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Determination of lasalocid residues in the tissues ofbroiler chickens by liquid chromatography-tandemmass spectrometrySoňa Tkáčiková a , Ivona Kožárová b , Ján Mačanga b & Martin Levkut c

a Department of Medical and Clinical Biophysics, Pavol Jozef Šafárik University in Košice,Trieda SNP, SK-040 11, Košice, Slovakiab Department of Food Hygiene and Technology, University of Veterinary Medicine andPharmacy in Košice, Komenského 73, SK-041 81 Košice, Slovakiac Department of Pathological Anatomy and Pathological Physiology, University of VeterinaryMedicine and Pharmacy in Košice, Komenského 73, SK-041 81 Košice, Slovakia

Available online: 06 Jan 2012

To cite this article: Soňa Tkáčiková, Ivona Kožárová, Ján Mačanga & Martin Levkut (2012): Determination of lasalocidresidues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry, Food Additives &Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment, 29:5, 761-769

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Food Additives and ContaminantsVol. 29, No. 5, May 2012, 761–769

Determination of lasalocid residues in the tissues of broiler chickens by liquid

chromatography-tandem mass spectrometry

Sona Tkacikovaa, Ivona Kozarovab*, Jan Macangab and Martin Levkutc

aDepartment of Medical and Clinical Biophysics, Pavol Jozef Safarik University in Kosice, Trieda SNP, SK-040 11, Kosice,Slovakia; bDepartment of Food Hygiene and Technology, University of Veterinary Medicine and Pharmacy in Kosice,Komenskeho 73, SK-041 81 Kosice, Slovakia; cDepartment of Pathological Anatomy and Pathological Physiology, University ofVeterinary Medicine and Pharmacy in Kosice, Komenskeho 73, SK-041 81 Kosice, Slovakia

(Received 22 June 2011; final version received 28 December 2011)

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribedconcentration and during a certain time interval. Due to a public health concern about the presence ofcoccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues inthe edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fedcommercially produced feed containing 100mgkg�1 of lasalocid in complete feed throughout the 5-daywithdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrosprayionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) andthe limit of quantification (LOQ) of the method were 0.47 and 1.44 mg kg�1, respectively. The average recoverybased on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was foundto accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowestconcentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrationsof lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL)established for lasalocid in poultry from 20 to 100 mg kg�1 by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residuesin the tissues of broiler chickens to the safe residue level.

Keywords: chromatography – LC/MS; veterinary drug residues – antibiotics; animal products – meat

Introduction

Coccidiosis is the most important parasitic diseaseaffecting the economics of poultry production. It iscaused by a highly host-specific, single-celled proto-zoan parasite belonging to the genus Eimeria. Theparasite multiplies in the intestines and causes tissuedamage, decreased feed intake, poor absorption ofnutrients from the feed, dehydration, blood loss andeven death (Goldova et al. 2000, 2001; Goldova 2002;http://attra.ncat.org/attra-pub/PDF/coccidiosis.pdf).

Current coccidiosis-control strategies depend heav-ily upon the addition of coccidiostats to the feed at theauthorised levels observing the prescribed hygienerequirements. Coccidiostats need to be administeredthroughout the animal life (in the case of fatteningchickens) in order to protect against re-infection fromthe ever-present oocyst stage of the disease (http://ec.europa.eu/food/food/animalnutrition/feedadditives/docs/Report-Coccs-233-2008-SK.pdf).

According to Regulation (EC) No. 1831/2003 ofthe European Parliament and of the Council of 22

September 2003 on additives for use in animalnutrition, only monensin, salinomycin, lasalocid,narasin, maduramycin, semduramycin (ionophoriccoccidiostats), and nicarbazin, robenidine, diclazuril,halofuginone and decoquinate (chemical coccidiostats)are allowed to be used as feed additives in theEuropean Union (European Commission 2003).Because these substances are used in food-producinganimals for a long time, their residues may be presentin the tissues of the respective animals and may poserisk to public health.

In order to protect public health, maximum residuelimits (MRLs) should be established in accordance

with generally recognised principles of safety assess-

ment, taking into account toxicological risks, environ-

mental contamination, as well as the microbiological

and pharmacological effects of residues (European

Commission 2009b). MRLs should be set for pharma-

cologically active substances used or intended to be

used in veterinary medicinal products placed on

the market in the European community, including

*Corresponding author. Email: kozarova@uvm.sk

ISSN 1944–0049 print/ISSN 1944–0057 online

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/19440049.2011.653987

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coccidiostats. The current regulation on MRLs –

Commission Regulation (EU) No. 37/2010 of 22

December 2009 on pharmacologically active sub-

stances and their classification regarding MRLs in

foodstuffs of animal origin – establishes the MRL only

for two coccidiostats belonging to the group of

polyether ionophoric antibiotics: for monensin in

bovine from 2 mg kg�1 in muscle, kidney and milk to

30 mg kg�1 in liver; and for lasalocid in poultry from

20 mg kg�1 in muscle, 50 mg kg�1 in kidney, 100 mg kg�1

in liver, skin and fat to 150 mg kg�1 in eggs (European

Commission 2010 b).Lasalocid (Figure 1) is a polyether ionophoric

coccidiostat produced by the mould Streptomyces

lasaliensis and authorised for use for the prevention

of coccidiosis in chickens for fattening, chickens reared

for laying (up to 16 weeks of age) and turkeys (up to 16

weeks of age) at a minimum/maximum content of

active substance in complete feed of 7–125 mgkg�1.

The withdrawal period (WP) for lasalocid is at least

5 days prior to slaughter (European Commission 2004,

2010 a).MRLs in foodstuffs of animal origin should be

carefully monitored and controlled in accordance with

Council Directive 96/23/EC of 29 April 1996 on

measures to monitor certain substances and residues

thereof in live animals and animal products (European

Commission 1996). Several analytical approaches have

been used to determine the coccidiostat residues in

different biological matrices; however, those methods

are suitable for the determination of one or more

coccidiostats only. Because lasalocid is the only

ionophoric coccidiostat that possess a useful chromo-

phore, it should be monitored by an ultraviolet (UV)

detector at a maximum absorbance of 205 nm. Besides

this, lasalocid is natively fluorescent, so it should be

also investigated by a fluorescence (FLD) detector with

an excitation wavelength of 305 nm and an emission

wavelength of 420 nm.

The analytical techniques used to detect ionophoricresidues not surprisingly date from the time of thediscovery of monensin. Despite the variety and depthof coccidiostat screening assays, the topic has not beenreviewed since 1985 when Weiss and MacDonaldsummarised detailed information on the bioassaysavailable at that time (Weiss and MacDonald 1985;Elliott et al. 1998). Vanderkop and MacNeil (1989)developed a multi-thin-layer chromatography-bioauto-graphy method that allowed for the identification oflasalocid, monensin, and salinomycin residues inchicken liver and kidney extracts. The analytes wereextracted from the sample with methanol and chloro-form, and silica gel plates were developed in ethylacetate and acetonitrile (50/50, v/v). Zone visualisationdetermining the presence of residue was based onutilising the organism Bacillus stearothermophilus.Sensitivities ranged from 450 to 1000 mg kg�1.Enzyme-linked immunosorbent assay (ELISA) meth-ods were developed to detect ionophoric residues in asensitive and specific manner using polyclonal andmonoclonal antibodies. Kennedy et al. (1995) used anELISA procedure for the analysis of lasalocid inchicken serum, liver and muscle. By using this proce-dure the authors were able to detect residues oflasalocid at concentrations lower than 15.0 ng g�1.Watanabe et al. (2004) developed an ELISA methodfor the simultaneous determination of lasalocid andsemduramycin. This method was quantitative inthe range of 0.1–50 ng ml�1 for lasalocid and at0.05–12.5 ngml�1 for semduramycin. The detectionlimits for lasalocid and semduramycin were 10 and5 ng g�1 in chicken liver and muscle, respectively.

Another way to detect lasalocid residues uses LC.Gerhardt et al. (1995) determined lasalocid togetherwith monensin, salinomycin and narasin, but lasalocidwas detected directly by fluorescence detection at anemission wavelength of 300 nm and an excitationwavelength of 420 nm. The LOD of the method was5 mg kg�1; the average recovery was 71%. Matabudulet al. (2001) utilised supercritical fluid extraction oflasalocid from poultry feed followed with HPLCanalysis with fluorescence detection with excitation at310 nm and emission at 430 nm. The results showedgood repeatability with a minimum quantification levelof 0.5mg g�1.

The increasing use of highly selective and sensitivetechniques, such as mass spectrometry (MS), tandemmass spectrometry (MS/MS) and time-of-flight massspectrometry (TOF/MS), together with advances in LCbrought about additional possibilities for the determi-nation of lasalocid residues in the spectrum of othercoccidiostats. The current methods for the determina-tion of lasalocid residues in biological matrices(muscle, liver, eggs) are usually the multi-residuemethods based on an LC-MS/MS technique(Matabudul et al. 2002; Dubois et al. 2004; Olejnik

CH3

OH

CH3

O

H3C

OH3C

H3CO

CH3

OH CH3

OH

CH3

OHO

Figure 1. Molecular structure of lasalocid – 6-[7-[5-ethyl-5 -(5-ethyl-5-hydroxy-6-methyl-oxan-2-yl)-3-methyl-oxolan-2-yl]-4-hydroxy-3,5-dimethyl-6-oxo-nonyl]-2-hydroxy-3-methyl –benzoic acid (Scientific Opinion of the Panel onContaminants in the Food Chain 2007).

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et al. 2009; Shao et al. 2009). Due to simple sample

preparation and short separation time on HPLC, theLC-MS/MS techniques are also suitable for screeningpurposes in large-scale studies. The LOD for most ofthe coccidiostats was below 1 mg kg�1; and the LOQ

was below 2 mg kg�1. Recently, Stubbings and Bigwood(2009) developed a novel rapid multi-residue/multi-class method suitable for the screening of 41veterinary drug residues, including ionophores, in

animal tissues. Because of ionophoric properties,lasalocid makes alkali metal complexes, especiallywith sodium. Its molecular complexity and ability torearrange and fragment makes this molecule a good

candidate for MS detection. Employment of MS forconfirmatory purposes is also recommended by theEuropean Union due to increased specificity of themethod and reduced likelihood of false-positive results.

According to the list of methods (Bohm et al. 2005)used by the National Reference Laboratories (NRL)

for residue control, edited by the CommunityReference Laboratory (CRL), and since 2010 renamedas The European Union Reference Laboratory (EU-RL), European Union member states use different

methods (LC with diode array detector or fluorescencedetector, LC-MS and LC-MS/MS) for screening andconfirmation of lasalocid residues in eggs, muscle andliver of individual food-producing animals. LC-MS/

MS is the most commonly used technique. The LOD ofthe method for lasalocid in various matrices rangesfrom 0.03 to 100mg kg�1.

Because lasalocid is one of the coccidiostatspreferred for the prevention of coccidiosis in poultryand the presence of coccidiostat residues in the tissues

of broiler chickens is still a matter of concern for publichealth, the objective of this paper was to build on theauthors’ previous study (Tkacikova et al. 2010) and todetermine the residual concentration of lasalocid in

edible chicken tissues (breast muscle, thigh muscle,heart, liver, gizzard, kidneys and skin/fat) after oraladministration in medicated feed by the methodutilised by the European Union Reference

Laboratory (BVL), Berlin (Bohm et al. 2005), andadopted and validated in the authors’ laboratoryaccording to Commission Decision 2002/657/EC(European Commission 2002) with respect to the day

of the WP.

Materials and methods

Experimental animals and pre-preparation of thesamples

Eighty 1-day-old broiler chickens (Gallus gallus, hybridRoss) were used. The broiler chickens were randomlydivided into two groups: experimental (n¼ 40) andcontrol (n¼ 40), placed in animal care-approved cages

with free access to feed and water. The feeding of

broiler chickens was as follows:

. The 1–18-day-old experimental broiler chick-ens were fed commercially produced feed

HYD-01 STARTER, medicated with

Cycostat 6, 6% premix (Alpharma BVBA,

Belgium) according to the recommendations

for use (33mg kg�1 of active substance robe-nidine hydrochloride in the complete feed);

between 19 and 35 days of age the experimen-

tal broilers were fed the commercially pro-

duced feed HYD-02-NORM-TYP medicated

with Avatec 15% premix (Alpharma BVBA)according to the recommendations for use

(100mgkg�1 of active substance lasalocid A

sodium in the complete feed); and between 36

and 41 days of age (WP) the experimental

broilers were fed unmedicated, commerciallyproduced feed HYD-03-FINAL.

. The broiler chickens from the control group

were fed the same way as the experimental

animal, but the feeds were unmedicated

throughout the experiment. On day 35 of age(day 0 of the WP), on days 1–5 of the WP, and

on the first day after elapse of the WP (day 6),

two chickens from the experimental group

were slaughtered and the respective samples of

muscle (breast and thigh), heart, liver, gizzard,kidneys and skin/fat were withdrawn and

stored at –20�C until analysis. The control

broiler chickens were slaughtered and pro-

cessed on day 5 of the WP.

The experiment was approved by the State

Veterinary and Food Administration of the Slovak

Republic under Decision No. 1859/09-221/3 and car-

ried out in the approved experimental facility at theUniversity of Veterinary Medicine in Kosice (SK P

52004).

Chemicals and reagents

Methanol gradient grade, acetonitrile gradient grade

and acetic acid glacial pro analysis were purchased fromMerck (Darmstadt, Germany). Water was purified by

reverse osmosis and deionised by Milli-Q Plus system

(Millipore S.A.S., Molshem, France). The lasalocid

standard was obtained from Chemos-group (American

Cyanamid Co., New York, USA). Standard stocksolution (500mg ml�1) and working standard solutions

(made at concentrations of 0.002, 0.005, 0.010, 0.020,

0.030, 0.040 and 0.060 mg ml�1) were prepared in

methanol. The stability of the stock solution is 6

months when stored under refrigeration at –18�C.Working solutions were prepared fresh prior to use.

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Sample preparation

A total of 2 g aliquots of homogenised tissue sampleswere extracted with 10ml of acetonitrile in an ultra-sonic bath for 15min. The samples were then shakenfor 30min on a horizontal shaker and centrifuged for10min at 4�C at 3000 rpm. The sample extract wasdried under a stream of nitrogen at 55�C in TurboVap(Zymark, Oftringen, Switzerland) and re-dissolved in5ml of deionised water. The supernatant was purifiedusing a solid-phase extraction (SPE, Strata-X, 200mg,6ml; Phenomenex, Torrance, USA). Firstly thecolumn was conditioned with 3ml of methanol fol-lowed by 3ml of deionised water. The re-dissolvedsample extract was allowed to pass through theconditioned column by gravity. The column waswashed with 3ml of deionised water. Subsequentlythe column was dried under vacuum for 5min.Lasalocid was eluted from the column with 5ml ofmethanol and the eluate was concentrated undernitrogen at 40�C to a final volume of 1ml. Theconcentrated eluate was filtered into an autosamplervial using 0.2 mm syringe filter (Whatman,International Ltd., Maidstone, UK). Before analysisthe samples were cooled to 10�C.

Matrix spiking

For the spiking procedure lasalocid-free tissues fromthe control group were used. A calculated amount oflasalocid standard in methanol was added to thehomogenised sample prior to extraction and methanolwas allowed to evaporate for about 30min at roomtemperature. The samples were then analysed asroutine samples.

LC-MS/MS analysis

Lasalocid residues were analysed with a WatersAlliance 2695 liquid chromatograph (Waters Co.,Dublin, Ireland) coupled with a MicroMass QuattroMicro spectrometer with a triple-quadrupole massanalyser (MicroMass Ltd, Manchester, UK).Electrospray data were acquired using Mass Lynxsoftware; and the instrument was operated in positive-ion reaction mode. HPLC separation was performedon an analytical column Luna C18, length 150mm,internal diameter 2mm, particle size 3 mm(Phenomenex, USA) at a controlled temperature of40�C. Mobile phase composition was methanol/deio-nised water/acetic acid glacial in a ratio of 900/99/1, v/v/v, delivered at a flow rate of 400 ml min�1. Theinjection volume was 10 ml. Lasalocid was eluted forabout 3.5min.

To achieve high output of the instrument, the triplequadrupole analyser was operated in multiple reactionmonitoring (MRM) mode and the parameters of the

system were optimised using a direct injection of thestandard solution at a concentration of 100 ngml�1.The optimised parameters were: ion source tempera-ture of 100�C, desolvation temperature of 400�C,capillary voltage of 3.5 kV, cone voltage of 50V,collision voltage of 38 eV, extractor voltage of 1V, andRF lens voltage of 0V. To obtain fragmentation oflasalocid molecules they were bombarded withargon atoms with purity 99.999% in a collision cell.Nitrogen, produced by a Domnik Hunter nitrogengenerator (Noblegen, Gateshead, UK), was used as anebuliser gas at a flow rate of 600 l h�1 and a cone gasat a flow rate of 50 l h�1. The investigated transitions oflasalocid were 613.2! 359.2 Da, and 613.2! 377.2Da, respectively. The mass spectra of lasalocid precur-sor and fragment ions are presented in Figures 2 and 3.

Method validation

The method was validated in compliance withCommission Decision 2002/657/EC (EuropeanCommission 2002). The following validation parame-ters were determined: linearity, LOD, LOQ, decisionlimit (CC�), detection capability (CC�), recovery,specificity, repeatability and robustness. The linearityof the LC-MS/MS response was proved with eightcalibration points in the concentration range0–100 ngml�1 (working standard solutions includingzero). With each set of investigated samples a newcalibration curve was made. The determination coef-ficients (R2) of the standard calibration curves were atleast 0.991 (Figure 4). LOD¼ 0.47mg kg�1, LOQ¼1.44mg kg�1, CC�¼ 0.27 mg kg�1 and CC�¼0.48 g kg�1. They were calculated from the calibrationcurve by measuring each calibration point three times.The average recovery from the spiked blank tissuesamples was 95%. Specificity of the LC-MS/MSresponse was proved by analysing lasalocid in thepresence of the other polyether ionophoric coccidio-stats (monensin, salinomycin, narasin, maduramycinand semduramycin) both in standard solution andspiked matrix. No interferences were detected.Repeatability was checked by using six muscle tissuesfortified with 100mg kg�1 of lasalocid standard. Thecoefficient of variation was 10.9%.

Results and discussion

The mean residual concentrations of lasalocid residues(mg kg�1) determined in respective edible chickentissues and the standard deviations (SD) of measure-ments are presented in Table 1. Due to the 5-day WPestablished for lasalocid, the residue concentrations oflasalocid were investigated on day 0 (the last day oflasalocid administration), days 1–5 (the days of theWP) and day 6 (the first day after elapse of the WP).

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Figure 4. Calibration curve of the lasalocid standard constructed with lasalocid standard solutions at concentrations from 2 to100 ngml�1 in methanol with R2

¼ 0.999441.

Figure 3. Mass spectra of fragment ions of lasalocid.

Figure 2. Mass spectra of the precursor ion of lasalocid.

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Recoveries of lasalocid from spiked control tissuesamples ranged from 79% to 98%. The resultspresented in Table 1 are not corrected for recovery.According to Table 1, detectable residues of lasalocidwere found in all examined matrices throughout theperiod of investigation. The liver contained the highestlevels of lasalocid residues, ranging from 1112.7�83.1 mg kg�1 (day 0 of the WP) to 7.7� 0.3 mg kg�1

(day 6). Relatively high levels of lasalocid residues werealso found in the heart and fat/skin. The levels oflasalocid residues in the heart ranged from610.2� 15.7 mg kg�1 (day 0 of the WP) to 5.9�1.6 mg kg�1 (day 6) and in the fat/skin from 532.9�29.7 mg kg�1 (day 0 of the WP) to 2.4� 0.1 mg kg�1

(day 6).Lower levels of lasalocid residues were found in the

kidneys (346.1� 21.9 mg kg�1/day 0 of the WP to1.9� 0.2 mg kg�1/day 6/) followed by the thigh muscle(217.6� 9.9mg kg�1/day 0 of the WP/ to 1.1� 0.1mgkg�1/day 6/) (Figure 5) and gizzard (94.0�31.6 mg kg�1/day 0 of the WP/ to 1.8� 0.1 mg kg�1/day 6/). The lowest lasalocid residues were found in the

breast muscle (58.3� 10.6mg kg�1/day 0 of the WP/to0.3� 0.03 mg kg�1/day 6/).

The MRL for lasalocid in poultry, established inthe European Union by Commission Regulation (EU)No. 37/2010, is 20 mg kg�1 in muscle, 50 mg kg�1 inkidneys, and 100 mg kg�1 in liver, skin and fat(European Commission 2010 b). The results showthat the concentrations of lasalocid residues in allinvestigated edible tissues of broiler chickens on thelast day (day 5) of the WP and on the first day aftertermination of the WP (day 6) were below the MRLlevels. The heart and breast muscle were positive forlasalocid residues up to day 3 of the WP and the thighmuscle, kidneys, liver, gizzard, and fat and skin up today 4 of the WP. No residues of lasalocid were foundin the tissues of broiler chickens obtained from thelasalocid-free control group (Figure 6).

The findings agree with the toxicological studieswhich reported that the primary target organs of theionophores are cardiac and skeletal muscles (Pressmanand Fahim 1983; Novilla 1992; Shier and Dubourdieu1992; Tkacikova et al. 2010). The relatively high

Figure 5. Typical ion chromatogram of a positive chicken thigh sample on day 4 of the WP containing lasalocid residue at aconcentration of 25.84 mg kg�1.

Table 1. Mean lasalocid residual concentrations (mg kg�1� SD) detected by LC-MS/MS in the edible tissues of broiler chickensthroughout the 5 days of the WP.

Matrix

Day of WP

0 1 2 3 4 5 6

Heart 610.2� 15.7 129.1� 6.9 7.7� 1.8 171.8� 27.5 12.7� 0.5 14.3� 5.7 5.9� 1.6Breast 58.3� 10.6 17.4� 3.7 4.6� 0.4 37.6� 13.4 11.5� 1.8 2.7� 0.1 0.3� 0.03Thigh 217.6� 9.9 79.9� 6.3 30.4� 9.1 85.7� 2.2 25.2� 5.6 4.7� 0.2 1.1� 0.1Kidney 346.1� 21.9 89.4� 4.9 2.2� 0.4 204.3� 23.2 111.9� 14.4 22.1� 4.8 1.9� 0.2Liver 1112.7� 83.1 131.5� 10.1 47.9� 7.2 250.5� 15.7 182.3� 4.2 19.0� 5.7 7.7� 0.3Gizzard 94.0� 31.6 10.8� 0.9 1.3� 0.2 89.4� 2.9 24.1� 4.9 4.3� 0.4 1.8� 0.1Fat/skin 532.9� 29.7 93.1� 32.2 54.3� 2.4 160.9� 3.5 72.1� 4.0 17.4� 1.0 2.4� 0.1

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concentrations of lasalocid residues in the skin/fatsupport the theory that most drugs and medicationsare lipophilic, tending to deposit in the areas rich in fat,especially in adipose as well as in other organs(Cecchini 2006; Tkacikova et al. 2010).

An interesting finding was that a gradual decline inthe residual levels of lasalocid in the tissues ofbroiler chickens throughout the WP was not seen, asobserved in previous studies (Tkacikova et al. 2010;Kozarova et al. 2011). Similar findings were

0

200

400

600

800

1000

0 1 2 3 4 5 6

Day of withdrawal period

Con

cent

ratio

n of

lasa

loci

d

Heart

Thigh muscle

Kidneys

Liver

Gizzard

Skin

Breast muscle

Figure 7. Comparison of lasalocid residues in examined chicken tissues (mg kg�1) on day 0 of the WP, during 5 days of the WPand on the first day after elapse of the WP.

Figure 6. MRM transitions of a lasalocid-free chicken breast sample from the control group.

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reported by Daeseleire et al. (2006) at thedetermination of levels of narasin residues in eggs oflaying hens consuming fed narasin-containing feed.A comparison of the levels of lasalocid residuesdetermined in examined chicken tissues throughoutthe period of investigation is presented in Figure 7.

The lasalocid residual levels in the tissues of thebroiler chickens on day 5 of the WP were in compli-ance with the MRLs established for lasalocid byCommission Regulation (EU) No. 37/2010. A 5-daywithdrawal period established for lasalocid ensured thedecline of its residues in the tissues of broiler chickensbefore slaughter to the levels of the set MRLs,although it should be mentioned that the levels oflasalocid residues in the tissues on the last day ofthe WP were several times higher than the LOD(0.47 mg kg�1) of the method. According toCommission Regulation (EC) No. 124/2009 of 10February 2009, which covers the cross-contaminationfrom non-target animals, the maximum content oflasalocid in food of animal origin from animal speciesother than fattening chickens and turkeys is 2 mg kg�1

(European Commission 2009 a). Comparison of thisvalue with the present results showed that lasalocidresidual levels in all examined samples with theexception of breast muscle on the last day of the WPwere several times higher.

LC-MS/MS represents a sensitive and effective toolapplicable for routine analysis of lasalocid residues inthe tissues of broiler chickens at the level of interest.

Conclusions

LC-MS/MS is a powerful analytical tool in residueanalysis due to its high sensitivity and selectivity. TheLC-MS/MS method proved to be a very effective wayof determining very low levels of lasalocid residues inthe tissues of broiler chickens, thus providing a highlevel of protection for human health. The methodmeets all the criteria of Commission Decision 2002/657/EC, and since 2007 it is a statutorily prescribedconfirmatory method for routine analysis and theofficial control of coccidiostat residues in poultry, eggs,rabbit and bovine executed under Council Directive96/23/EC.

Acknowledgements

This study was supported by VEGA Grant Number1/0658/09.

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