Eur. Commun. L15 (2010) 1.

[

[[

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