Chemistry Research Article Received 29 October 2012; Accepted 5 April 2013 Abstract: Keywords: 1. Introduction

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1 Cent. Eur. J. Chem. 11(8) DI: /s y Centrl Europen Journl of Chemistry Determintion of some frequently used ntibiotics in wste wters using solid phse extrction followed by high performnce liquid chromtogrphy with diode rry nd mss spectrometry detection csn priş 1, Mri-Loredn Sorn 1*, Virgini Comn 2, Florin Copciu 2, Dumitru Ristoiu 3 1 tionl Institute for Reserch nd Development of Isotopic nd Moleculr Technologies, Cluj-poc , Romni 2 Rluc Ripn Institute for Reserch in Chemistry, Bbeş-Bolyi University, Cluj-poc , Romni 3 Fculty of Environmentl Sciences nd Engineering, Bbeş-Bolyi University, Cluj-poc , Romni Reserch Article Received 29 ctober 2012; Accepted 5 April 2013 Abstrct: This study is focused on the determintion of some importnt ntibiotics from different clsses in wste wter smples using solid phse extrction followed by high performnce liquid chromtogrphy with two detectors, diode rry nd mss spectrometer in positive ionistion mode. The investigted ntibiotics include three penicillins (moxicillin, mpicillin, penicillin G), two cephlosporins (ceftzidime, ceftrixone), nd two tetrcyclines (tetrcycline, doxycycline). The studied ntibiotics were extrcted from wste wter smples using hydrophilic-lipophilic blnced crtridges. The extrction of ntibiotics from wter mtrices ws tested t severl ph vlues. The best recoveries were obtined t ph 3 nd 7 respectively. Depending on the nture of ntibiotic, the limits of detection nd quntifiction were obtined in the rnge of µg ml 1 nd µg ml 1 respectively. Influent nd effluent wste wter smples were collected from Wste Wter Tretment Plnt from Romni in order to detect the studied ntibiotics. The ntibiotics detected in the influent wste wter smples were ceftrixone (334 µg L 1 ), tetrcycline (146 µg L 1 ) nd doxycycline (110 µg L 1 ). In effluent wste wter smples no trget ntibiotics were detected. Keywords: Antibiotics Wste wters Solid-phse extrction High performnce liquid chromtogrphy Mss spectrometry Versit Sp. z o.o. 1. Introduction Antibiotics re chemicl substnces with biologicl ctivity ginst some microorgnisms being considered the most productive fmily of drugs so fr developed for meliorting humn helth. Besides this bsic ppliction, ntibiotics (ntimicrobils t lrge) re used for preventing nd treting nimls, different plnt infections nd lso for dvncing growth in niml frming [1]. After dministrtion to humns nd nimls, through excretion, ntibiotics rech the sewge tretment plnts, where they re not totl removed. Hence, t the end ntibiotics cn get into the nturl wter courses. A direct entrnce of veterinry ntibiotics into soil is the fertiliztion of the soil with niml mnure. Afterwrds, depending on the mobility of the compounds, some ntibiotics cn rech * E-mil: loredn.sorn@itim-cj.ro 1343

2 Determintion of some frequently used ntibiotics in wste wters using solid phse extrction followed by high performnce liquid chromtogrphy with diode rry nd mss spectrometry detection ground or surfce wters [2]. Due to their spordic enter into the environment nd permnent presence, this ctegory of phrmceuticls is considered to be persistent nd pseudo-persistent contminnts [3]. These issues of ntibiotics in the qutic environment hve conducted to rising preoccuptions cused by the potentil environmentl risks nd the extension of ntibcteril resistnce mong the microorgnisms [4]. To prevent these risks, the monitoring of ntibiotics tht rech the environment, lthough they re in smll mounts, is necessry. The literture presents different nlyticl techniques tht hve been used for the determintion of ntibiotics in vrious environmentl smples such s cpillry electrophoresis [5], thin lyer chromtogrphy [6] nd high performnce liquid chromtogrphy (HPLC) [7-10]. Usully, the nlysis of ntibiotics in wter smples includes pre-concentrtion step, like solid phse extrction (SPE), followed by liquid chromtogrphic (LC) determintion. The chromtogrphic system coupled with mss spectrometer (MS) cn give very selective nd sensitive detection [11]. Ltely, the hydrophilic-lipophilic blnced mterils, such s sis HLB (styrenedivinylbenzene-vinylpyrrolidone copolymer) hve been found to be suitble for the SPE extrction of wide rnge of ntibiotics [12]. Mny ppers reported good recoveries for the ntibiotics from environmentl smples with this type of crtridge [13-16]. Thus, SPE-LC, SPE- LC-MS or SPE-LC-MS/MS methods hve been reported for tetrcyclines, β-lctms [14], quinolones, penicillins [17], fluoroquinolones [18] nd mcrolides [19]. The ntibiotics investigted in this study tke prt from two different clsses. The first clss contins β-lctm ntibiotics (BLAs) which re considered the most importnt group of ntibiotics used now for more thn 80 yers. BLAs include lrge vriety of nturl penicillins (e.g. penicillin G), semi-synthetic penicillins (e.g. moxicillin, mpicillin) nd cephlosporins (e.g. ceftzidime, ceftrixone). The bsic chemicl structure of BLAs is β-lctm ring which is responsible for their ntibcteril ctivity. The difference between BLAs consists in the vrible side chins responsible for their chemicl nd phrmcologicl properties. The first recognizing structurl difference between cephlosporins nd penicillins is the ring system merged to the β-lctm ring. Cephlosporins re chrcterised by the presence in their molecule of six-member dihydrothizine ring while penicillins by five-member thizolidine ring (Tble 1) [8]. The second clss of ntibiotics re tetrcyclines (TCs) which re tody widely used in humn nd veterinry medicine prctice. TCs (e.g. doxycycline, tetrcycline) re ctive bcteriosttic gents with lrge spectrum ginst Grm-positive nd Grm-negtive bcteri tht serve to inhibit protein synthesis. The bsic structures of TCs consist of hydronphthcene bckbone which contins four fused rings (Tble 1) [9]. The im of the present study is to determine simultneously some ntibiotics from wste wters by mens of solid phse extrction followed by high performnce liquid chromtogrphy with two detectors, nmely diode rry nd electrospry ioniztion mss spectrometry in positive mode (SPE-LC-DAD- ESI(+)-MS). The trget ntibiotics re three penicillins (moxicillin, mpicillin, penicillin G), two cephlosporins (ceftzidime, ceftrixone), nd two tetrcyclines (tetrcycline, doxycycline) (Tble 1). 2. Experimentl procedure 2.1. Chemicls nd mterils The working ntibiotic stndrds (Tble 1) were powders for injection, s cidic (CFZ) or slt (AMX with clvulnic cid, AMP, PEG, CFX) form nd cpsules, s hydrochloride (TET, DXY) form. AMX, AMP, PEG, CFX, nd TET were purchsed from Antibiotice (Romni), CFZ from GlxoSmithKline (Romni) nd DXY from Sndoz (Romni). Stock solutions of ll compounds were prepred in ultrpure wter. Acetonitrile of HPLC grde ws purchsed from Sigm-Aldrich (Germny) nd formic cid from Cristl R Chim (Romni). For the SPE method, methnol (MeH) ws bought from Merck (Germny), the mmoni from Primexchim (Romni) nd the hydrochloric cid (HCl) from Poch (Polnd). Ethylenedimine tetrcetic cid disodium slt dihydrte ( 2 EDTA 2H 2 ) ws purchsed from Fluk (Germny). Wter of high purity used in ll experiments ws prepred using Milli-Q Ultrpure wter purifiction system (Millipore, USA). The extrction of ntibiotics from spiked nd wste wter smples ws crried out on SPE crtridges sis HLB (6 ml, 200 mg, Wters, USA) using SPE device model Supelco vcuum mnifold from Sigm-Aldrich (Germny) Smple collection nd preprtion In order to void the free silnol, influent nd effluent wste wter smples were collected in clen polyethylene terephtlt bottles from Wste Wter Tretment Plnt (WWTP) from Romni which serves residentil popultion round of inhbitnts. The smples were filtered through Mcherey-gel filters (M 640 d ø 125 mm) nd stored t 4 C in refrigertor. For minimizing the microbil degrdtion the ntibiotics were extrcted, in less thn 24 hours from smpling. 1344

3 . priş et l. Tble 1. Dt of the studied ntibiotics. Generic nme /Abbrevition /Trde nme Chemicl formul /Moleculr weight (g mol 1 ) Chemicl structure Penicillins / Bctericidl usge Amoxicillin / AMX / Amoxiplus C 16 H S / H H H H 2 H S CH 3 CH 3 C Ampicillin / AMP / Ampicillin C 16 H S/ H 2 H H H S CH 3 CH 3 C Penicillin G / PEG / Penicillin G potssium slt C 16 H SK/ H H H S CH 3 CH 3 CK Cephlosporins / Bctericidl usge Ceftzidime / CFZ / Fortum C 22 H S 2 / S H 2 H 3 C H 3 C H H H H S + - Ceftrixone / CFX / Cefort C 18 H S 3 2 / H 2 S H H CH 3 H S C CH 3 S Tetrcyclines / Bcteriosttic usge Tetrcycline / TET / Tetrcycline C 22 H HCl/ H H CH 3 H (CH 3 ) 2 H.HCl CH 2 H H H Doxycycline / DXY / Doxycycline C 22 H HCl/ H H CH 3 H (CH 3 ) 2 H.HCl CH 2 H H H Amoxicillin with clvulnic cid 1345

4 Determintion of some frequently used ntibiotics in wste wters using solid phse extrction followed by high performnce liquid chromtogrphy with diode rry nd mss spectrometry detection 2.3. SPE method The sis HLB crtridges were pre-conditioned with 10 ml MeH followed by 10 ml ultrpure wter nd then loded with 100 ml wste wter smples prepred for the extrction by dding 5% (w/v) 2 EDTA 2H 2 to chelte residul metls [15]. In order to find the optiml ph for the extrction of ntibiotics from rel smples, the selected ntibiotics were extrcted from 100 ml ultrpure wter spiked with 120 µg of ech ntibiotic, t different ph vlues (2, 3, 7 nd 9). From these four ph vlues tested, the best recoveries were obtined t ph 3 for CFZ, CFX, TET nd t ph 7 for the other ntibiotics studied AMP, PEG, nd DXY. AMX showed recovery less thn 50% for both ph vlues. Further, the wste wter smples were subjected to two different extrctions, t ph 3 nd 7. The wste wter smples were djusted to ph 3 using 0.5 HCl or to ph 7 with 5% H 4 H immeditely prior to extrction. The wste wter smples were pssed through the SPE crtridges with flow rte of 1.2 ml min 1. The nlytes retined on crtridges were eluted with 6 ml MeH. The obtined extrcts were concentrted to dryness with rotry evportor t 40 C nd then solubilised in 2 ml ultrpure wter. All the procedures were performed three times LC-DAD-ESI(+)-MS method The nlyses were crried out on Shimdzu HPLC with DAD nd MS detectors, using ESI in positive mode (LC-DAD-ESI(+)-MS). The MS instrumenttion used ws single qudrupole. The chromtogrphic seprtion ws performed using Grce Alltim C18 column (100 3 mm, 3 µm) thermostted t 26 C with grdient elution. The used eluents were ultrpure wter with formic cid t ph 3 (A) nd cetonitrile (B). The progrm of grdient elution ws: from 0 to 6 min, the decresing of eluent A from 90% concentrtion to 30% nd in the dditionl 4 min to 20% followed by suddenly incresing of eluent A to the initil 90% concentrtion nd column equilibrtion of 5 min. The injection volume ws 20 µl nd the flow rte ws set t 0.5 ml min 1. All compounds were eluted up to 7.5 min. For MS detection ws used ESI in positive mode with cpillry voltge of 1.5 kv, curved desolvtion line (CDL) temperture of 250 C nd interfce temperture of 250 C. Peks res from totl ion chromtogrms (TICs) were used for quntifiction. DAD detection ws initilly used for preliminry estblishing of seprtion conditions nd identifiction of ntibiotics. Peks were detected t ll wvelengths nd presented mximum bsorption s follow: AMX, AMP, PEG, CFZ nd CFX t 197 nm, TET nd DXY t 272 nm. To llow the quntifiction of the selected ntibiotics in environmentl smples with the LC-ESI(+)-MS proposed method, studies of linerity, limits of detection, quntifiction respectively, nd precision were done for ech ntibiotic. Accurcy ws crried out for the ntibiotics found in wste wters (CFX, TET nd DXY). For linerity studies, stock solution of 1 mg ml 1 concentrtion of ech studied ntibiotic ws prepred in ultrpure wter nd then stored in refrigertor t 4 C in the drk. To obtin the clibrtion curves, working stndrd solutions in concentrtion rnge of µg ml 1 were prepred from the stock solution by successive dilutions with ultrpure wter. A volume of 20 µl of ech ntibiotic solution ws injected under the operting chromtogrphic conditions previously described. The clibrtion curves were estblished by plotting the pek re versus the corresponding concentrtion of ech ntibiotic. The correltion coefficients were lso clculted. The limits of detection (LD) nd quntifiction (LQ) were determined using the stndrd devition of the response (σ) nd the slope of clibrtion curves (S) nd were clculted by Eqs. 1 nd 2 [20]: LD = 3.3 σ/s (1) LQ = 10 σ/s (2) The precision of the quntittive determintion of studied ntibiotics ws performed by the evlution of the repetbility. In this purpose stndrd solutions of 40 µg L 1 concentrtion were prepred. All the determintions were done in triplictes nd the Reltive Stndrd Devitions (RSD, %) were clculted. The ccurcy ws evluted by dding n mount of ntibiotic to the initil content of ntibiotic detected in the wste wter smples. Both, the wste wter smples nd the smples with the dded ntibiotic mount were pre-concentrted using the sis HLB crtridges nd nlyzed using LC-DAD-ESI(+)-MS method previously estblished. Accurcy studies were done only for the ntibiotics tht hve been detected in influent wter smples (CFX, TET nd DXY). The dded ntibiotic mounts were 8.5, 2.5, nd 1.5 µg ml 1 respectively. Three replictes from ech smple were performed. The percent recovery ws clculted with Eq. 3 [21]: Recovery (%) = 100 x (found mount initil mount) / dded mount (3) 1346

5 . priş et l. Figure 1. Effects of ph vlues on SPE recoveries of the selected ntibiotics. 3. Results nd discussions 3.1. SPE of studied ntibiotics Polymeric crtridges sis HLB with hydrophilic nd lipophilic blnce chrcteristics were selected for simultneous extrction of penicillins, cephlosporins nd tetrcyclines from wter mtrices becuse such sorbents do not contin free silnols to which the trget compounds my directly bind or through metl ion complexes [15]. This type of crtridge hs excellent wetting properties due to the hydrophilic -vinylpyrrolidine monomer [10]. The effect of ph on the extrction efficiency ws studied by djusting the ph vlue of the spiked ultrpure wter t 2, 3, 7 nd 9 (Fig. 1). The obtined results show tht the extrction recoveries for mny of selected ntibiotic were higher t ph 3 (CFZ 68.7%, CFX 110.1% nd TET 102.6%) nd respectively 7 (AMP 88.8%, PEG 98.7% nd DXY 84.4%), excepting AMX tht showed recovery less thn 50% for both ph vlues (42.9% ph 3 nd 31.7% ph 7) (Fig. 1, Tble 2). The different recovery rnges under vrious ph conditions cn be ssigned to the mphoteric properties tht most of the nlytes hve [16]. For exmple, penicillins hve crboxylic cid nd one or more mine functionl groups (pk1 2 3 nd pk2 7) [7] while TCs show three pk vlues (pk1 3, pk2 7, pk3 9) [22]. Throughout the rnge of ph, TCs lwys hve locl chrge nd they re zwitterionic in the ph rnges mentioned bove. Thus in the nlysis of TCs the ph djustment is very importnt step [23]. The recovery dt of the trget ntibiotics obtined t ph vlues 3 nd 7 re presented in Tble 2 with the RSD vlues. 3.2 LC-DAD-ESI(+)-MS nlysis All nlytes were confirmed bsed on the retention time from chromtogrm nd the moleculr ion (m/z) from MS spectrum obtined with ESI in positive mode s it is shown in Tble 3. The results of these experiments in terms of moleculr ion were in greement with the vlues obtined by clcultion nd with those previously reported in the literture [3,15,17,24-26]. The clibrtion curves were constructed on six-eight points, bsed on pek res obtined from the injection of the stndrds. The correltion coefficients rnging from to were obtined for ll the nlytes. The vlues of LD nd LQ obtined re between µg ml 1, respectively µg ml 1. The results re given in Tble 4. Precision studies conducted to RSD vlues for repetbility in the rnge of % which re well within the limit of 2.0%, confirming the good precision of the LC-DAD-ESI(+)-MS proposed method (Tble 5). 1347

6 Determintion of some frequently used ntibiotics in wste wters using solid phse extrction followed by high performnce liquid chromtogrphy with diode rry nd mss spectrometry detection Tble 2. Men recovery rtes for the selected ntibiotics with the reltive stndrd devitions, t ph 3 nd 7. Antibiotic Men recovery ± RSD (%) ph 3 ph 7 AMX 42.9 ± ± 2.3 AMP 81.2 ± ± 2.0 PEG 21.2 ± ± 1.7 CFZ 68.7 ± ± 4.3 CFX ± ± 2.1 TET ± ± 4.3 DXY 80.9 ± ± 0.4 Averge of three extrctions Tble 3. The typicl retention time, the moleculr ion nd the reltive bundnce for the selected ntibiotics. Antibiotic Retention time (min) Moleculr ion (m/z) Reltive bundnce (%) AMX AMP PEG <10 CFZ CFX TET DXY Tble 4. Clibrtion rnges, clibrtion curves, correltion coefficients, limits of detection nd quntifiction of the selected ntibiotics. Antibiotic Clibrtion rnge Clibrtion curve Correltion coefficient LD LQ AMX 1 50 y = x b AMP y = x PEG y = x CFZ y = 88447x CFX y = 73750x TET y = 42047x DXY y = 63852x Are of ntibiotic. b Concentrtion of ntibiotic The percent recoveries of the ntibiotics from the spiked smples re between %. The results re presented in Tble Environmentl nlysis ppliction In order to determine the ntibiotics in the wste wters of the WWTP plnt nd lso its efficiency we nlyzed the trget compounds in three smples of influents nd in their corresponding effluents. For ll collected wste wter smples the SPE-LC- DAD-ESI(+)-MS procedure proposed in this study ws pplied. In the influent smples only three ntibiotics were detected in different concentrtion levels: 334 µg L 1 CFX, 146 µg L 1 TET, 110 µg L 1 DXY (Tble 7, Fig. 2). Wste wter smples were concentrted nd nlyzed in order to determine the content of ntibiotics. 1348

7 . priş et l. For clculting the concentrtion of ntibiotics presented in Tble 7, were tken into ccount both the volume of wste wter smple extrcted nd the volume of ultrpure wter used for solubilising the trget compounds. Tble 5. The repetbility of the selected ntibiotics. Antibiotic Concentrtion Men of mesured concentrtion ± RSD (%) AMX ± 1.4 AMP ± 0.9 PEG ± 1.3 CFZ ± 1.0 CFX ± 0.7 TET ± 0.9 DXY ± 0.9 Averge of three determintions In the collected wste wter smples only three ntibiotics from those selected were found, becuse their presence is in concordnce with the consumption of ntibiotics nd with the dischrges of wste wters contining ntibiotics in the sme period in which the smples were collected. To increse the probbility of detecting ntibiotics in wste wter smples will need to be collected smples more often nd from different smpling points. In the corresponding effluents ws not detected ny ntibiotic found in the influents from the WWTP which mens tht the tretment nd purifiction systems for wste wters re efficient regrding the studied ntibiotics. 4. Conclusions This pper describes sensitive nd reproducible nlyticl SPE-LC-ESI(+)-MS procedure for the simultneously extrction nd quntifiction of some Tble 6. Accurcy studies for ceftrixone, tetrcycline nd doxycycline. Antibiotic Added mount Initil mount Found mount Recovery (%) Men recovery ± RSD (%) CFX TET DXY Averge of three determintions ± ± ± 1.2 Tble 7. Amount of the studied ntibiotics in wste wter smples collected in the sme dy t intervls of three hours from influents nd effluents of WWTP respectively. Smpling time Wste wter smple Amount (µg L 1 ) AMX AMP PEG CFZ CFX TET DXY 9 Influent n.d. n.d. n.d. n.d. n.d Influent n.d. n.d. n.d. n.d. n.d. n.d. n.d. 15 Influent n.d. n.d. n.d. n.d. 334 n.d. n.d. 16 Effluent n.d. n.d. n.d. n.d. n.d. n.d. n.d. 19 Effluent n.d. n.d. n.d. n.d. n.d. n.d. n.d. 22 Effluent n.d. n.d. n.d. n.d. n.d. n.d. n.d. ot detected 1349

8 Determintion of some frequently used ntibiotics in wste wters using solid phse extrction followed by high performnce liquid chromtogrphy with diode rry nd mss spectrometry detection Figure 2. SPE-LC-DAD-ESI(+)-MS chromtogrms of some wste wter smples collected from WWTP: A - stndrd mixture of the studied ntibiotics; B - influent t 9 o clock; C - influent t 15 o clock. Pek identifiction: (1) moxicillin, (2) ceftzidime, (3) mpicillin, (4) ceftrixone, (5) tetrcycline, (6) doxycycline, (7) penicillin G. ntibiotics, five β-lctms nd two tetrcyclines, in influents nd effluents from WWTP. The SPE of the studied ntibiotics were performed using hydrophiliclipophilic blnced crtridges obtining good recoveries t ph 3 (CFZ 68.7%, CFX 110.1% nd TET 102.6%) nd t ph 7 (AMP 88.8%, PEG 98.7% nd DXY 84.4%), excepting AMX tht showed recovery less thn 50% for both ph vlues (42.9% ph 3 nd 31.7% ph 7). Studies of linerity, limits of detection nd quntifiction, precision nd ccurcy were lso done. The method presented in this pper is tht the seven selected ntibiotics belong to three different clsses, nd the time of nlysis is up to 7.5 min, resulting lower nlysis costs. Using the performed SPE-LC-ESI(+)-MS procedure, CFX, TET nd DXY ntibiotics were successfully detected in the influent wste wter smples t concentrtion rnges between µg L 1. In the corresponding effluents ny ntibiotic found in influents were not detected. The bsence of ntibiotics studied in the effluent wste wter smples is due to the efficiency of the purifiction process. The proposed method ws developed becuse there is no monitoring of ntibiotics in this re nd will continue to be used in future studies in the collbortion with WWTP. The occurrence of humn nd veterinry phrmceuticl compounds in WWTP influent nd effluent wste wters is significnt environmentl issue continuously developing, even if in our specific cse study, occurrence of ntibiotics in effluent wste wter smples ws not detected. Acknowledgements This pper ws relized with the support of EURDC Doctorl Scholrships for reserch performnce t Europen level project, finnced by the Europen Socil Fund nd Romnin Government. The uthors would like to thnk Assoc. Prof. Dr. Costel Sârbu for the helpful suggestions during the development of experiments. 1350

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