抗生素检测技术英文版

Liquid chromatography–tandem mass spectrometry multiclass method for the determination of antibiotics residues in water samples from water supply systems in food-producing animal

farms

Malgorzata Gbylik-Sikorska ?,Andrzej Posyniak,Tomasz Sniegocki,Jan Zmudzki

National Veterinary Research Institute (NVRI),al.Partyzantow 57,24-100Pulawy,Poland

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

water sample

CONTROL

OUT OF CONTROL

LC-MS/MS

water with antibiotics

residues

adsorption

constant exposure

environment

Veterinary inspection

a r t i c l e i n f o Article history:

Received 7February 2014

Received in revised form 25April 2014Accepted 28April 2014

Handling Editor:Klaus Kümmerer Keywords:Antibiotics Water Multiclass LC–MS/MS

a b s t r a c t

A sensitive liquid–chromatography–electrospray tandem mass spectrometry multiclass method for determination of 45veterinary compounds belonging to 9different antibiotic groups,including amino-glicosides (4),b -lactams (13),diaminopyrimidines (1),?uoroquinolones (10),lincosamides (1),macro-lides (5),pleuromutilins (1),sulfonamides (6)and tetracyclines (4),in water from breeding animal watering supply system has been developed.Isolation of the analytes was carried out by solid phase extraction with hepta?uorobutyric acid as an ion-pair agent on the Strata-X reversed phase cartridges.All analytes were determined simultaneously in one single run on a C18column with gradient elution and short analysis time (13min).Method was validated,average relative recoveries were in the range of 84.3–109.3%with satisfactory precision are repeatability for all compounds are in the range of 4.7–12.2%,within-laboratory reproducibility are in the range of 4.4–13.5%for.The limit of quantitation (LOQ)of the method was in the range of 0.02–10l g L à1,depending of analyte.The applicability of the method was tested by determining antimicrobial compounds in real water samples collected from water supply systems in breeding animal farms.The average antibiotics concentration in real water samples were,respectively,in the range of 0.14–1670l g L à1.

ó2014Elsevier Ltd.All rights reserved.

1.Introduction

Despite recommendations,the usage of antibiotics in food-producing animals therapy is still increasing (Kemper,2008).

https://www.360docs.net/doc/3b4956891.html,/10.1016/j.chemosphere.2014.04.1050045-6535/ó2014Elsevier Ltd.All rights reserved.

?Corresponding author.Tel.:+48818893127;fax:+48818862595.

E-mail address:malgorzata.gbylik@piwet.pulawy.pl (M.Gbylik-Sikorska).

Obviously,excluding the use of antibiotics in bacterial diseases in breed animal farms is impossible.Therefore,many classes of anti-biotics,such as aminoglicosides,b-lactams,diaminopyrimidines,?uoroquinolones,lincosamides,macrolides,pleuromutilins,sul-fonamides and tetracyclines are widely used in current veterinary practice to treat animal diseases or illegally to promote animal growth.In poultry and swine therapy,the antimicrobial com-pounds are commonly added to animal feed or drinking water sources.Due to the fact that many antibiotics are added to water, some water supply systems can be contaminated and can spread to the farm environment.Not using regular cleaning of water sup-ply systems,especially after?nish antibiotic treatment,deliberate illegal administration of low doses of antibiotics in water to ani-mals stimulation,as well as remains of unconscious application of antibiotics,can lead to undesirable consequences.The constant exposure of breeding animals to these compounds may lead to res-idues in animal tissues and antimicrobial resistance of animal and environment pathogens.Moreover,exposure of animal for trace amounts of antibiotics can also interfere with the pharmacokinet-ics of other compounds and can in?uence on action of other anti-bacterial agents,which were administered intentionally in the treatment of infection in animals.This may result in prolongation of the withdrawal period of antibiotic,which was administrated to treat animals diseases.In water supply systems,the same phys-ical phenomenon as in the natural aquatic environment,such as antibiotics accumulation,may also occur.Physicochemical proper-ties of some antibacterial agents(tetracyclines,?uoroquinolones and sulfonamides)enable them to absorb onto sewage sludge,soil and sediments(Kemper,2008;Kummerer,2009).Thus,can adsorb onto water supply system pipes and after then can be systemati-cally eluted after animal treatment?nish.This situation may be harmful to the animals and consumers of food of animal origin health and can lead to increase of allergy and bacteria resistance to antibiotics use in human medicine.Non-controlled antibacterial residues in water supply systems can also spread to the natural environment with the farm sludge.

The aim of this study was to develop a non-invasive form of control of antibiotic residues,which are present in water supply systems after?nishing their administration in water for the treat-ment and the illegal administration in subliminal doses of antibac-terial compounds in water for health animals.Therefore,it was necessary to develop an analytical method for the determination of antimicrobial compounds residues in water samples collected from water supply systems in food-producing animals.Until now,there have been many analytical methods for determining antimicrobial compounds in different biological matrices,animal tissues(Chico et al.,2008;Hurtaud-Pessel D.et al.,2011;Granelli and Branzell,2007;Smith et al.,2009;Gbylik et al.,2013)and feed (Chafer-Pericas et al.,2011).In addition,there are many approaches contributing to the realisation of studies on the occur-rence of antibiotics in environmental water samples,such as wastewater,river water,lake water,?sh farms and ground water (Kim and Carlson,2007;Chang et al.,2010;Borecka et al.,2013; Gros et al.,2013).Some of the reported methods included the anal-ysis of only one group of the most important antibiotics used in veterinary and human therapy,such as sulfonamides(Raich-Montiu et al.,2007)or?uoroquinolones(Xiao et al.,2008).The separate determination of antibiotics from different groups in one sample is more expensive and time consuming.Furthermore, more frequent detection of the trace amounts,as well as high con-centrations of antimicrobial compounds from different classes in the same water sample are observed.The multiclass analytical methods for determination of antibacterial compounds belonging to groups such as macrolides,sulfonamides,?uoroquinolones and tetracyclines(Carlson and Kim,2007;Tong et al.,2009),b-lactams (Campagnolo et al.,2002),or lincozamides and thrimethoprim,additionally(Silvia et al.,2005;Kasprzyk-Horden et al.,2007)in environmental water samples have been described in the litera-ture.However,most of the methods do not include the analysis of aminoglycosides in the same analytical protocol(Campagnolo et al.,2002;Carlson and Kim,2007;Tong et al.,2009).Most of them used the solid phase extraction(SPE)followed by liquid chro-matography coupled with the tandem mass spectrometry(LC–MS/ MS).Typically,these methods used ethylenediaminetetraacetic acid(EDTA)as a chelating agents and OASIS HLB SPE cartridges (Campagnolo et al.,2002;Silvia et al.,2005;Karthikeyan and Meyer,2006;Carlson and Kim,2007;Tong et al.,2009).Unfortu-nately,none of them relates to determination and analysis of anti-biotics in water samples from food-producing animal water supply systems,which aim to control systems contaminations and there are no published methods for simultaneous determination of aminoglicosides,b-lactams,diaminopyrimidines,?uoroquino-lones,lincosamides,macrolides,pleuromutilins,sulfonamides and tetracyclines.

This study presents a simple and ef?cient multi-class method for determination of45analytes belonging to9different antibacte-rial compound groups in water collected from food-producing ani-mals water supply systems.The sample preparation step contains the ion-par agent addition and the Strata-X SPE cartridges usage. The method was validated according to selected requirements of the European Decision2002/657/EC(C.D.2002/657/EC),(linearity, precision,recovery and selectivity),for analytical limits of the method,limit of detection(LOD)and limit of quantitation(LOQ) were estimated.A developed method is dedicated to determination of antibiotics residues(trace amounts,in the range of0.02–1000l g Là1)after?nishing the antibiotics application on breeding-animal farms,but it cannot be used to determine of the antibiotic concentrations during treatment(therapeutic doses). The applicability of the method was evaluated by determining antimicrobial compounds in water supply systems from food-producing animals collected from a few different farms in Poland.

2.Materials and methods

2.1.Reagents

All reagents used were of analytical grade,>95%purity.Sodium acetate and sodium hydroxide were from POCH(Gliwice,Poland), acetonitrile and methanol were obtained from J.T.Baker(Deventer, the Netherlands).Hepta?uorobutyric acid(HFBA)was from Fluka, (Newport News,VA,the United States).Water was deionised (>18M X cmà1)by the Millipore system.Amoxicillin(AMOX), ampicillin(AMPI),penicillin G(PEN G),nafcillin(NAF),dicloxacillin (DICLOX),oxacillin(OXA),cephapirin(CFPI),ceftiofur(CFT),cef-operazone(CFPE),cephalexin(CFLE),cefquinome(CFQ),cefazolin (CFZ),cefalonium(CFLO),dano?oxacin(DAN),di?oxacin(DIF), enro?oxacin(ENR),cipro?oxacin(CIP),nor?oxacin(NOR),marbo-?oxacin(MAR),?umequine(FLU),sara?oxacin(SAR),oxolinic acid (OXO),nalidix acid(NAL),chlortetracycline(CTC),tetracycline(TC), doxycycline(DC),oxytetracycline(OTC),streptomycin(STRP), dihydrostrepromycin(DISTRP),spectinomycin(SPEC),neomycin (NEO),sulfamerazine(SME),sulfamethazine(SMT),sulfamethoxa-zole(SMA),sulfamonomethoxine(SMM),sulfadimethoxine (SDMX),sulfathiazole(SFT),trimethoprim(TMP),tylosin(TYL), erythromycin(ERY),spiramycin(SPI),tilmicosin(TIL),josamycin (JOS),lincomycin(LIN),tiamuline(TIM),sulfafenazole(SFF)–internal standard(IS),were from Sigma–Aldrich(St.Louis,MO, the United States).Strata X(100mg,6mL)cartridges were obtained from Phenomenex(Torrance,CA,the United States),syr-inge?lters0.22l m PVDF were from Restek(Bellefonte,PA,the United States).

M.Gbylik-Sikorska et al./Chemosphere119(2015)8–159

2.2.Standard solutions

Individual stock standard solutions(1mg mLà1)for macrolides (TYL,ERY,SPI,TIL,JOS),tetracyclines(TC,CTC,DC,OTC),sulfona-mides(SME,SMT,SMA,SMM,SDMX,SFT)diaminopyrimidine (TMP),pleuromutylines(TIM)and lincozamides(LIN)were pre-pared in methanol and stored in amber volumetric?asks at à18°C.Whereas for?uoroquinolones(DAN,DIF,ENR,CIP,NOR, FLU,SAR,OXO,NAL,MAR)standard solutions were prepared in methanol with addition of sodium hydroxide and stored in amber volumetric?asks atà18°C.For aminoglycosides(STRP,DISTRP, SPEC,NEO)and b-lactams(AMOX,AMPI,OXA,DIKLOX,PEN G, NAF,CFPI,CFT,CFLE,CFQ,CFZ)standard solutions were prepared in water;cefalosporines(CFLO and CFPE)were prepared in aceto-nitrile and water(1:1,v/v)and stored in amber volumetric?asks atà18°C.Mixtures of working standard solutions were prepared in water and stored in plastic?asks at4°C.Individual stock inter-nal standard(IS)solution(1mg mLà1)for SFF was prepared in water and stored in amber volumetric?asks atà18°C.Working internal standard solution(2l g mLà1)was prepared in deionised water and stored in amber volumetric?ask at4°C.

2.3.Water samples

For validation experiments,water samples were collected from water supply systems on local food-producing animal farm.Before the experiment,water samples were respectively checked to be free of the antibiotics.For the purpose of the matrix effect,water samples from water supply systems and wastewater samples were collected.For the main study,water samples were collected from water supply systems of25food-producing animal farms in Poland.Water samples were collected into dark plastic bottles and kept in a cooler with ice until transportation to the laboratory. Once back to the laboratory,water samples were stored atà18°C until the analysis.

2.4.Sample preparation

Before extraction,50l L of IS was added to a250mL water sam-ple in polypropylene bottle(500mL),mixed and left at room temperature in a dark place for15min,after that6mL of0.5M sodium acetate pH=5.6and30l L of HFBA were added and shaken brie?y for5min.Such prepared samples were transferred to pre-conditioned Strata-X SPE cartridges(sequentially conditioned with 5mL of MeOH,5mL H2O and5mL of0.05M HFBA).The?ow was no faster than1drop/5s.After then cartridges were vacuum-dried for5min at a pressure ranged from12mmHg to18mmHg.The analytes were eluted with3mL mixture of acetonitrile and 0.05M HFBA(9:1,v/v)twice.Eluates were collected in10mL glass tubes and evaporated to dryness under a stream of nitrogen at 45±5°C.The residues were dissolved in500l L of0.025%HFBA and?ltered through0.22l m PVDF syringe?lters into LC vials. 2.5.Liquid chromatography–mass spectrometry

The LC–MS/MS analysis was performed on the Agilent1200 HPLC system(Agilent Technologies,Germany)with an automatic degasser,a binary pump and an autosampler connected to the AB Sciex API4000triple quadrupole mass spectrometer(AB Sciex, Canada).The chromatographic separation was performed on the Luna C18(2)100A column(50?3.0mm,particle size3l m,Phe-nomenex),which was maintained at30°C.The?ow rate of mobile phase was400l L minà1,the injection volume–30l L.The optimal composition of mobile phase A and B was acetonitrile(A)and 0.025%HFBA(B).The mobile phase gradient program started at 85%of B,60%B at1min,40%B at3min and then5%at4min,held for3min.The column returned to the initial composition and re-equilibrated within6min before the next injection.The MS instrument was operated in the positive ESI mode.The following parameters were used in the tune mode:resolution Q1and Q3–unit;temperature–500°C,nebuliser gas(N2)–40;curtain gas (N2)–20;collision gas(N2)–3;auxiliary gas–50;ion spray volt-age–5500V.The Analyst1.5software controlled the LC–MS/MS system and processed the data.The data acquisition was in multi-ple reactions monitoring(MRM)mode.The ion transitions and mass parameters monitored for each analyte are listed in Table1.

2.6.Method validation

Water samples free from antibiotics were used in the validation process.Linearity was performed by the matrix-matched calibra-tion curve which had been prepared by fortifying blank water sam-ples at9concentration levels(0.02,0.05,1,10,50,100,250,500 and1000l g Là1).The50l L of IS was added to each sample. Quantitative results evaluation was performed by comparing the analyte/internal standard peak area ratio from matrix-matched calibration curve to the analyte/internal standard peak area ratio in analysed samples.

The repeatability was calculated as the coef?cients of variation (CV,%)of results obtained after fortifying of six blank samples at three concentration levels:10,100and500l g Là1.The spiked samples were analysed on the two subsequent days with the same instrument and the same operators.

The within-laboratory reproducibility was calculated as the overall coef?cients of variation(CV,%)of the results obtained after fortifying another two sets of blank samples at the same concen-tration levels of analysed compounds as for the repeatability and analysing on2d with the same instrument and another operators.

Selectivity was checked by analysis of20blank water samples collected from different sources which allows to verify the appear-ance of possible presence of interfering substances around the retention times of the compounds of interest.

The percentage relative recovery was evaluated in the same experiment as repeatability by comparing the mean measured con-centration with the forti?ed concentration of the samples.

The method detection limit(LOD)and the quanti?cation limit (LOQ)were estimated after analysis of20forti?ed blank samples at the minimum detectable concentration level of each analyte and measured the signal to noise ratios at3and10respectively.

The stability of antibiotics in water samples were evaluated by analysing samples forti?ed at the10l g Là1concentration level concentration for each of45compounds.The stability of individual stock standard solution for all analytes were estimated in the following order:1d,1and2weeks,1,3,6and10months.The standard solutions were stored in a dark place at two different temperatures:4°C andà18°C.The prepared samples were stored atà18°C and analysed after1,7and14d,1,2,3and 6months.

The matrix effect was calculated by analysing6water samples from different sources spiked at10l g Là1concentration level for each analyte and it was calculated by means of the Eq.(1).The matrix effect was evaluated as a percentage of signal intensity of water sample extract forti?ed after extraction(IW M)in relation to signal intensity of deionised water forti?ed(IW D)at the same concentration level.

Matrix effecte%T?

IW M

IW D

?100e1T

The robustness of the method was estimated for the four different possible factors that could have an in?uence on the method results. Among possible factors are:the volume of solvent for conditioning

10M.Gbylik-Sikorska et al./Chemosphere119(2015)8–15

of SPE cartridges and volume of elution solvent,the temperature of the evaporation of the extracts,the size of syringe?lters.The study was performed with eight water samples spiked at the level of 10l g Là1.The results were evaluated on the basis of the Youden approach,described by Plackett and Burman,1946and Van der Heyden et al.,2001.

3.Results and discussion

3.1.LC–MS/MS conditions

The mass spectrometer settings were optimised with a direct infusion of working standard solutions.For all compounds,the two most abundant product ions produced from each precursor ion were chosen as the ion transition in order to comply with the criteria needed for qualitative and quantitative methods.Accord-ing to the EU criteria(C.D.2002/657/EC),two MRM transitions were monitored.The characteristic MS/MS parameters:collision energy(CE),declustering potential(DP)and dwell time have been optimised separately for each analyte(Table1).The analyses were performed in the positive ionisation mode.

In order to improve the chromatographic separation of the selected antibiotics from the possible matrix interferences,the LC conditions were optimised.In the majority of the published meth-ods,for the determination of the antibacterial compounds in water samples,the suf?cient chromatography separation was obtained with a different C18column(Raich-Montiu et al.,2007;Carlson and Kim,2007;Kasprzyk-Horden et al.,2007;Xiao et al.,2008; Tong et al.,2009).This can be explained by the fact that highly polar compounds are not retained and elute in the void volume of column when reversed-phase separation is used.Ion-pair chro-matography can be the solution to this problem,however,asym-metric peak shape,low signal intensity also ion suppression may occur.For this reason,C18columns with different parameters(col-umn size and particle size)were chosen for this experiment.It was found that the best result(high selectivity,good peak shape,higher intensity of most compounds and shorter retention times),was obtained with the Luna C18(2)100A column(50?3.0,3l m).At the second step,different compositions of the mobile phase includ-ing ion-pair agents,were tested.Mobile phase compositions of methanol and acetonitrile coupled with formic acid or water including one of two ion-pair agents such as hepla?uorobutyric acid and penta?uoropionic acid were tested.The use of the ion-pair

Table1

MS/MS ion transitions and parameters.

Analyte Ion transition1(m/z)Ion transition2(m/z)Dwell time(ms)DP(V)CE(Ev)

SPEC351.1/333.2351.1/207.2203267 STRP582.0/263.0582.0/246.02052166 DISTRP584.3/263.2584.3/246.22042150 NEO615.3/161.0615.3/163.22010942 AMOX366.1/349.1366.1/208.0201445 PEN G335.1/160.0335.1/176.1201760 AMPI350.1/106.0350.1/160.0155827 DICLOX470.0/160.0470.0/311.0155022 NAF415.0/199.0415.0/171.0154820 OXA402.0/160.0402.0/243.0155225 CFPI424.0/152.0424.0/124.0203550 CFT524.0/241.0524.0/125.0202550 CFQ529.0/134.0529.0/125.0202550 CFLO459.0/337.1459.0/152.0201646 CFZ455.0/323.0455.0/156.0201550 CFLE348.0/158.0348.0/106.0201050 CFPE646.0/530.0646.0/530.0201760 TMP292.1/262.2292.1/231.3155236 CIP332.0/314.0332.0/231.0152865 ENR360.0/342.0360.0/286.01533100 DIF400.5/382.1400.5/356.0153050 DAN358.0/340.0358.0/255.0156033 FLU262.1/244.0262.1/202.0154425 OXO262.0/244.0262.0/216.0155325 NAL233.0/215.0233.0/187.0154230 MAR363.0/345.0363.0/320.0157030 SAR385.8/368.1385.8/348.0155031 NOR320.0/302.0320.0/231.0153050 ERY734.0/576.5734.0/158.2152875 TYL916.0/174.0916.0/772.51552110 TIL869.6/696.5869.6/174.21513561 JOS828.2/173.9828.2/229.0158046 SPI843.5/540.4843.5/174.21512044 LIN407.2/126.1407.2/359.3157428 TIM494.4/192.2494.4/118.81512830 SMT279.2/156.0279.2/108.0102550 SME265.0/156.0265.0/108.0102750 SDMX311.0/156.0311.0/108.0102350 SMA254.0/107.8254.0/155.9102440 SMM281.0/156.0281.0/108.0103550 SFT256.0/156.0256.0/108.0105320 DC445.0/428.0445.0/154.0152350 OTC461.0/426.0461.0/444.0152840 TC445.0/410.0445.0/427.0152755 CTC479.0/444.0479.0/462.0152960

M.Gbylik-Sikorska et al./Chemosphere119(2015)8–1511

chromatography was necessary because of the polar character of aminoglycosides(Karthikeyan and Meyer,2006).Unfortunately, the addition of ion-pair agent caused a signi?cant reduction of sig-nal intensity of ERY,but lower concentration of ion-pair agent caused the lost of aminoglycosides elution from column.The best results such as better aminoglycosides elution from chromatogra-phy column,satisfactory peak shapes and reproducible retention time were obtained with0.025%HFBA combined with acetonitrile. Because of this compromise between adequate ion-pair agent vol-ume and pH of mobile phase,therefore the LOQ for ERY,NEO, AMOX and PEN G are in the range of5l g Là1to10l g Là1. Furthermore,the mobile phase gradient was also optimised in order to achieve the best chromatographic result with the mini-mum analysis time.The last eluting analyte was detected at 7.66min,the total run time was13min.In Fig.1,total ion current (TIC)chromatogram of real sample spiked on5l g Là1of mixture containing45analytes is displayed.

3.2.Optimisation sample preparation

Water samples from water supply systems from breeding-animals farms are speci?c kind of matrix,signi?cantly differing from the environmental water samples.Besides the antibiotics,different pH values of the solution,?ltration step,ion-pair agent and chelating agent addition prior SPE extraction were conducted.

At?rst,the necessity of the?ltration step was tested and no sig-ni?cant in?uence on the SPE extraction was detected.Similarly to reports of other authors,it is very important to adjust the pH val-ues to acidic and neutrals compounds.The pH value is a critical part of the sample preparation and SPE extraction process because the pKa values of analytes are in the range of about2.6–9.7.The additions of a different buffer prepared with non-organic acid like, hydrochloric acid and organic acids(formic acid,acetic acid)and sodium acetate were tested.All buffers were tested with pH value in the range of3.0–6.0.It was dif?cult to?nd the compromise between very sensitive to the presence of acid b-lactams and mac-rolides,and tetracyclines,?uoroquinolones and sulfonamides,for which the isolation is better in acidic solution.The most satisfac-tory recoveries were achieved with0.5M sodium acetate pH=5.6.

In the previous studies,addition of EDTA to water sample to improve tetracyclines isolation was used(Xiao et al.,2008; Chang et al.,2010;Gros et al.,2013).During the experiment,there was no signi?cant impact on tetracyclines recoveries with EDTA addition into the sample.

In the next step the employing of the ion-pair agents which are commonly recommended in the sample extraction to enable the aminoglicosides extraction,took place.The selection of ion-pair

Representative total ion current(TIC)chromatogram of real water sample spiked on5l g Là1concentration level by mixture 12M.Gbylik-Sikorska et al./Chemosphere119(2015)8–15

agent were checked.The best choice was found with Strata-X car-tridges and the mixture of acetonitrile with HFBA (9:1,v/v)as the elution solvent,because only these cartridges usage with ion pair addition,enable the aminoglycosides elution.Overall,the Strata-X (100mg and 200mg)cartridges gave the same recoveries better then Strata-XL (500mg).Finally,Strata-X (100mg)was selected to be used at this study because of shorter time of the SPE sample loading step then Strata-X 200mg.

To enhance the puri?cation of the samples,?nal extract syringe ?lters (PVDF and NYLON)were tested.PVDF syringe ?lters gave the best ‘‘purity’’of the extract without any in?uence on the analytes recoveries.

3.3.Method validation

The validation results of the developed method are reported in Table 2.During the validation study parameters such as precision (repeatability and within –laboratory reproducibility),selectivity and linearity were established according to the European Decision 2002/657/EC (C.D.2002/657/EC ).The matrix-matched calibration curves of 9points achieved good linearity,greater than r 2=0.99for all analytes.As can be seen in Table 2,the overall coef?cient of variation (CV)for all compounds is in the range of 4.7–12.2%

Table 2

Validation report of developed method.Analyte Repeatability (CV,%)Within-lab reproducibility (CV,%)LOD (l g L à1)LOQ (l g L à1)Recovery (%)SPEC 6.1±0.77.6±0.70.35194.1±3.6STRP 5.3±0.87.1±0.80.36196.4±2.4DISTRP 6.0±1.17.6±0.50.73291.3±4.2NEO 7.3±0.7 6.8±0.8 3.791097.1±3.5AMOX 5.6±1.0 4.0±0.5 3.541097.7±5.1PEN G 9.4±2.112.7±0.3 3.721097.1±3.1AMPI 7.9±0.612.2±0.60.020.05105.7±2.5DICLOX 5.7±1.18.7±0.60.020.05104.8±3.1NAF 4.3±0.49.2±0.70.020.05100.3±1.7OXA 9.8±1.79.3±0.40.020.0590.3±2.4CFPI 8.0±0.613.6±0.40.020.0592.1±2.8CFT 7.4±0.58.8±0.50.020.05102.7±1.9CFQ 6.2±0.89.7±0.60.010.02103.2±3.2CFLO 5.5±1.011.2±0.40.010.0296.4±1.7CFZ 8.2±0.87.9±0.70.010.0299.4±2.4CFLE 7.8±0.913.9±1.50.020.0592.7±1.2CFPE 7.5±1.29.9±0.60.010.0291.1±2.2TMP 5.4±0.712.5±0.80.020.0595.5±2.6CIP 6.5±0.310.5±0.20.010.0287.7±2.7ENR 7.3±0.37.1±0.50.010.0289.5±3.2DIF 6.3±0.5 6.1±0.80.010.0295.2±1.4DAN 8.4±0.28.3±0.60.010.0296.0±1.9FLU 11.5±0.310.2±0.30.010.02101.7±3.1OXO 9.1±0.79.1±0.40.010.02105.1±1.6NAL 9.2±0.211.1±0.10.010.02109.3±2.8MAR 5.2±0.710.9±0.30.010.0285.7±1.1SAR 10.0±0.79.1±0.80.010.0284.3±1.7NOR 10.2±0.511.6±0.70.010.0285.3±2.7ERY 11.4±0.49.4±0.3 2.03596.4±4.6TYL 11.3±0.610.5±0.30.010.0291.4±1.6TIL 6.2±0.412.2±0.30.020.05103.3±3.1JOS 7.7±0.411.3±0.60.020.05103.6±2.7SPI 8.1±0.48.0±0.60.020.0595.7±2.9LIN 4.7±0.38.5±0.20.010.0298.9±1.6TIM 5.3±0.67.3±0.40.010.0296.8±1.6SMT 6.3±0.910.1±0.20.010.02106.9±2.1SME 8.2±0.711.4±0.60.010.02104.5±1.8SDMX 8.3±0.38.1±0.30.010.0298.9±2.3SMA 10.0±0.77.0±0.20.010.02102.1±1.6SMM 9.1±0.89.1±0.50.010.0291.0±2.6SFT 5.8±0.87.9±0.50.020.02105.1±2.2DC 4.4±0.613.3±0.40.020.0595.8±1.8OTC 11.4±0.712.9±0.30.010.0299.3±1.6TC 11.0±0.68.0±0.30.020.0595.9±2.9CTC

5.3±0.5

10.5±0.4

0.02

0.05

98.8±2.3

Table 3

Matrix effects:negative values characterise the ion suppressions,and positive ones –the ion enhancement (à)–ion suppression,(+)–ion enhancement.Analyte Matrix effect (%)Analyte Matrix effect (%)SPEC à11.6±1.4OXO à8.6±0.5STRP 10.2±0.7NAL à5.3±0.4DISTRP 8.8±0.6MAR à7.8±0.6NEO 9.8±0.4SAR à6.3±0.2AMOX à9.6±1.1NOR à5.2±0.4PEN G à6.3±0.4ERY à12.4±1.3AMPI à6.8±0.2TYL à11.8±0.8DICLOX à5.2±0.6TIL à10.7±0.6NAF à7.1±1.4JOS à9.1±1.6OXA à6.4±0.4SPI à10.3±0.4

CFPI à3.6±0.9LIN 17±0.5CFT à2.1±1.1TIM à8.2±0.6CFQ à4.4±0.2SMT à7.5±0.5CFLO à3.6±0.3SME à8.9±0.4CFZ à4.9±0.6SDMX à7.9±1.3CFLE à2.9±0.4SMA à8.4±0.9CFPE à5.4±0.5SMM à10±1.6TMP 4.9±0.6SFT à8.3±1.0CIP à8.3±0.7DC 11.9±1.3ENR à9.9±0.4OTC 13.6±1.2DIF à7.1±0.9TC 10.5±0.9DAN à8.9±0.5CTC

11.3±0.5

FLU

à9.2±0.3

M.Gbylik-Sikorska et al./Chemosphere 119(2015)8–1513

for repeatability and4.4–13.5%for within–laboratory reproduc-ibility.All analytes provided the acceptable results in the agree-ment with the criteria of the European Decision2002/657/EC (C.D.2002/657/EC).The percentage of the relative recoveries was calculated in terms of the internal standard and is in acceptable range of the EU requirement with recovery values ranged from 70%to120%respectively.The average recoveries for three valida-tion levels are listed in Table2.

The selectivity of the method improves no interfering peaks co-eluted at the same retention time of interest for analytes in any sample extracts.

The LOD values are in the range of0.01–3.79l g Là1and the LOQ values are in the range of0.02–10l g Là1,depending of analyte.

It was found that the stability of proven individual stock stan-dard solution for?uoroquinolones,sulfonamides,pleuromutilins, diaminopyrimidines and tetracyclines stored atà18°C remains unchanged for at least6months.The aminoglicosides individual stock standard solutions stored under the same conditions are stable for3months.The stability of penicillines,cephalosporins and macrolides is2months at least because of the considerable decrease of concentration levels.All standard stock solution stored at4°C was stable not longer than for3months with the exception for penicillines,cephalosporins and macrolides which are stable for 1month.The stability results for water samples spiced with?uoro-quinolones,sulfonamides,pleuromutilins,diaminopyrimidines at the10l g Là1concentration level for all tested antibiotics remain stable at the similar level stored atà18°C at least for6months. For aminoglicosides,tetracyclines,macrolides,cefalosporines and penicillines,signi?cant reductions of concentrations of fresh extracts were observed after2weeks.

The water samples from food-producing animal watering sys-tems are known to be rich in mechanical impurities,nutrients,feed residues,vitamin and minerals,which can greatly affect the perfor-mance of the method,especially in the ESI-MS analysis with signal suppression and signal enhancement.The matrix effect was calcu-lated for all analytes in different water samples(from water supply systems and waste waters).The matrix effect is reported as the average results in Table3.

As it was found,the average matrix effect expressed as ion sup-

Chromatograms of real water samples with mostly detected analytes:a–water sample contaminated with ENR=22.6l g Là1and DC=75.1l g Là1,b–water contaminated with ENR=64.4l g Là1and TIM=68.8l g Là1,c–water sample contaminated with SMT=22.5l g Là1and TMP=44.5l g Là1,d–contaminated water contaminated with LIN=304l g Là1.

14M.Gbylik-Sikorska et al./Chemosphere119(2015)8–15

water and0.05M HFBA for SPE cartridges conditioning step were tested.Then,different values of elution mixture(2?3mL, 2?4mL,2?5mL)and the temperature evaporation(35–50°C) were checked.Additionally,some minor changes like different size of syringe?lters(0.22l m and0.45l m)were investigated,too. There were no signi?cant differences observed at the evaluation of robustness.In addition,no degradation of the compounds was noted at low and higher temperature of evaporation.The syringe ?lters resize was not affected for analytes concentration level. 3.4.Application of real samples

In order to check suitability of the proposed method,50water samples obtained from different food-producing animal farms in Poland were tested.Water samples were only collected on farms provided with documentation of non-use of antibiotics during sample collecting.Matrix-matched calibration curves were used for the quanti?cation of the antibiotics compounds in these sam-ples,whereas retention times,comparing analyte/internal stan-dard peak area ratio from matrix-matched calibration curve to analyte/internal standard peak area ratio in analysed samples, were also used for identi?cation.The results of this study indicate that8out of45antibiotics were detected in the analysed samples of food-producing animal farms(Table4).

Speci?cally,ENR was one of the most frequently detected anti-biotics(in19samples),DC was found in13out of analysed sam-ples.Twenty-six out of the analysed water samples were contained antibiotics residues.The sample chromatograms of real water samples are shown in Fig.2.The presence of antibiotics was found in52%out of analysed water samples.This may be caused by a wrong dosage of pharmaceuticals,as well as antibiot-ics accumulation in water supply systems because of no cleaning systems.Both cases provided the occurrence of antibiotics residues in farm environment and constant exposure of animals to antibac-terial agents.This situation can cause negative effect on the farm environment,pass into animals organisms and promote dissemi-nation of not only antibiotics resistant bacteria,but also the resis-tance genes in bacterial populations.This is con?rm of the necessity to research and control the degree of water supply sys-tems contamination by antibiotics.

4.Conclusion

The invented method,allows for a fast quantitative and qualita-tive analysis of45veterinary antibiotics in water from food-pro-ducing animals water supply systems.The method is based on the SPE extraction and clean up subsequently analysed by LC–MS/MS.The proposed method is simple,fast and easy to perform and allows to determine a wide variety of antibiotics belonging to9different groups in a single run method.The application of the proposed method allows for a new and non-invasive control of antibiotic residues in water supply systems.The concentration of antibiotics residues in real water samples were respectively in the range of0.14–1670l g Là1.

Acknowledgements

The authors wish to thank Malgorzata Kotulska for her work in the validation process,Anna Gajda and Marta Piatkowska for their participation in sample analysis.References

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