Simultaneous analysis of chlorophenols, alkylphenols, nitrophenols and cresols in wastewater effluents, using solid phase extraction and further determination by gas chromatography-tandem mass spectrometry

Else ie Edi o ial Sys em( m) o Talan a Manusc ip D a Manusc ip Numbe : TAL-D-11-00529R1 Ti le: Simul aneous analysis o chlo ophenols, alkylphenols, ni ophenols and c esols in was ewa e e luen s, using solid phase ex ac ion and u he de e mina ion by gas ch oma og aphy- andem mass spec ome y A icle Type: Full Leng h A icle Keywo ds: Phenols; Was ewa e ; Solid-phase ex ac ion (SPE); Gas ch oma og aphy-mass spec ome y (GC-MS); S anda d addi ion calib a ion; Ma ix-ma ched calib a ion. Co esponding Au ho : D . A. Ga ido F enich, Ph.D Co esponding Au ho 's Ins i u ion: Resea ch G oup Analy ical Chemis y o Con aminan s Fi s Au ho : Juan An onio Padilla-Sánchez O de o Au ho s: Juan An onio Padilla-Sánchez; Pa icia Plaza-Bolaños; Robe o Rome o-González; Nie es Ba co-Bonilla; José Luis Ma ínez-Vidal; A. Ga ido F enich, Ph.D Abs ac : An analy ical me hodology has been de eloped o he simul aneous ex ac ion o 13 phenolic compounds, including chlo ophenols (CPs), ni ophenols (NTPs), c esols and alkylphenols (APs) in di e en ypes o was ewa e (WW) e luen s. A solid-phase ex ac ion (SPE) me hod has been op imized p io o he de e mina ion by gas ch oma og aphy coupled o iple quad upole andem mass spec ome y (GC-QqQ-MS/MS). Due o he complexi y o he ma ix, a compa ison s udy o ma ix-ma ched-calib a ion (MMC) and s anda d addi ion calib a ion (SAC) was ca ied ou o quan i ica ion pu poses. The op imized p ocedu e was alida ed using he SAC app oach since i p o ided he mos adequa e quan i ica ion esul s (in e ms o eco e y and p ecision alues). Reco e ies we e in he ange 60- - and 78- wi h p ecision alues (exp essed as ela i e s anda d de ia ion, RSD) ≤ 30% (excep o 2-ni ophenol) in ol ing in a-day and in e -day p ecision s udies. Limi s o de ec ion (LODs) and quan i ica ion (LOQs) we e also e alua ed, an - -1. The p oposed me hod was applied o he analysis o 8 eal WW e luen samples, inding some phenolic compounds (e.g. 2- chlo ophenol, 2,4,6- ichlo ophenol and 4- e -oc ylphenol) a concen a ions highe han he es ablished LOQs. 1 Simul aneous analysis o chlo ophenols, alkylphenols, ni ophenols and c esols 1 in was ewa e e luen s, using solid phase ex ac ion and u he de e mina ion 2 by gas ch oma og aphy– andem mass spec ome y 3 4 J. A. Padilla-Sáncheza,1, P. Plaza-Bolañosa,b, R. Rome o-Gonzáleza, N. Ba co-5 Bonillaa, J. L. Ma ínez-Vidala, N. Ba co-Bonillaa,1, A. Ga ido-F enicha* 6 7 aDepa men o Analy ical Chemis y, Uni e si y o Alme ia, Ca e e a Sac amen o 8 s/n, E-04071 Alme ia, Spain 9 bDepa men o Analy ical Chemis y, Uni e si y o G anada, E-18071 G anada, 10 Spain 11 12 13 * Co espondence o: An onia Ga ido F enich, Depa men o Analy ical Chemis y, 14 Alme ia Uni e si y o Alme íia, 04120, Alme iía, Spain. 15 Tel: +34950015985; Fax: +34950015483; e-mail: [email p o ec ed] 16 17 1 Bo h au ho s con ibu ed equally o his wo k. 18 19 Ma ked-up manusc ip 2 20 Abs ac 21 An analy ical me hodology has been de eloped o he simul aneous ex ac ion o 22 13 phenolic compounds, including chlo ophenols (CPs), ni ophenols (NTPs), c esols 23 and alkylphenols (APs) in di e en ypes o was ewa e (WW) e luen s. A solid-24 phase ex ac ion (SPE) me hod has been op imized p io o he de e mina ion by gas 25 ch oma og aphy coupled o iple quad upole andem mass spec ome y (GC-QqQ-26 MS/MS). Due o he complexi y o he ma ix, a compa ison s udy o ma ix-ma ched-27 calib a ion (MMC) and s anda d addi ion calib a ion (SAC) was ca ied ou o 28 quan i ica ion pu poses. The op imized p ocedu e was alida ed using he SAC 29 app oach since i p o ided he mos adequa e quan i ica ion esul s (in e ms o 30 eco e y and p ecision alues). Reco e ies we e in he ange 60–135% (0.5 g L−1), 31 70–115% (1 g L−1), and 78–120% (5 g L−1), wi h p ecision alues (exp essed as 32 ela i e s anda d de ia ion, RSD) ≤ 30% (excep o 2-ni ophenol) in ol ing in a-33 day and in e -day p ecision s udies we e ob ained. Limi s o de ec ion (LODs) and 34 quan i ica ion (LOQs) we e also e alua ed, and LOQs anged om 0.03 g L-1 o 2.5 35 g L-1. The p oposed me hod was applied o o he analysis o 8 eal WW e luen 36 samples, inding some phenolic compounds (e.g. 2-chlo ophenol, 2,4,6-37 ichlo ophenol and 4- e -oc ylphenol) a concen a ions highe han he es ablished 38 LOQs es ablished du ing he me hod alida ion. 39 Keywo ds: Phenols, was ewa e , solid-phase ex ac ion (SPE), gas ch oma og aphy-40 mass spec ome y (GC-MS), s anda d addi ion calib a ion, ma ix-ma ched 41 calib a ion. 42 43 Fo ma ed: None 3 44 1. In oduc ion 45 Phenolic compounds can be ound in was ewa e (WW) e luen s ia di e en 46 sou ces. They can be de ec ed in his ype o samples because o hei use in plas ics 47 [1], d ug manu ac u ing, phy osani a y p oduc s o lea he colo ing [2], by 48 an h opogenic emission [2] o and by he use o ea men s wi h ae obic o anae obic 49 mic oo ganisms [4]. Some phenols show high oxici y, es ogenic [5] and an i-50 and ogenic ac i i y [6], andas well as hey can ac as endoc ine dis up e s [7]. 51 Phenols can be classi ied in a wide ange o amilies. The mos s udied analy es in 52 wa e a e chlo ophenols (CPs) [1] and alkylphenols (APs) [8]. Howe e , he Uni ed 53 S a es En i onmen al P o ec ion Agency (US EPA) classi ies CPs, ni ophenols and 54 APs as p io i y pollu an s [9] and i has es ablished a maximum con amina ion le el 55 (MCL) o pen achlo ophenol (PCP) o 1 µg L-1 in d inking wa e s [10]. On he o he 56 hand, he Eu opean Union (EU) has adop ed a lis o p io i y subs ances in he ield o 57 wa e policy, including 4-n-nonylphenol (4-n-NP), 4- e -oc ylphenol (4- e OP) and 58 PCP [11]. Fu he mo e, maximum allowable concen a ions (MAC) ha e been 59 es ablished o NP (2 g L-1) and PCP (1 g L-1) in inland and o he su ace wa e s 60 [12]. Howe e , i mus be poin ed ou ha legisla ion o WWs is s ill e y sca ce, and 61 he alues es ablished in d inking wa e a e usually used as guide in WWs. Bea ing in 62 mind hese ac s, he de elopmen o sensi i e analy ical me hodologies o he 63 simul aneous de e mina ion o phenols belonging o di e en g oups, such as CPs, 64 APs, ni ophenols (NTPs) and c esols (also known as me hyl-phenols) wi h di e en 65 pola i y ange (log Kow 1.77-5.01) is needed in o de o p o ide a comple e o e iew 66 o he occu ence o phenolic compounds in WW e luen s. 67 4 Se e al ex ac ion echniques ha e been applied o he ex ac ion o phenols om 68 aqueous samples, such as solid-phase ex ac ion (SPE) [8,13-15] and liquid-liquid 69 ex ac ion (LLE) [16]. Recen ly, mic oex ac ion echniques, such as solid-phase 70 mic oex ac ion (SPME) [17-19], s i ba so p i e ex ac ion (SBSE) [19-21], liquid 71 phase mic oex ac ion (LPME) [22] o dispe si e liquid-liquid mic oex ac ion 72 (DLLME) [23] ha e been appliedused. Howe e , mos o hem ha e only been used 73 o he simul aneous analysis o only one o ew phenols belonging o he same amily 74 such as APs [18,20] and CPs [17]. I is well-known ha SPE is he mos used 75 echnique in wa e analysis [24] due o he less educed exposi ionu e and 76 con amina ion by o ganic sol en s, he high p e-concen a ion ac o s ha make 77 possible o a oididing e apo a ion s eps, he semi-au oma ion o he ex ac ion 78 p ocess, educing he sample handling, and i allows he ex ac ion o compounds wi h 79 di e en physico-chemical p ope ies. The applica ion o mic oex ac ion echniques 80 is inc easing bu se e al disad an ages, such as cos and li e ime o ibe s and ba s, o 81 he limi ed scope o a wide pola i y ange can hinde hei u iliza ion. 82 83 Fo he de e mina ion o phenolic compounds, gas ch oma og aphy (GC) [13,25] o 84 liquid ch oma og aphy (LC) [26,27] a e he p edominan he mos used echniques, 85 mainly coupled o andem mass spec ome y (MS/MS) [28-31]. When GC is used, a 86 de i a iza ion s ep is equi ed in o de o imp o e he ch oma og aphic pe o mance 87 and sensi i i y o he selec ed compounds, and se e al de i a iza ing eagen s can be 88 applied [32,33]. 89 A well-known c i ical poin in he analysis o WW is he ma ix e ec [34]. In o de 90 o minimize i , di e en calib a ion me hods such as ma ix-ma ched calib a ion 91 5 (MMC) [33,35], s anda d addi ion calib a ion (SAC) [34] and he use o iso ope-92 labeled in e nal s anda ds [36,37] ha e been employed o complex ma ixes. 93 Quan i ica ion based on iso ope-labeled in e nal s anda ds has disad an ages due o 94 he expensi eness o hese s anda ds and hei limi ed a ailabili y. MMC is o en he 95 mos used quan i ica ion me hod in ace analysis. Howe e , he lack o blank 96 ma ixes and he need o s o ing hem can make his app oach logis ically one ous 97 and no necessa ily accu a e. SAC is he mos adequa e echnique o use when i is 98 di icul o ind a blank samples o he s udied ma ix s udied, bu a calib a ion se is 99 equi ed o each sample, inc easing he o al numbe o injec ions and he ime spen 100 in da a p ocessing. 101 Ano he p oblem ela ed o he de e mina ion o phenols in WW is ha depending 102 on he ype o WW ea men , WW e luen s can ha e di e en amoun s o suspended 103 pa icula e ma e (SPM). This SPM is no mally disca ded du ing he ex ac ion 104 p ocess by il a ion in mos o he analy ical me hods epo ed in li e a u e [38]. 105 Howe e , a ecen s udy [35] has demons a ed ha ce ain analy es can be e ained in 106 he SPM, depending on i s pola i y. The e o e, i should be necessa y o e alua e he 107 p esence o phenols in bo h phases in o de o de e mine whe he he SPM mus be 108 disca ded o no . 109 Fu he mo e, i mus be poin ed ou ha many a icles epo ing simul aneous 110 ex ac ion and de e mina ion o di e en classes o phenols (including APs, CPs and, 111 NTPs) in wa e [39,40] can be ound. Howe e , hey ha e been de eloped o he 112 analysis o his ype o compounds in su ace wa e , and hey a e no alid o he 113 analysis in WW samples, due o hey a e mo e complex ma ices wi h di e en 114 physico-chemical cha ac e is ics (SPM le els, o ganic ma e , e c.). 115 6 The e o e, Iin his s udy, a simul aneous SPE ex ac ion by SPE and de e mina ion 116 o di e en phenolic amilies (, namely CPs, NTPs, c esols and APs), has been 117 de eloped o WW e luen samples. In addi ion wo no el aspec s o his wo k 118 mus be poin ed ou : (i) a s udy o he p esence o phenolic compounds in he 119 SPM acco ding o he s a egy ecen ly p oposed by Ba co-Bonilla e al [35], and 120 (ii) a compa ison o MMC and SAC in o de o e alua e he bes quan i ica ion 121 s a egy o phenolic compounds in complex ma ices such as WWs. Fo ha , A 122 s udy o he p esence o phenolic compounds in he SPM has been ca ied ou 123 acco ding o he s a egy ecen ly p oposed by Ba co-Bonilla e al [35]. Due o he 124 complexi y o he ma ix and he di icul y o ob ain blank samples, a s udy o 125 quan i ica ion s udy using MMC and SAC was de eloped in o de o e alua e he bes 126 quan i ica ion s a egy. T wo di e en WW e luen s we e s udied indi idually: 127 memb ane bio eac o (MBR, low SPM con en ) and anae obic pond (ANAP, high 128 SPM con en ). The op imized SPE and quan i ica ion me hod was alida ed in bo h 129 ypes o WWs e luen samples. 130 131 2. Expe imen al 132 2.1. Chemicals and ma e ials 133 Phenolic compounds s anda ds, 2-ni ophenol (2-NTP), 4-ni ophenol (4-NTP), 2,4-134 dime hylphenol (2,4-DMP), 2-CP, 4-chlo ophenol (4-CP), 2,4-dichlo ophenol (2,4-135 diCP), 2,4,5- ichlo ophenol (2,4,5- iCP), 2,4,6- ichlo ophenol (2,4,6- iCP) and 4-136 n-NP we e ob ained om Fluka (Buchs, Swi ze land). On he o he hand, 3-137 ni ophenol (3-NTP), 4-chlo o-3-me hylphenol (4-C-3-MP), 4- e OP and PCP we e 138 supplied by Supelco (Belle on e, PA, USA). Pu i ies we e always >97%. A s anda d 139 7 solu ion (100 mg L−1) o iso opically labeled PCP ([13C6]-PCP) was used as in e nal 140 s anda d (IS) and i was ob ained om D . E hens o e (Augsbu g, Ge many). S ock 141 s anda d solu ions o indi idual compounds (wi h concen a ions anging om 200 o 142 450 mg L-1) we e p epa ed by exac weighing o he powde o liquid and dissolu ion 143 in 50 mL o ace one. These solu ions we e hen s o ed unde e ige a ion (T <5 ºC). 144 A wo king s anda d solu ion o he 13 phenolic compounds (2 mg L-1 o each 145 compound) was p epa ed by app op ia e dilu ion o he s ock solu ions wi h ace one, 146 and i was s o ed unde e ige a ion (T <5 ºC). A wo king s anda d solu ion o [13C6]-147 PCP (22 mg L-1) was p epa ed by app op ia e dilu ion o he s anda d solu ion wi h 148 ace one and s o ed unde he a o emen ioned condi ions. HPLC-g ade me hanol 149 (MeOH), anhyd ide ace ic acid (AAA) (99.9%), and py idine (Py) (99.8%) we e 150 pu chased om Sigma-Ald ich (Mad id, Spain). Ace one and hyd ochlo ic acid (HCl) 151 we e ob ained om J.T. Bake (De en e , Ne he lands). Dichlo ome hane (DCM) was 152 pu chased om Riedel-de Haën (Seelze-Hanno e , Ge many). Ul apu e wa e was 153 ob ained om a Milli-Q G adien wa e sys em (Millipo e, Bed o d, MA, USA). 154 Thi y mm cellulose il e s and 47-mm glass mic o ibe il e s om Wha man 155 (Maids one, England, UK) and 0.45-µm HNWP nylon memb ane il e s om 156 Millipo e (Ca ig wohill, Coun y Co k, I eland) we e also a ailable o il a ion 157 s ages. Fo SPE, Oasis HLB (200 mg, 6 cm3) ca idges we e ob ained om Wa e s 158 (Mil o d, MA, USA). 159 160 2.2. Appa a us 161 A GC sys em Va ian 3800 (Va ian Ins umen s, Sunny ale, CA, USA) equipped wi h 162 elec onic low con ol was in e aced o a 1200L iple quad upole (QqQ) mass 163 8 spec ome e . Samples we e injec ed in o an SPI/1079 spli /spli less p og ammed-164 empe a u e injec o using a Combi Pal (CTC Analy ics AG, Zwingen, Swi ze land) 165 wi h a 100-µL sy inge. A used-silica un ea ed capilla y column (2 m × 0.25 mm i.d.) 166 om Supelco was used as p e-column connec ed o a VF-5 ms Fac o Fou capilla y 167 column (30 m × 0.25 mm i.d. × 0.25 µm ilm hickness) pu chased om Va ian. 168 Helium was used as ca ie gas (99.9999%) a a cons an low a e o 1 mL min−1, and 169 a gon (99.999%) was used as collision gas. The mass spec ome e was ope a ed in 170 elec on ioniza ion (EI) mode a 70 eV. The mass spec ome e was calib a ed e e y 171 ou days wi h pe luo o ibu ylamine. Va ian Wo ks a ion so wa e was used o 172 ins umen con ol and da a analysis. 173 A Reax-2 o a y agi a o om Heidolph (Schwabach, Ge many) was used o 174 agi a ion o he de i a iza ion mix u e. An analy ical balance AB204-S om Me le 175 Toledo (G ei ensee, Swi ze land) and a o a y e apo a o R-114 (Büchi, Flawil, 176 Swi ze land) we e used du ing ex ac ion and s anda d p epa a ion. The ho izon al 177 shake used in he dis ibu ion s udy was ob ained om P-Selec a (Selec a, Ba celona, 178 Spain). 179 180 2.3. Sampling 181 WW u ban e luen s om wo di e en ea men s, namely, MBR and ANAP, wi h 182 low and high SPM con en espec i ely, we e collec ed om WW ea men plan 183 (WWTP) o he ounda ion Cen e o New Wa e Technologies (“Cen o de las 184 Nue as Tecnologías del Agua”, CENTA, Se ille, Spain). This WWTP has 41,000 m2 185 and i cu en ly holds mo e han 20 sys ems wi h di e en echnologies. Addi ional 186 physicochemical da a ela ed o he ea men s e alua ed in his s udy can be ound in 187 15 Reco e y and p ecision we e e alua ed using bo h quan i ica ion app oaches. I can 329 be obse ed ha in WW e luen s wi h high SPM, such as ANAP, MMC did no 330 p o ided adequa e esul s o he lowe spiked concen a ions (0.5 and 1 g L-1). 331 Reco e ies and in a and in e -day p ecision o mos o compounds we e below 60% 332 and o e 30%, espec i ely o hese wo concen a ion le els. On he con a y, o 5 333 g L-1, eco e y alues we e in he ange 60-120%, excep o 4-n-NP (51%) and 334 in a and in e -day p ecision we e <12%. These esul s (Table 2) sugges ed ha MMC 335 is no a sui able op ion o he adequa e quan i ica ion o a e y low concen a ions o 336 phenols in WWs e luen s wi h high SPM. On he o he hand, when SAC was used, 337 eco e ies o all compounds we e in ange 60-125%, excep o 4- e OP (135%) a 338 he lowes o i ica ion le el (0.5 g L-1). In a and in e -day p ecision alues we e 339 <27% and <31% o all compounds, espec i ely. As i is shown in Table 2, he SAC 340 app oach is mo e app op ia e o WW e luen s wi h high SPM con en . Linea i y was 341 s udied in he ange 10-150 g L-1 (excep o NTPs which was 100-300 g L-1) and 342 he ob ained de e mina ion coe icien s (R2) we e in he ange 0.9912 (3-NTP)-0.9999 343 (2-CP, 2,4,5- T iCP, PCP and 4-n-NP a ious compounds) o ANAP (Table 4). 344 Fo WW e luen s wi h low SPM, such as MBR (Table 3), he eco e ies ob ained 345 when MMC was used o he h ee le els assayed anged om 62-119%, excep o 4-346 n-NP, wi h eco e ies lowe han 56%. Despi e he adequa e eco e y esul s p o ided 347 by MMC o all he s udied o i ica ion le els, in gene al, RSD alues we e <30% 348 only o he highes spiked le el s udied (5 g L-1), as i can be obse ed in Table 3, 349 whe eas a he lowes concen a ion le els e alua ed (0.5 and 1 g L-1), in a and 350 in e -day p ecision anged om 22 o 113%. On he o he hand, he applica ion o 351 SAC on MBR WW samples yielded eco e y alues in he ange 70-120%, excep o 352 4-CP (125%) a 0.5 g L-1. Besides, RSD alues we e always <28% o in a-day 353 16 p ecision and <27% o in e -day p ecision in all cases, excep o 2-NTP, which was 354 41% a 0.5 g L-1. In consequence, i can be concluded ha o MBR ea ed WW 355 e luen s, SAC was also he mos sui able me hod o an adequa e quan i ica ion o 356 WW e luen s wi h low SPM con en , such as MBR WW samples (Table 3). 357 Fu he mo e, linea i y was also e alua ed o MBR and R2 alues anged om 0.9943 358 (4-NTP) o 0.9999 (2-CP, 4-CP, 2,4,6- iCP and 4-n-NP). 359 Conside ing hese esul s, he SAC me hod should be applied o a eliable 360 quan i ica ion o phenols in WW e luen s samples o compensa e ma ix e ec s on 361 he signal a ia ion du ing de ec ion and his does no depend on he SPM con en o 362 he WW. The SAC me hodology was he e o e applied o he quan i ica ion o 363 phenols in eal samples. 364 365 3.4. Es ima ion o he lowe limi s o he me hodology 366 Despi e o he es ima ion o he ueness and p ecision ca ied ou in he p e ious 367 sec ion, o he pe o mance cha ac e is ics o he me hod, such as limi s o de ec ion 368 (LODs) and quan i ica ion (LOQs) we e s udied. LODs and LOQs we e de e mined 369 as he lowes concen a ion le el ha yielded a signal- o-noise (S/N) a io o 3 and 10, 370 and hey a e shown in Table 4. LODs and LOQs we e de e mined in WW sample 371 blanks o each phenolic compound s udied. LODs we e om 0.01 o 1 g L-1 and 372 LOQs anged om 0.03 g L-1 o 2.5 g L-1 o ANAP and MBR (Table 4). I mus 373 be no iced ha simila alues we e ob ained o bo h ypes o WW e luen s, excep 374 o 2-NTP and 4-C-3-MP, which showed highe LOD and LOQ alues in ANAP han 375 in MBR. This could be explained aking in o accoun ha he SPM con en is highe 376 17 in ANAP, inc easing he amoun o co-ex ac ed ma e ial and a ec ing he es ima ion 377 o he lowe limi s o he me hod. 378 379 3.5. Applica ion o he analysis o eal WW e luen samples 380 The de eloped me hodology was applied o he analysis o 8 WW e luen samples 381 om he CENTA, ob ained a e he applica ion o di e en WW ea men s 382 employed in his WWTP. To assu e he quali y o he esul s and a oid e o s, he 383 quan i ica ion o he phenolic compounds was achie ed using he SAC app oach. An 384 in e nal quali y con ol (IQC) was pe o med consis ing o he analysis o spiked 385 blank WW samples a 1 g L-1 (excep o 3-NTP and 4-NTP a 5 g L-1), which we e 386 used o assess he ex ac ion e iciency and a SAC calib a ion cu e o check linea i y 387 and sensi i i y. Se e al phenolic compounds we e ound o e he LOQs es ablished 388 by he me hod, showing he ob ained esul s in Table 5. 2-CP and 2,4,6- iCP we e 389 ound in six and i e samples, espec i ely, wi h concen a ions anging om 0.04 o 390 0.20 g L-1 o 2-CP and om 0.05 o 0.10 g L-1 o 2,4,6- iCP. 4-CP and 4- e OP 391 we e ound in ou samples, and he concen a ions anged om 0.04 o 0.08 g L-1 392 and 0.04 o 0.16 g L-1 espec i ely. 2-CP, 2,4-DiCP, 4- e OP, PCP and 4-n-NP we e 393 ound simul aneously in one o he samples (Table 5). I mus be highligh ed ha 394 phenolic compounds we e no ound o e he MCLs and MACs es ablished by he 395 EPA and he EU o hese compounds [10,12]. Finally, Figu e 4 shows a posi i e 396 sample o 4- e OP de ec ed in a WW e luen sample a 0.12 g L-1. 397 398 5. Conclusions 399 18 A single ex ac ion me hod o he simul aneous ex ac ion o CPs, APs, NTPs and 400 c esols in WW e luen samples has been de eloped using SPE. A dis ibu ion s udy 401 o he phenolic compounds be ween he aqueous phase and he SPM was ca ied ou , 402 e i ying ha he SPM could be in ac disca ded du ing he ex ac ion since only 403 phenolic compounds wi h high log Kow we e ound in he SPM a a negligible 404 pe cen age. Due o he di icul y o ind WW blank samples and o ha e good 405 accu acy in he quan i ica ion, a s udy using MMC e sus SAC was pe o med in wo 406 di e en ea ed WW e luen samples (ANAP and MBR) showing ha SAC is he 407 mos sui able quan i ica ion app oach. The me hod was alida ed s udying eco e y, 408 in a and in e -day p ecision, lowe limi s (LODs and LOQs) and linea i y. 409 De e mina ion o he analy es was ca ied ou using GC-QqQ-MS/MS ope a ing in 410 SRM mode. The me hod was applied o WW e luen samples wi h sa is ac o y 411 esul s, obse ing ha phenols o se e al amilies we e simul aneously de ec ed in 412 WW e luen s, highligh ing he po en ial o analy ical me hods ha allows he 413 simul aneous de e mina ion o se e al classes o phenolic compounds. 414 415 Acknowledgmen s 416 The au ho s g a e ully acknowledge he Andalusian Regional Go e nmen (Regional 417 Minis y o Inno a ion, Science, and En e p ise-FEDER) o inancial suppo 418 (P ojec Re . P08-RNM-03892). PPB acknowledges o pe sonal unding h ough 419 Juan de la Cie a P og am (Spanish Minis y o Science and Inno a ion-Eu opean 420 Social Fund). RRG is also g a e ul o pe sonal unding h ough Ramón y Cajal 421 P og am (Spanish Minis y o Science and Inno a ion-Eu opean Social Fund). NBB 422 is g a e ul o he p e-doc o al g an om he a o emen ioned p ojec . CENTA is 423 g a e ully acknowledged o p o iding WWs e luen s samples. 424 19 425 426 20 Re e ences 427 [1] S. Eke , K.J. Fik e , En i on. 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Compa ison o he eco e y alues ob ained applying di e en elu ion sol en s 507 o he ex ac ion o spiked WW samples a 0.5 g L-1. Abb e ia ions: DCM: 508 dichlo ome hane; Sequen ial: sequen ial elu ion. 509 Fig. 2. To al ion ch oma og am (TIC) o an ex ac ed spiked WW sample (5 g L-1) 510 ob ained by GC-QqQ-MS/MS. Fo compound ab e ia ions, see Table 1. 511 Fig. 3. Calib a ion cu es in he ange 10-150 g L-1 o 4- e OP when SAC and 512 MMC we e used: a) ANAP; b) MBR. Abb e ia ions: ANAP: anae obic pond; MBR: 513 memb ane bio eac o ; MMC: ma ix-ma ched calib a ion; SAC: s anda d addi ion 514 calib a ion; 4- e OP: 4- e oc ylphenol 515 Fig. 4. Selec ed- eac ion moni o ing (SRM) (a) ch oma og am and (b) MS/MS 516 spec um o 4- e OP (0.12 g L-1) ound in a eal WW sample and (c) SRM 517 ch oma og am and (d) MS/MS spec um o a SAC s anda d (50 g L-1). 518 519 520 521 3 1. In oduc ion 39 Phenolic compounds can be ound in was ewa e (WW) e luen s ia di e en 40 sou ces. They can be de ec ed in his ype o samples because o hei use in plas ics 41 [1], d ug manu ac u ing, phy osani a y p oduc s o lea he colo ing [2], by 42 an h opogenic emission [2] and by he use o ea men s wi h ae obic o anae obic 43 mic oo ganisms [4]. Some phenols show high oxici y, es ogenic [5] and an i- 44 and ogenic ac i i y [6], and hey can ac as endoc ine dis up e s [7]. 45 Phenols can be classi ied in a wide ange o amilies. The mos s udied analy es in 46 wa e a e chlo ophenols (CPs) [1] and alkylphenols (APs) [8]. Howe e , he Uni ed 47 S a es En i onmen al P o ec ion Agency (US EPA) classi ies CPs, ni ophenols and 48 APs as p io i y pollu an s [9] and i has es ablished a maximum con amina ion le el 49 (MCL) o pen achlo ophenol (PCP) o 1 µg L-1 in d inking wa e s [10]. On he o he 50 hand, he Eu opean Union (EU) has adop ed a lis o p io i y subs ances in he ield o 51 wa e policy, including 4-n-nonylphenol (4-n-NP), 4- e -oc ylphenol (4- e OP) and 52 PCP [11]. Fu he mo e, maximum allowable concen a ions (MAC) ha e been 53 es ablished o NP (2 g L-1) and PCP (1 g L-1) in inland and o he su ace wa e s 54 [12]. Howe e , i mus be poin ed ou ha legisla ion o WWs is s ill e y sca ce, and 55 he alues es ablished in d inking wa e a e usually used as guide in WWs. Bea ing in 56 mind hese ac s, he de elopmen o sensi i e analy ical me hodologies o he 57 simul aneous de e mina ion o phenols belonging o di e en g oups, such as CPs, 58 APs, ni ophenols (NTPs) and c esols (also known as me hyl-phenols) wi h di e en 59 pola i y ange (log Kow 1.77-5.01) is needed in o de o p o ide a comple e o e iew 60 o he occu ence o phenolic compounds in WW e luen s. 61 4 Se e al ex ac ion echniques ha e been applied o he ex ac ion o phenols om 62 aqueous samples, such as solid-phase ex ac ion (SPE) [8,13-15] and liquid-liquid 63 ex ac ion (LLE) [16]. Recen ly, mic oex ac ion echniques, such as solid-phase 64 mic oex ac ion (SPME) [17-19], s i ba so p i e ex ac ion (SBSE) [19-21], liquid 65 phase mic oex ac ion (LPME) [22] o dispe si e liquid-liquid mic oex ac ion 66 (DLLME) [23] ha e been applied. Howe e , mos o hem ha e been used o he 67 simul aneous analysis o only one o ew phenols belonging o he same amily such 68 as APs [18,20] and CPs [17]. I is well-known ha SPE is he mos used echnique in 69 wa e analysis [24] due o he educed exposi ion and con amina ion by o ganic 70 sol en s, he high p e-concen a ion ac o s a oiding e apo a ion s eps, he semi- 71 au oma ion o he p ocess, and i allows he ex ac ion o compounds wi h di e en 72 physico-chemical p ope ies. The applica ion o mic oex ac ion echniques is 73 inc easing bu se e al disad an ages, such as cos and li e ime o ibe s and ba s, o 74 he limi ed scope o a wide pola i y ange can hinde hei u iliza ion. 75 Fo he de e mina ion o phenolic compounds, gas ch oma og aphy (GC) [13,25] o 76 liquid ch oma og aphy (LC) [26,27] a e he p edominan echniques, mainly coupled 77 o andem mass spec ome y (MS/MS) [28-31]. When GC is used, a de i a iza ion 78 s ep is equi ed in o de o imp o e he ch oma og aphic pe o mance and sensi i i y 79 o he selec ed compounds, and se e al de i a iza ing eagen s can be applied [32,33]. 80 A well-known c i ical poin in he analysis o WW is ma ix e ec [34]. In o de o 81 minimize i , di e en calib a ion me hods such as ma ix-ma ched calib a ion (MMC) 82 [33,35], s anda d addi ion calib a ion (SAC) [34] and he use o iso ope-labeled 83 in e nal s anda ds [36,37] ha e been employed o complex ma ixes. Quan i ica ion 84 based on iso ope-labeled in e nal s anda ds has disad an ages due o he 85 expensi eness o hese s anda ds and hei limi ed a ailabili y. MMC is o en used 86 5 quan i ica ion me hod in ace analysis. Howe e , he lack o blank ma ixes and he 87 need o s o ing hem can make his app oach logis ically one ous and no necessa ily 88 accu a e. SAC is he mos adequa e echnique o use when i is di icul o ind blank 89 samples o he s udied ma ix, bu a calib a ion se is equi ed o each sample, 90 inc easing he o al numbe o injec ions and he ime spen in da a p ocessing. 91 Ano he p oblem ela ed o he de e mina ion o phenols in WW is ha depending 92 on he ype o WW ea men , WW e luen s can ha e di e en amoun s o suspended 93 pa icula e ma e (SPM). This SPM is no mally disca ded du ing he ex ac ion 94 p ocess by il a ion in mos o he analy ical me hods epo ed in li e a u e [38]. 95 Howe e , a ecen s udy [35] has demons a ed ha ce ain analy es can be e ained in 96 he SPM, depending on i s pola i y. The e o e, i should be necessa y o e alua e he 97 p esence o phenols in bo h phases in o de o de e mine whe he he SPM mus be 98 disca ded o no . 99 Fu he mo e, i mus be poin ed ou ha many a icles epo ing simul aneous 100 ex ac ion and de e mina ion o di e en classes o phenols (including APs, CPs and, 101 NTPs) in wa e [39,40] can be ound. Howe e , hey ha e been de eloped o he 102 analysis o his ype o compounds in su ace wa e , and hey a e no alid o he 103 analysis in WW samples, due o hey a e mo e complex ma ices wi h di e en 104 physico-chemical cha ac e is ics (SPM le els, o ganic ma e , e c.). 105 The e o e, in his s udy, a simul aneous SPE ex ac ion and de e mina ion o 106 di e en phenolic amilies (CPs, NTPs, c esols and APs) has been de eloped o WW 107 e luen samples. In addi ion wo no el aspec s o his wo k mus be poin ed ou : 108 (i) a s udy o he p esence o phenolic compounds in he SPM acco ding o he 109 s a egy ecen ly p oposed by Ba co-Bonilla e al [35], and (ii) a compa ison o 110 6 MMC and SAC in o de o e alua e he bes quan i ica ion s a egy o phenolic 111 compounds in complex ma ices such as WWs. Fo ha , wo di e en WW 112 e luen s we e s udied indi idually: memb ane bio eac o (MBR, low SPM con en ) 113 and anae obic pond (ANAP, high SPM con en ). The op imized SPE and 114 quan i ica ion me hod was alida ed in bo h ypes o WWs e luen samples. 115 116 2. Expe imen al 117 2.1. Chemicals and ma e ials 118 Phenolic compounds s anda ds, 2-ni ophenol (2-NTP), 4-ni ophenol (4-NTP), 2,4- 119 dime hylphenol (2,4-DMP), 2-CP, 4-chlo ophenol (4-CP), 2,4-dichlo ophenol (2,4- 120 diCP), 2,4,5- ichlo ophenol (2,4,5- iCP), 2,4,6- ichlo ophenol (2,4,6- iCP) and 4- 121 n-NP we e ob ained om Fluka (Buchs, Swi ze land). On he o he hand, 3- 122 ni ophenol (3-NTP), 4-chlo o-3-me hylphenol (4-C-3-MP), 4- e OP and PCP we e 123 supplied by Supelco (Belle on e, PA, USA). Pu i ies we e always >97%. A s anda d 124 solu ion (100 mg L−1) o iso opically labeled PCP ([13C6]-PCP) was used as in e nal 125 s anda d (IS) and i was ob ained om D . E hens o e (Augsbu g, Ge many). S ock 126 s anda d solu ions o indi idual compounds (wi h concen a ions anging om 200 o 127 450 mg L-1) we e p epa ed by exac weighing o he powde o liquid and dissolu ion 128 in 50 mL o ace one. These solu ions we e hen s o ed unde e ige a ion (T <5 ºC). 129 A wo king s anda d solu ion o he 13 phenolic compounds (2 mg L-1 o each 130 compound) was p epa ed by app op ia e dilu ion o he s ock solu ions wi h ace one, 131 and i was s o ed unde e ige a ion (T <5 ºC). A wo king s anda d solu ion o [13C6]- 132 PCP (22 mg L-1) was p epa ed by app op ia e dilu ion o he s anda d solu ion wi h 133 ace one and s o ed unde he a o emen ioned condi ions. HPLC-g ade me hanol 134 7 (MeOH), anhyd ide ace ic acid (AAA) (99.9%), and py idine (Py) (99.8%) we e 135 pu chased om Sigma-Ald ich (Mad id, Spain). Ace one and hyd ochlo ic acid (HCl) 136 we e ob ained om J.T. Bake (De en e , Ne he lands). Dichlo ome hane (DCM) was 137 pu chased om Riedel-de Haën (Seelze-Hanno e , Ge many). Ul apu e wa e was 138 ob ained om a Milli-Q G adien wa e sys em (Millipo e, Bed o d, MA, USA). 139 Thi y mm cellulose il e s and 47-mm glass mic o ibe il e s om Wha man 140 (Maids one, England, UK) and 0.45-µm HNWP nylon memb ane il e s om 141 Millipo e (Ca ig wohill, Coun y Co k, I eland) we e also a ailable o il a ion 142 s ages. Fo SPE, Oasis HLB (200 mg, 6 cm3) ca idges we e ob ained om Wa e s 143 (Mil o d, MA, USA). 144 145 2.2. Appa a us 146 A GC sys em Va ian 3800 (Va ian Ins umen s, Sunny ale, CA, USA) equipped wi h 147 elec onic low con ol was in e aced o a 1200L iple quad upole (QqQ) mass 148 spec ome e . Samples we e injec ed in o an SPI/1079 spli /spli less p og ammed- 149 empe a u e injec o using a Combi Pal (CTC Analy ics AG, Zwingen, Swi ze land) 150 wi h a 100-µL sy inge. A used-silica un ea ed capilla y column (2 m × 0.25 mm i.d.) 151 om Supelco was used as p e-column connec ed o a VF-5 ms Fac o Fou capilla y 152 column (30 m × 0.25 mm i.d. × 0.25 µm ilm hickness) pu chased om Va ian. 153 Helium was used as ca ie gas (99.9999%) a a cons an low a e o 1 mL min−1, and 154 a gon (99.999%) was used as collision gas. The mass spec ome e was ope a ed in 155 elec on ioniza ion (EI) mode a 70 eV. The mass spec ome e was calib a ed e e y 156 ou days wi h pe luo o ibu ylamine. Va ian Wo ks a ion so wa e was used o 157 ins umen con ol and da a analysis. 158 8 A Reax-2 o a y agi a o om Heidolph (Schwabach, Ge many) was used o 159 agi a ion o he de i a iza ion mix u e. An analy ical balance AB204-S om Me le 160 Toledo (G ei ensee, Swi ze land) and a o a y e apo a o R-114 (Büchi, Flawil, 161 Swi ze land) we e used du ing ex ac ion and s anda d p epa a ion. The ho izon al 162 shake used in he dis ibu ion s udy was ob ained om P-Selec a (Selec a, Ba celona, 163 Spain). 164 165 2.3. Sampling 166 WW u ban e luen s om wo di e en ea men s, namely, MBR and ANAP, wi h 167 low and high SPM con en espec i ely, we e collec ed om WW ea men plan 168 (WWTP) o he ounda ion Cen e o New Wa e Technologies (“Cen o de las 169 Nue as Tecnologías del Agua”, CENTA, Se ille, Spain). This WWTP has 41000 m2 170 and i cu en ly holds mo e han 20 sys ems wi h di e en echnologies. Addi ional 171 physicochemical da a ela ed o he ea men s e alua ed in his s udy can be ound in 172 [35]. WW e luen samples we e s o ed a 4 ºC and p ocessed wi hin 5 days a e 173 collec ion. In he MMC expe imen s, and due o he di icul y o inding WW e luen 174 blank samples, he co esponding signal o he blank was emo ed om he MMC 175 plo in hose samples whe e analy e signal was obse ed. 176 177 2.4. Dis ibu ion s udy 178 Non- il e ed WW e luen samples we e spiked wi h 0.5 µg L-1 o he s udied 179 phenolic compounds, and hen, hey we e agi a ed o e nigh a a a e o 100 180 oscilla ions pe min o allow a ho oughly in e ac ion be ween he analy es and bo h 181 9 phases o WW (aqueous phase and SPM). A e his, samples we e il e ed o sepa a e 182 and analyze bo h phases. The aqueous phase was ex ac ed by SPE, whe eas o he 183 analysis o he SPM, a me hod de eloped by Padilla-Sánchez e al. [33] o he 184 ex ac ion o phenolic compounds in ag icul u al soils was employed. The dis ibu ion 185 o he compounds be ween bo h phases was de e mined as he pe cen age o hem 186 p esen in each phase. 187 188 2.5. GC-QqQ-MS/MS 189 Aliquo s o 10 µL we e injec ed in o he GC sys em ope a ing a a sy inge injec ion 190 low a e o 10 µL s−1. The injec o empe a u e p og am was as ollows: 70 ºC (hold 191 o 0.5 min) → 310 ºC (100 ºC min−1, hold o 10 min). The injec o spli a io was 192 ini ially se a 10:1. Spli less mode was swi ched on a 0.5 min un il 3.5 min. A 3.5 193 min, he spli a io was 100:1 and a 10 min, 20:1. The column o en p og am was as 194 ollows: 70 ºC (hold o 3.5 min) → 300 ºC (20 ºC min−1) → 300 ºC (hold 4 min). 195 C yogenic cooling wi h CO2 was applied when he injec o empe a u e was 170 ºC. 196 The o al unning ime was 19 min. 197 The QqQ mass spec ome e was mainly ope a ed in he selec ed eac ion 198 moni o ing (SRM) mode, al hough selec ing ion moni o ing (SIM) mode was also 199 used o con i ma ion pu poses. The elec on mul iplie was se +200 V abo e he 200 op imal alue indica ed by he so wa e ins umen . The empe a u es o he ans e 201 line, mani old and ioniza ion sou ce we e se a 300, 40 and 265 °C, espec i ely. The 202 op imal alues o he scan ime anged om 0.132 o 0.240 s. Peak wid hs o m/z 2.0 203 and 1.5 we e se in he i s (Q1) and hi d quad upole (Q3), espec i ely. The 204 op imized MS/MS pa ame e s a e indica ed in Table 1. 205 10 2.6. SPE ex ac ion and de i a iza ion p ocedu e 206 WW e luen samples we e il e ed consecu i ely (250 mL) using wo di e en po e- 207 size il e s (47-mm glass mic o ibe il e s and 0.45-µm nylon memb ane il e s). The 208 il e ed WW e luen s showed pH alues be ween 7.7 and 8.3. Then, pH was adjus ed 209 o 2.5-2.7 wi h HCl (2 M) o ensu e he p o ona ed o m o he phenolic compounds, 210 acili a ing he abso p ion in o he solid phase, and an adequa e p ese a ion o he 211 samples. The Oasis HLB ca idges we e condi ioned wi h 5 mL o ace one ollowed 212 by 5 mL o MeOH and 3 x 5 mL o ul apu e wa e wi hou allowing he ca idges o 213 d y ou . Then, he il e ed WW sample (250 mL) was passed h ough he ca idges 214 unde acuum a a low a e o 10 mL min-1. The ca idges we e d ied o 2 h and he 215 phenolic compounds we e elu ed sequen ially wi h 3 mL o ace one and 2 mL o 216 DCM. The ex ac s we e collec ed in o 5-mL olume ic lasks, adjus ing he o al 217 olume wi h DCM, wi hou any e apo a ion s ep. Then, he de i a iza ion s age was 218 pe o med acco ding o he p ocedu e desc ibed by Padilla-Sánchez e al. [33]. 219 B ie ly, 860 µL o he ex ac we e ans e ed o a 2-mL ial and 20 µL o [13C6]- 220 PCP (IS), 20 µL o Py and 100 µL o AAA we e added o ca y ou he de i a iza ion 221 eac ion. The mix u e was shaking in a o a y agi a o o 2 min and hen injec ed 222 di ec ly o he GC-QqQ-MS/MS sys em. 223 224 3. Resul s and discussion 225 WWs can be submi ed o di e en ea men s, ob aining e luen s wi h a a ie y o 226 SPM con en s, and hus, WW e luen s can p esen di e en physico-chemical 227 p ope ies. When an analy ical me hod is de eloped o his ype o samples, his 228 di e si y should be aken in o accoun . In o de o co e a wide ange o WW 229 11 e luen s, wo ypes o hem we e e alua ed, MBR and ANAP, which ha e low and 230 high SPM con en [35], espec i ely. The op imiza ion o he ex ac ion p ocedu e as 231 well as he quan i ica ion me hods, we e e alua ed in bo h ypes o WW e luen s. Fo 232 ha pu pose, a GC-QqQ-MS/MS me hod ecen ly de eloped [33] was applied. 233 234 3.1. Ex ac ion me hod 235 Fo he op imiza ion o he SPE p ocedu e, a me hodology epo ed by Po hi ou e al. 236 [8] was i s conside ed. This s udy epo ed he de e mina ion o only one amily o 237 phenolic compounds, APs, using Oasis HLB ca idges and ace one as elu ion sol en . 238 Besides, ce ain p oblems ega ding he e apo a ion s ages ha e been p e iously 239 epo ed [33], and he e o e, he ex ac ion me hod was designed wi hou any 240 e apo a ion s ep. Since he amilies o phenolic compounds included in his s udy 241 showed a wide pola i y ange, se e al elu ion sol en s we e es ed o achie e a 242 simul aneous ex ac ion [8]. Ace one (5 mL), DCM (5 mL) and a sequen ial elu ion 243 wi h ace one (3 mL) and DCM (2 mL) we e es ed. Bea ing in mind ha e apo a ion 244 s eps we e no included in he ex ac ion p ocedu e and aliquo s o he ex ac s a e 245 di ec ly injec ed in he ch oma og aphic sys em, he elu ion sol en could be pa ially 246 e ained in he solid phase o e apo a ed du ing he elu ion s ep. This can p o oke an 247 o e es ima ion o he inal concen a ion in ela ion o he heo e ical alue, ob aining 248 high eco e y alues. In o de o a oid his, 5-mL olume ic lasks we e used o 249 collec he ex ac s and he inal olume was adjus ed o 5 mL wi h he co esponding 250 sol en used du ing he elu ion s ep. The ob ained esul s a e shown in Figu e 1 and i 251 can be obse ed ha ace one p o ided adequa e esul s o all compounds, excep o 252 2,4-dMP and 4-n-NP. When DCM was used, eco e ies highe han 120% we e 253 12 ob ained o 2-CP, 2-NTP and 4-CP, al hough eco e y o 4-n-NP was imp o ed. 254 Consequen ly, in o de o ob ain good eco e ies o all he compounds, a sequen ial 255 elu ion wi h ace one and DCM was es ed. In gene al, his elu ion imp o ed he 256 eco e y a es, especially o 2,4-dMP, 2-CP, 2-NTP and 4-CP. None heless, 257 eco e ies be ween 50-60% may be accep ed ex ao dina ily in en i onmen al 258 analysis whene e he p ecision alues a e adequa e (<30%). The e o e, u he 259 expe imen s we e ca ied ou using he sequen ial elu ion wi h ace one (3 mL) and 260 DCM (2 mL) as elu ion sol en s. Finally, a o al ion ch oma og am (TIC) o an 261 ex ac ed spiked WW sample a 50 g L-1 is showed in Figu e 2. 262 263 3.2. Dis ibu ion s udy 264 Once he ex ac ion me hod was op imized o he analysis o he aqueous phase o 265 WW e luen samples, a dis ibu ion s udy is needed o e i y whe he he phenolic 266 compounds a e also p esen in he SPM. I phenolic compounds a e p esen 267 quan i a i ely in he SPM, he analysis o WW e luen s should no be limi ed o he 268 aqueous phase. The dis ibu ion s udy was he e o e ca ied ou , applying he 269 app oach desc ibed in Sec ion 2.4 o bo h ype o samples. I was obse ed ha only 270 he phenolic compounds wi h high log Kow we e ound in he SPM, bu a negligible 271 pe cen ages (<5%). On he con a y, phenolic compounds wi h lowe log Kow whe e 272 no ound in he SPM (da a no shown). Taking in o accoun his esul , u he 273 expe imen s we e limi ed o he analysis o he a ge analy es in he aqueous phase, 274 disca ding he SPM phase. These esul s a e in acco dance wi h a p e ious s udy [35] 275 epo ing ha pola compounds we e no e ained in he SPM. 276 277 19 Re e ences 405 [1] S. Eke , K.J. Fik e , En i on. Manage. 90 (2009) 692-698 406 [2] M. Conce a-Tomei, M. C is ina-Annesini, En i on. Sci. Technol. 39 (2005) 5059- 407 5065. 408 [3] C. Li, S. Taneda, A.K. Suzuki, C. Fu u a, G. Wa anabe, K. Taya, Toxicol. Appl. 409 Pha m. 217 (2006) 1–6. 410 [4] D. Li, J. Pa k, J.R. Oh, Anal. Chem. 73 (2001) 3089-3095. 411 [5] M. Solé, M.J. López-De-Alda, M. Cas illo, C. Po e, K. Ladegaa d-Pede sen, D. 412 Ba celó, En i on. Sci. 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Calib a ion cu es in he ange 10-150 g L-1 o 4- e OP when SAC and 489 MMC we e used: a) ANAP; b) MBR. Abb e ia ions: ANAP: anae obic pond; MBR: 490 memb ane bio eac o ; MMC: ma ix-ma ched calib a ion; SAC: s anda d addi ion 491 calib a ion; 4- e OP: 4- e oc ylphenol 492 Fig. 4. Selec ed- eac ion moni o ing (SRM) (a) ch oma og am and (b) MS/MS 493 spec um o 4- e OP (0.12 g L-1) ound in a eal WW sample and (c) SRM 494 ch oma og am and (d) MS/MS spec um o a SAC s anda d (50 g L-1). 495 496 497 498 Depa amen o de Hid ogeología y Química Analí ica C a . S a c a m e n o L a C a ñ a d a d e S a n U b a n o 0 4 1 2 0 A l m e í a ( E s p a ñ a ) T e l . : 9 5 0 0 1 5 4 8 3 F A X : 9 5 0 0 1 5 4 8 3 w w w . u a l . e s Alme ia, 12 July 2011 Dea Edi o : Please, ind enclosed he e ised e sion o he manusc ip en i led “Simul aneous analysis o chlo ophenols, alkylphenols, ni ophenols and c esols in was ewa e e luen s, using solid phase ex ac ion and u he de e mina ion by gas ch oma og aphy– andem mass spec ome y”, Manusc ip No. TAL-D-11-00529. We e ised ou manusc ip aking in o accoun he e iewe ’s commen s (changes a e indica ed by using “T ack Changes”). Al hough we ied o cla i y he e iewe ’s commen s, we ha e conside ed ha i was also necessa y o indica e some aspec s, which can be seen in he documen uploaded as “Response o Re iewe s”. I hope ha he e iewing p ocess inds he e ised manusc ip accep able o publica ion in he jou nal. You s Since ely, P o . An onia Ga ido F enich Co e Le e Figu e(s) Click he e o download high esolu ion image Figu e(s) Click he e o download high esolu ion image Figu e(s) Click he e o download high esolu ion image Figu e(s) Click he e o download high esolu ion image