Ionic liquid-based homogeneous liquid–liquid microextraction for the

Journal of Chromatography A,1218 (2011) 7254–7263Contents lists available at SciVerse ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maIonic liquid-based homogeneous liquid–liquid microextraction for thedetermination of antibiotics in milk by high-performance liquid chromatographyShiqian Gao,Haiyan Jin,Jingyan You,Yi Ding,Nan Zhang,Ying Wang,Ruibing Ren,Rui Zhang,Hanqi Zhang ∗College of Chemistry,Jilin University,Qianjin Street 2699,Changchun 130012,Chinaa r t i c l ei n f oArticle history:Received 5January 2011Received in revised form 15August 2011Accepted 17August 2011Available online 25 August 2011Keywords:Ionic liquid-based homogeneousliquid–liquid microextraction (IL-based HLLME)Fluroquinolones Sulfonamides Milk samplesa b s t r a c tIonic liquid-based homogeneous liquid–liquid microextraction (IL-based HLLME)high-performance liq-uid chromatography was developed and applied to the extraction,separation and determination of some antibiotics in milk.The proteins and lipids were removed by adding salt and adjusting the pH value.The homogeneous extraction was applied to the improvement of recoveries for IL phase and analytes.The experimental parameters of the IL-based HLLME,including salt concentration in sample solution,pH value of sample solution,volume of [C 6MIM][BF 4],amount of ion-pairing agent (NH 4PF 6),and extrac-tion time,were evaluated.The limits of detection for enoxacin,pefloxacin,norfloxacin,enrofloxacin,sulfamethoxazole and sulfadimethoxine were 15.8,7.07,5.13,4.00,7.79and 8.33␮g L −1,respectively.When the proposed method was applied to the analysis of milk samples the recoveries of the analytes ranged from 92.5to 118.6%and relative standard deviations were lower than 7.00%.© 2011 Elsevier B.V. All rights reserved.1.IntroductionFluroquinolones (FQs)and sulfonamides (SAs)are a class of rela-tively new synthetic antibiotics.The antibiotics are commonly used as veterinary drugs to promote the growth of livestock and prevent the bacterial infection.However,the antibiotic residues in livestock products,such as meat,milk and eggs,can endanger human health [1–3].Considering the food safety,some antibiotics are prohibited from breeding livestock by a number of countries,such as China,Australia,Canada,USA and EU member states [4–6].Meanwhile,to protect the health of consumers and minimize the potential harm,maximum residue limits (MRLs)of the antibiotic residues have been established by the European Union (Commission Decision 37/2010/CE)and USA Food and Drug Administration.The contents of enrofloxacin and ciprofloxacin residues in milk were not higher than 100␮g kg −1.The MRLs for the sulfamethazine and the SAs established by the Ministry of Agriculture of the People’s Republic of China,were 25␮g kg −1and 100␮g kg −1,respectively [7–12].Some sensitive,simple and reliable analytical methods were developed to determine the antibiotic residues in dairy products,such as capillary electrophoresis (CE)[13,14],ultra high-performance liquid chromatographic (UHPLC)separation coupled∗Corresponding author.Tel.:+8643185168399;fax:+8643185112355.E-mail addresses:gaosq08@ ,analchem@ (H.Zhang).with diode array detection (DAD),fluorescence detection (FLD)or mass spectrometric (MS)[7,14–23]detection.The extraction,sep-aration and concentration of the analytes in the sample matrices were very important before chromatographic analysis.The solid-phase extraction (SPE)and matrix solid-phase dispersion (MSPD)were considered as the most effective extraction methods and widely applied to the extraction of antimicrobial residues in bovine milk [7,15,16].However,the two extraction methods were compli-cated and required a large amount of organic solvent.In the last few years,some new extraction methods were developed to sim-plify sample preparation steps and save organic solvents.On-line column-switching and molecularly imprinted (MI)SPE were devel-oped to extract antibiotic residues from milk [7,20].The advantage of the methods was that the concentration and purification can be completed simultaneously.Solid-phase microextracion (SPME),stir bar sorptive extraction (SBSE),liquid-phase microextraction (LPME)and dispersive liquid–liquid microextraction (DLLME)have been applied for the extraction of antibiotic residues in livestock products [12,21,22].Ionic liquids (ILs)are semi-organic molten salts,which consist of organic cations and various anions with melting points at or below 100◦C [24–26].They have attracted extensive attention because of their low vapour pressure,high thermal stability,tunable viscosity,moderate dissolvability of organic compounds and environmental friendliness.The structure adjustability is the typical advantage of ILs and the functionalized ionic liquids are easily obtained.There-fore,ILs are considered as replacements for conventional organic0021-9673/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2011.08.063S.Gao et al./J.Chromatogr.A 1218 (2011) 7254–72637255OONNHNNFOHENOONNNFOHPEFO NHNNFOHNOROONNN FOHENRNOS OONHNH 2SMOS OONHNH 2NNOOSDMFig.1.Chemical structure of the antibiotics.solvents and found wide application in analytical chemistry,such as chromatography [27,28],selective microextraction [29]and elec-troanalytical chemistry [30].Herrera-Herrera et al.studied the separation of fluoroquinolone antibiotics in water samples using ILs as mobile phase additives [31].High temperature headspace IL-based LPME,IL-based ultrasound-assisted DLLME and IL-based aqueous two-phase system (IL-ATPS)have been developed for the extraction of pesticides and antibiotic residues in matrix samples [32–35].Moreover,the IL-ATPS has also been successfully applied to the food analysis by Zeng et al.[36]for the concentration of three quinolones antibiotics in milk.However,a large amount of IL was required when IL-ATPS was applied.The homogeneous liquid–liquid extraction (HLLE)was applied to the multi-residue analysis in water samples [37,38].Baghdadi and Shemirani [39]determined the inorganic species in saline solutions by the in situ solvent formation microextraction (ISFME)coupled with UV–vis spectrometric detection.However,to date,the HLLE has not been applied for the extraction of antibiotic residues in dairy products.In this paper,IL-based HLLME was developed for extracting FQs and SAs from milk.The proteins and lipids were removed with some inorganic agents.After the [C 6MIm][BF 4]was added in the sample solution,the NH 4PF 6was added in the sample.The resulting [C 6MIM][PF 6]is not dissolvable in the sample solu-tion.After the extraction phase separation was performed bycentrifugation.The proposed method was applied to the analysis of some milk samples.2.Experimental2.1.Reagents and chemicalsFQs,including enoxacin (ENO),pefloxacin (PEF),norfloxacin (NOR)and enrofloxacin (ENR),and SAs,including sulfamethox-azole (SMO)and sulfadimethoxine (SDM)were obtained from Sigma–Aldrich Chemical Company (St.Louis,MO,USA).The purity of all the compounds was higher than 98%.The chemical struc-tures and properties of the compounds are shown in Fig.1and Table 1,respectively.Standard stock solutions for the antibiotics at the concentration level of 500␮g mL −1were prepared in acetoni-trile containing 5.24mmol L −1acetic acid.The working solutions were obtained by diluting the stock solutions with acetonitrile.Chromatographic grade acetonitrile was purchased from Fisher Scientific Company (UK)and pure water was obtained with a Milli-Q water purification system (Millipore Co.,USA).Acetic acid,orthophosphoric acid (99%),ammonium acetate and sodium chlo-ride are of analytical-reagent grade and purchased from Beijing Chemical Factory (Beijing,China).1-Hexyl-3-methylimidazolium tetrafluoroborate ([C 6MIM][BF 4]),1-ethyl-3-methylimidazolium7256S.Gao et al./J.Chromatogr.A1218 (2011) 7254–7263Table1Physicochemical properties of the FQs and SAs.ENO PEF NOR ENR SMO SDMMolecular weight320.3333.36319.3359.4253.28310.3m.p.(◦C)246–248270–272220–228221–226168–172198–201 log K ow a0.82 1.2450.93 1.780.89 1.63p K a1b 6.00 6.21±0.12 6.36±0.12 5.86±0.05 1.85±0.30 2.13±0.30 p K a2b8.507.87±0.108.62±0.088.24±0.07 5.60±0.04 6.08±0.09 max(nm)c266276276276267264a log K ow values are from literature[40–43].b p K a values are from literature[44].c Wavelengths of maximum absorption( max)were obtained from this study.Table2Physicochemical properties of the ILs.[C2MIM][BF4][C6MIM][BF4][C6MIM][PF6][C6MIM]ClChemical name1-Ethyl-3-methylimidazoliumtetrafluoroborate 1-Hexyl-3-methylimidazoliumtetrafluoroborate1-Hexyl-3-methylimidazoliumhexafluorophosphate1-Hexyl-3-methylimidazoliumchlorideMolecular weight197.97254.08312.24202.72 Density(g mL−1) 1.24 1.15 1.29 1.05 Viscosity(25◦C,cP)37279.8658618.089 Water content(mol L−1)Miscible0.880.68Miscible Solubility in water(g/100mL)Soluble Partly soluble0.75Solubletetrafluoroborate([C2MIM][BF4])and ammonium hexafluorophos-phate(NH4PF6)were obtained from Chengjie Chemical Co.Ltd. (Shanghai,China).The physicochemical properties of the ILs are listed in Table2.The milk samples were purchased from local large-scale supermarket and stored at4◦C.2.2.SamplesIn the study,five milk samples,including pure(sample1), skim(sample2),low-fat(sample3),pasteurization(sample4)and ultra high temperature sterilization(sample5)milk were analyzed. Except for the experiments mentioned in Section3.4.2,which were performed with allfive samples,all other results were obtained with sample1.The spiked samples containing FQs and SAs were prepared by spiking the working solutions into milk samples and shaking for10min.2.3.InstrumentsThe1100series liquid chromatograph(Agilent Technologies Inc.,USA)equipped with photodiode-array detector was used. Chromatographic separation of antibiotics was performed on Zor-bax Eclipse XDB-C18column(150mm×4.6mm,3.5␮m,Agilent, USA)with a C18guard column(7.5mm×2.1mm I.D.,5␮m). The mobile phase consisted of acetonitrile(A)and aqueous solution(2.90mmol L−1[C2MIM][BF4],9.70mmol L−1ammonium acetate–acetic acid,pH=2.70)(B).The gradient program is as fol-lows:0–10min,11–15%A;10–15min,15%A;15–18min,15–20% A;18–20min,20–28%A;20–25min,28–35%A and25–35min, 35–11%A.Theflow rate of mobile phase was set at0.5mL min−1 and column temperature was kept at30◦C.The injection vol-ume was20␮L.The monitoring wavelengths were270nm for ENO,SMO and SDM and280nm for PEF,NOR and ENR.The refer-ence wavelength and bandwidth were360and4nm,respectively. The output power of ultrasonic generator(KQ2200E Kunshan Ultrasonic Instrument Co.Ltd.,Kunshan,China)was100W.Ger-hardt Kjeldahl Divice(QSY,Beijing Powerful Analytical Instruments Manufacturing Center,Beijing,China)was used to determine the proteins in milk.The phase separation was performed on high-speed freezing centrifuge(Allegra64R,Beckman Coulter, Inc.,USA).2.4.Preparation of sample solution2mL of milk sample was placed into the5mL polyterafluo-roethylene(PTFE)tube.2mL of50g L−1NaCl solution and60␮L of orthophosphoric acid were added into the tube.The mixture was ultrasonically shaken for5min,heated in water bath for10min at70◦C and then ultrasonically shaken for2min.The resulting solution was centrifuged at20◦C for10min at15,000rpm.The supernatant was decanted into a5mL PTFE tube.The residue was dissolved with1mL of50g L−1NaCl solution,shaken for2min,and then centrifuged at15,000rpm for10min.The supernatants were combined and referred to as the sample solution.2.5.Ionic liquid-based HLLME60␮L of[C6MIM][BF4]was added into the sample solution and the mixture was intensely shaken for10min.0.3157g of NH4PF6 was added into the solution and a cloudy solution was formed.The cloudy solution was ultrasonically shaken for5min and kept refrig-erated at−20◦C for15min.After centrifugation at5◦C for10min at15,000rpm,the IL phase was deposited at the bottom of the tube. Then the upper aqueous phase was removed completely.In order to reduce the loss of IL,acetonitrile was added to dilute the IL to 150␮L.The resulting analytical solution was homogenized ultra-sonically andfiltered with0.22␮m PTFEfilter membrane before HPLC analysis.3.Results and discussion3.1.Chromatographic conditionsFQs and SAs belong to strongly polar amphoteric compounds. FQs contain carboxylic acid and piperazinyl amino group at the N4-position.SAs have one active primary amine group and sul-fonylamino group[45].The studies showed that the interaction between the two groups(amidocyanogen and piperazinyl amino) and the residual silanol group of C18filler might be hydrogen bond and ion-exchange,which forms the secondary reserve effects, resulting in the chromatographic peak broadening and tailing [46,47].The chromatographic method was developed for the deter-mination of the FQs in the milk[15].As shown in Fig.2,whenS.Gao et al./J.Chromatogr.A 1218 (2011) 7254–72637257Fig.2.Chromatogram of spiked milk sample 1obtained in isocratic elution condition.Mobile phase,aqueous solution (3.0mmol L −1[C 2MIM][BF 4]and 10.0mmol L −1ammonium acetate–acetic acid,pH =3.0)–acetonitrile (v/v,87/13);flow rate of mobile phase,0.5mL min −1.1,ENO;2,PEF;3,NOR;4,ENR.the experimental conditions mentioned in the literature [15]were applied to the separation of the analytes,the retention times are prolonged and the SAs are not eluted.Considering the difference of the analytes determined by the proposed method and the method reported in the literature [15]and in order to obtain the satisfac-tory experimental results,the gradient elution was applied and the effect of the composition of the mobile phase also was examined in the proposed method.The mobile phase consists of acetonitrile and aqueous solution.The aqueous solution contains [C 2MIM][BF 4]and ammonium acetate–acetic acid.The effects of the concentra-tions of [C 2MIM][BF 4]and ammonium acetate–acetic acid in the aqueous solution on the chromatographic peaks were investigated.When ILs were used as the additives of the mobile phase,the reten-tion times can be shortened and the peak shapes can be improved.As shown in Fig.3A and B,the retention times of target ana-lytes are prolonged and the peak shapes are improved effectively with the increase of the concentration of [C 2MIM][BF 4].As can be seen from Fig.3B and C,when the concentration of ammonium acetate-acetic acid in the aqueous solution decreases the retention times are prolonged and the peak shapes are improved.Based on the experimental results,the mixture of acetonitrile and aqueous solution (2.90mmol L −1[C 2MIM][BF 4],9.70mmol L −1ammonium acetate–acetic acid,pH =2.70)was used as the mobile phase.3.2.Sample preparationBecause of the effect of the high protein and lipid content in milk,the methods reported for the determination of FQs and SAs in milk involve protein precipitation with an organic solvent (e.g.methanol,ethanol,acetonitrile)under acidic (e.g.trichloroacetic acid)condition.However,a large amount of organic solvent was required.The proteins and lipids in milk were removed by adding the salt and adjusting the pH value in the proposed method.The solubility of the proteins decreases with the increase of concen-tration of salt and the hydrophilic colloid system containing the proteins and water is destroyed.In the presence of the salt the pro-teins may be aggregated and precipitated.The isoelectric points (pIs)of most proteins are in the range of pH 4.0–6.0.The proteins are aggregated and precipitated when the pH value is lower than the pI.Meanwhile,the precipitation of the proteins is related to temperature.Roca et al.[48]investigated the resistance of FQs in milk sample for different heat treatments and the results showed that quinolones were stable under high temperature.Carretero et al.[49]reported the suitable extraction temperature for FQs andSAs.Therefore,in order to avoid the degradation of target analytes,the optimum demulsifying temperature was selected as 70◦C.The octanol–water partition coefficient (K ow )is an essential parameter of the lipophilicity of the antibiotics.The hydrophobic compounds have high log K ow ,whereas the low log K ow signifies a compound soluble in water.The log K ow and p K a values of the FQs and SAs are listed in Table 1.As can be seen,the log K ow values of antibiotics are lower than 2.0,which means that the antibiotics are easily soluble in water and not easily miscible with proteins and lipids.To remove the proteins and lipids,the effects of the concentrations of NaCl (10,20,40,50,60,70g L −1)and pH values (1.0–11.0)were inves-tigated when the volume of [C 6MIM][BF 4]was 40␮L and amount of NH 4PF 6was 0.25g,and the optimum concentration of NaCl and pH value were selected.3.2.1.Determination of proteins and lipidsTo evaluate if the proteins and lipids can be removed completely from the matrix of milk,the Kjeldahl’s method and GB 5413.3-2010method,which is a standard method of China,were applied to the determination of the proteins and lipids in the milk sample and the precipitate obtained in preparation of sample solution.The results listed in Table 3demonstrate that 95.6%of proteins and 91.4%of lipids are removed from the milk sample.3.2.2.The effect of salt concentration in sample solutionTo evaluate the interference of salt on the determination of antibiotics,the water was used as the sample.The sample solu-tion was prepared by the procedure mentioned in Section 2.4and analytical solution was obtained by the procedure mentioned in Section 2.5.To examine the effect of NaCl on the determina-tion of the analytes,NOR was selected in the experiment and the absorption spectra of NOR in the analytical solutions in the pres-ence and absence of NaCl were obtained.As can be seen from Fig.4,the absorbance increases and the shape of the spectrum does not change in the presence of NaCl.The experimental results indicate that if NaCl enters into IL phase,NaCl in IL phase can-not affect the maximum absorption wavelength of NOR and can enhance the signal,which is in agreement with the result obtained by HPLC.The effect of the concentrations of NaCl on the recov-eries of target analytes was investigated.The recoveries increase with the increase of NaCl concentration,reach the maximum at NaCl concentration of 50g L −1and then decrease.The effect of salt out is enhanced with the increase of the salt concentration,which results in the transfer of the target analytes from aqueous7258S.Gao et al./J.Chromatogr.A1218 (2011) 7254–7263Fig.3.Effect of the mobile phase composition.(A)Acetonitrile–aqueous solution(2.50mmol L−1[C2MIM][BF4]and10.0mmol L−1ammonium acetate–acetic acid, pH=2.70);(B)acetonitrile–aqueous solution(2.90mmol L−1[C2MIM][BF4]and10.0mmol L−1ammonium acetate–acetic acid,pH=2.70);(C)acetonitrile–aqueous solution (2.90mmol L−1[C2MIM][BF4]and9.70mmol L−1ammonium acetate-acetic acid,pH=2.70);flow rate:0.5mL min−1.1,ENO;2,PEF;3,NOR;4,ENR;5,SMO;6,SDM.S.Gao et al./J.Chromatogr.A1218 (2011) 7254–72637259 Table3The content of proteins and lipids in precipitate and milk sample1(n=3).Sample Content of protein(g100g−1)RSD,%Content of lipid(g100g−1)RSD,%Precipitate 2.82 1.01 3.31 1.74 Milk sample 2.950.52 3.62 1.40phase into the IL phase.However,when the salt concentration istoo high,the ion exchange between[PF6]−in[C6MIM][PF6]andCl−in aqueous solution occurs and the resulting[C6MIM]Cl is sol-uble in water,which may lead to the decrease of the amount ofIL phase and the poor extraction performance[44,50].50g L−1was selected as NaCl concentration for extracting antibiotics by acomprehensive.3.2.3.The effect of pH values in sample solutionThe Carboxylic acid group of FQs directly affects the p K a1value.Protonation at N4of the piperazine ring of FQs is the significantfactor to influence p K a2value.Meanwhile,the p K a1(below2.5)and p K a2(5–8.5)of SAs are related to the protonation of the ani-line group and deprotonation of the sulfonylamido group[51–53].Because the pH values of solutions influenced the species of the tar-get analytes,the recoveries of target analytes were related directlyto the pH values.As can be seen from Fig.5,the recoveries of thetarget analytes decrease significantly with the increase of pH values340330320310300290280270260-0.0050.0000.0050.0100.0150.0200.0250.0300.0350.040Absorbanceλ (n m)12Fig.4.The UV–vis absorption spectra of NOR in water sample containing(1)andnot containing(2)NaCl,spiked concentration,20␮g L−1.Recovery(%)pHFig.5.Effect of pH valve.Volume of[C6MIm][BF4],40␮L;volume of sample,2mL;spiked concentration,100␮g L−1;extraction time,5min.ranging from1.0to9.0and then increase slightly with the increaseof the pH values ranging from9.0to11.0.On the one hand,theinteraction between PO43−and water at high concentration wasstronger than that between water molecules,which was beneficialto the phase separation,and the enrichment factor of target ana-lytes should be higher at low pH value[34].On the other hand,the species of molecule may different at different pH solutions.Finally,the pIs of target analytes are nearly neutral and the sol-ubility is higher in acidic and alkaline solution than that in nearneutral solution,which results in low solubility of the analytes atthe pH values of6.0–8.0.Based on the experimental results,the pH1.0was selected.3.3.IL-based HLLMEIn the IL-based HLLME,the effects of experimental parameters,such as volume of ILs,amount of ion-pairing agent(NH4PF6)andextraction time,were investigated in order to obtain good sensitiv-ity,precision and selectivity.3.3.1.Volume of ILsOne of the critical steps in ILs-based HLLME is to select a suit-able water-miscible IL and ion-pairing agent for enrichment of thetarget analytes,[C6MIM][BF4]and NH4PF6were used according tothe report by Baghdadi and Shemirani[39].The effect of volumeof[C6MIM][BF4]on extraction recoveries was studied when theamount of NH4PF6was0.25g.The recoveries increase with theincrease of the volume of IL from20to60␮L.The recoveries arealmost unchanged when the volume increases from60to70␮L,which is due to the resulting amount of[C6MIM][PF6]is unchanged.Therefore,60␮L of[C6MIM][BF4]was selected in the work.3.3.2.Amount of NH4PF6The effect of amount of ion-pairing agent on the recoveries of theanalytes was studied when the amount of[C6MIM][BF4]was60␮L.As shown in Fig.6,the recoveries of the target analytes increasewith the increase of amount of NH4PF6from0.0902to0.3157g,andRecovery(%)NH4PF6 amount (g)ENOFig.6.Effect of NH4PF6amount.Volume of[C6MIm][BF4],60␮L;volume of sample,2mL;spiked concentration,100␮g L−1;pH,1.0;extraction time,5min.S.Gao et al./J.Chromatogr.A 1218 (2011) 7254–72637261T a b l e 6A n a l y t i c a l r e s u l t s o f m i l k s a m p l e s (n =5).S a m p l eA d d e d (␮g L −1)E N OP E FN O RE N RS M OS D MR e c o v e r y (%)R S D (%)R e c o v e r y (%)R S D (%)R e c o v e r y (%)R S D (%)R e c o v e r y (%)R S D (%)R e c o v e r y (%)R S D (%)R e c o v e r y (%)R S D (%)S a m p l e 150100.53.85115.45.64109.83.94110.31.54111.03.39100.22.13100110.86.30118.01.41113.64.93118.65.01101.95.18106.86.18150108.56.93118.22.30110.52.33113.52.52118.66.9598.45.81S a m p l e 250104.54.93108.52.70109.64.93107.24.47100.54.60104.45.24100118.34.74113.50.69111.96.62112.96.32107.15.14104.35.43150108.56.93112.91.48108.51.64110.41.2196.16.76109.33.61S a m p l e 350104.56.53109.42.70112.13.63106.35.41110.25.3697.27.00100109.73.64101.93.59109.03.04111.46.60108.15.28113.14.14150108.75.13115.86.14115.83.95113.41.0099.93.02103.44.19S a m p l e 450106.36.91116.54.91112.06.35108.24.93114.24.53103.56.07100114.95.83101.11.72107.62.79108.21.58112.06.7496.35.10150113.22.32110.76.96107.93.67108.43.55101.76.59116.02.13S a m p l e 550114.41.28111.53.48118.44.18113.14.5992.54.50106.33.46100106.22.28105.54.52110.72.67111.43.84110.33.32101.76.94150108.53.21101.13.70107.02.68107.73.17112.52.94102.36.02when the amount of NH 4PF 6is 0.3608g,the recoveries do not obvi-ously change.So the amount of ion-pairing agent NH 4PF 6selected was 0.3157g.That is to say,the molar ratio of [C 6MIM][BF 4]to NH 4PF 6was 1:7.3.3.3.Extraction timeTheoretically,the increase of extraction time was beneficial to the partition equilibrium of target analytes in [C 6MIM][PF 6]/water system and the improvement of the extraction yields of ana-lytes.The recoveries of the target analytes increase gradually with the increase of the extraction time from 1to 5min.The extrac-tion time longer than 5min resulted in a negligible increase of the recoveries and the equilibrium seemed to be reached.Based on the experimental results,the extraction time was selected to be 5min.3.4.Evaluation of the method3.4.1.Analytical performancesThe working curves were constructed by plotting the peak areas measured versus the concentrations of analytes.The slope and intercept of the linear regression equations,the residual standard deviations (Sy/x )and correlation coefficients are listed in Table 4.The limits of detection (LODs)and quantification (LOQs)indicated in Table 4are determined as the lowest con-centration yielding a signal-to-noise (S/N)ratio of 3and 10,respectively.The concentrations of the target analytes in the extract are higher than the LOQs and lower than upper limits of determination for the proposed method.So the LOQs and linear regression equations are appropriate to the goal of the proposed method.Repeatability was evaluated by determining target analytes in spiked milk samples.The intra-day precision was determined by analyzing the samples five replicates in one day.The inter-day precision was achieved by analyzing the samples once a day in five consecutive days.The results are presented in Table 4and the results indicate that the proposed method has good repeatability.Long-term stability of analytes in milk during sample storage was evaluated.The spiked samples were prepared according to the method mentioned in Section 2.2,kept in sealed bottle and then stored for 1,10,20and 30days at 4◦C,respectively.All experiments were performed in five replicates.The results are listed in Table 5.The recoveries and RSD values range from 87.7to 117.7%and 0.83to 6.99%,respectively.It can be concluded that the antibiotics in the milk samples were stable for at least one month.3.4.2.Analysis of samplesIn order to evaluate the applicability of the proposed method,the proposed method was applied to determine the residues of the FQs and SAs in some milk samples.The FQs and SAs in the spiked samples were determined and the results are listed in Table 6.As can be seen,the proposed method provides good recoveries (92.5–118.6%)and acceptable precision (≤7.00%).The chromatograms of blank and spiked milk samples are shown in Fig.7.parison of IL-based HLLME with other methodsThe proposed method was compared with the SPE reported for the extraction of antibiotics from milk samples (Table 7)[19,54].The results indicate that there is no significant difference in the recoveries.However,compared with the SPE,the proposed method has some advantages in the experimental cost,organic solvent con-sumption,and operation simplicity.。