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

Elsevier Editorial System(tm) for Talanta Manuscript Draft Manuscript Number: TAL-D-11-00529R1 Title: Simultaneous analysis of chlorophenols, alkylphenols, nitrophenols and cresols in wastewater effluents, using solid phase extraction and further determination by gas chromatography-tandem mass spectrometry Article Type: Full Length Article Keywords: Phenols; Wastewater; Solid-phase extraction (SPE); Gas chromatography-mass spectrometry (GC-MS); Standard addition calibration; Matrix-matched calibration. Corresponding Author: Dr. A. Garrido Frenich, Ph.D Corresponding Author's Institution: Research Group Analytical Chemistry of Contaminants First Author: Juan Antonio Padilla-Sánchez Order of Authors: Juan Antonio Padilla-Sánchez; Patricia Plaza-Bolaños; Roberto Romero-González; Nieves Barco-Bonilla; José Luis Martínez-Vidal; A. Garrido Frenich, Ph.D Abstract: An analytical methodology has been developed for the simultaneous extraction of 13 phenolic compounds, including chlorophenols (CPs), nitrophenols (NTPs), cresols and alkylphenols (APs) in different types of wastewater (WW) effluents. A solid-phase extraction (SPE) method has been optimized prior to the determination by gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS). Due to the complexity of the matrix, a comparison study of matrix-matched-calibration (MMC) and standard addition calibration (SAC) was carried out for quantification purposes. The optimized procedure was validated using the SAC approach since it provided the most adequate quantification results (in terms of recovery and precision values). Recoveries were in the range 60- - and 78with precision values (expressed as relative standard deviation, RSD) ≤ 30% (except for 2-nitrophenol) involving intra-day and inter-day precision studies. Limits of detection (LODs) and quantification (LOQs) were also evaluated, an - -1. The proposed method was applied to the analysis of 8 real WW effluent samples, finding some phenolic compounds (e.g. 2chlorophenol, 2,4,6-trichlorophenol and 4-tert-octylphenol) at concentrations higher than the established LOQs. 1 Simultaneous analysis of chlorophenols, alkylphenols, nitrophenols and cresols 1 in wastewater effluents, using solid phase extraction and further determination 2 by gas chromatography–tandem mass spectrometry 3 4 J. A. Padilla-Sáncheza,1, P. Plaza-Bolañosa,b, R. Romero-Gonzáleza, N. Barco-5 Bonillaa, J. L. Martínez-Vidala, N. Barco-Bonillaa,1, A. Garrido-Frenicha* 6 7 aDepartment of Analytical Chemistry, University of Almeria, Carretera Sacramento 8 s/n, E-04071 Almeria, Spain 9 bDepartment of Analytical Chemistry, University of Granada, E-18071 Granada, 10 Spain 11 12 13 * Correspondence to: Antonia Garrido Frenich, Department of Analytical Chemistry, 14 Almeria University of Almeríia, 04120, Almeriía, Spain. 15 Tel: +34950015985; Fax: +34950015483; e-mail: [email protected] 16 17 1 Both authors contributed equally to this work. 18 19 Marked-up manuscript 2 20 Abstract 21 An analytical methodology has been developed for the simultaneous extraction of 22 13 phenolic compounds, including chlorophenols (CPs), nitrophenols (NTPs), cresols 23 and alkylphenols (APs) in different types of wastewater (WW) effluents. A solid-24 phase extraction (SPE) method has been optimized prior to the determination by gas 25 chromatography coupled to triple quadrupole tandem mass spectrometry (GC-QqQ-26 MS/MS). Due to the complexity of the matrix, a comparison study of matrix-matched-27 calibration (MMC) and standard addition calibration (SAC) was carried out for 28 quantification purposes. The optimized procedure was validated using the SAC 29 approach since it provided the most adequate quantification results (in terms of 30 recovery and precision values). Recoveries were in the range 60–135% (0.5 g L−1), 31 70–115% (1 g L−1), and 78–120% (5 g L−1), with precision values (expressed as 32 relative standard deviation, RSD) ≤ 30% (except for 2-nitrophenol) involving intra-33 day and inter-day precision studies were obtained. Limits of detection (LODs) and 34 quantification (LOQs) were also evaluated, and LOQs ranged from 0.03 g L-1 to 2.5 35 g L-1. The proposed method was applied tofor the analysis of 8 real WW effluent 36 samples, finding some phenolic compounds (e.g. 2-chlorophenol, 2,4,6-37 trichlorophenol and 4-tert-octylphenol) at concentrations higher than the established 38 LOQs established during the method validation. 39 Keywords: Phenols, wastewater, solid-phase extraction (SPE), gas chromatography-40 mass spectrometry (GC-MS), standard addition calibration, matrix-matched 41 calibration. 42 43 Formatted: None 3 44 1. Introduction 45 Phenolic compounds can be found in wastewater (WW) effluents via different 46 sources. They can be detected in this type of samples because of their use in plastics 47 [1], drug manufacturing, phytosanitary products or leather coloring [2], by 48 anthropogenic emission [2] orand by the use of treatments with aerobic or anaerobic 49 microorganisms [4]. Some phenols show high toxicity, estrogenic [5] and anti-50 androgenic activity [6], andas well as they can act as endocrine disrupters [7]. 51 Phenols can be classified in a wide range of families. The most studied analytes in 52 water are chlorophenols (CPs) [1] and alkylphenols (APs) [8]. However, the United 53 States Environmental Protection Agency (US EPA) classifies CPs, nitrophenols and 54 APs as priority pollutants [9] and it has established a maximum contamination level 55 (MCL) for pentachlorophenol (PCP) of 1 µg L-1 in drinking waters [10]. On the other 56 hand, the European Union (EU) has adopted a list of priority substances in the field of 57 water policy, including 4-n-nonylphenol (4-n-NP), 4-tert-octylphenol (4-tertOP) and 58 PCP [11]. Furthermore, maximum allowable concentrations (MAC) have been 59 established for NP (2 g L-1) and PCP (1 g L-1) in inland and other surface waters 60 [12]. However, it must be pointed out that legislation for WWs is still very scarce, and 61 the values established in drinking water are usually used as guide in WWs. Bearing in 62 mind these facts, the development of sensitive analytical methodologies for the 63 simultaneous determination of phenols belonging to different groups, such as CPs, 64 APs, nitrophenols (NTPs) and cresols (also known as methyl-phenols) with different 65 polarity range (log Kow 1.77-5.01) is needed in order to provide a complete overview 66 of the occurrence of phenolic compounds in WW effluents. 67 4 Several extraction techniques have been applied for the extraction of phenols from 68 aqueous samples, such as solid-phase extraction (SPE) [8,13-15] and liquid-liquid 69 extraction (LLE) [16]. Recently, microextraction techniques, such as solid-phase 70 microextraction (SPME) [17-19], stir bar sorptive extraction (SBSE) [19-21], liquid 71 phase microextraction (LPME) [22] or dispersive liquid-liquid microextraction 72 (DLLME) [23] have been appliedused. However, most of them have only been used 73 for the simultaneous analysis of only one or few phenols belonging to the same family 74 such as APs [18,20] and CPs [17]. It is well-known that SPE is the most used 75 technique in water analysis [24] due to the lessreduced expositionure and 76 contamination by organic solvents, the high pre-concentration factors that make 77 possible to avoididing evaporation steps, the semi-automation of the extraction 78 process, reducing the sample handling, and it allows the extraction of compounds with 79 different physico-chemical properties. The application of microextraction techniques 80 is increasing but several disadvantages, such as cost and lifetime of fibers and bars, or 81 the limited scope for a wide polarity range can hinder their utilization. 82 83 For the determination of phenolic compounds, gas chromatography (GC) [13,25] or 84 liquid chromatography (LC) [26,27] are the predominantthe most used techniques, 85 mainly coupled to tandem mass spectrometry (MS/MS) [28-31]. When GC is used, a 86 derivatization step is required in order to improve the chromatographic performance 87 and sensitivity of the selected compounds, and several derivatizating reagents can be 88 applied [32,33]. 89 A well-known critical point in the analysis of WW is the matrix effect [34]. In order 90 to minimize it, different calibration methods such as matrix-matched calibration 91 5 (MMC) [33,35], standard addition calibration (SAC) [34] and the use of isotope-92 labeled internal standards [36,37] have been employed for complex matrixes. 93 Quantification based on isotope-labeled internal standards has disadvantages due to 94 the expensiveness of these standards and their limited availability. MMC is oftenthe 95 most used quantification method in trace analysis. However, the lack of blank 96 matrixes and the need for storing them can make this approach logistically onerous 97 and not necessarily accurate. SAC is the most adequate technique to use when it is 98 difficult to find a blank samples of the studied matrix studied, but a calibration set is 99 required for each sample, increasing the total number of injections and the time spent 100 in data processing. 101 Another problem related to the determination of phenols in WW is that depending 102 on the type of WW treatment, WW effluents can have different amounts of suspended 103 particulate matter (SPM). This SPM is normally discarded during the extraction 104 process by filtration in most of the analytical methods reported in literature [38]. 105 However, a recent study [35] has demonstrated that certain analytes can be retained in 106 the SPM, depending on its polarity. Therefore, it should be necessary to evaluate the 107 presence of phenols in both phases in order to determine whether the SPM must be 108 discarded or not. 109 Furthermore, it must be pointed out that many articles reporting simultaneous 110 extraction and determination of different classes of phenols (including APs, CPs and, 111 NTPs) in water [39,40] can be found. However, they have been developed for the 112 analysis of this type of compounds in surface water, and they are not valid for the 113 analysis in WW samples, due to they are more complex matrices with different 114 physico-chemical characteristics (SPM levels, organic matter, etc.). 115 6 Therefore, Iin this study, a simultaneous SPE extraction by SPE and determination 116 of different phenolic families (, namely CPs, NTPs, cresols and APs), has been 117 developed for WW effluent samples. In addition two novel aspects of this work 118 must be pointed out: (i) a study of the presence of phenolic compounds in the 119 SPM according to the strategy recently proposed by Barco-Bonilla et al [35], and 120 (ii) a comparison of MMC and SAC in order to evaluate the best quantification 121 strategy of phenolic compounds in complex matrices such as WWs. For that, A 122 study of the presence of phenolic compounds in the SPM has been carried out 123 according to the strategy recently proposed by Barco-Bonilla et al [35]. Due to the 124 complexity of the matrix and the difficulty to obtain blank samples, a study of 125 quantification study using MMC and SAC was developed in order to evaluate the best 126 quantification strategy. Ttwo different WW effluents were studied individually: 127 membrane bioreactor (MBR, low SPM content) and anaerobic pond (ANAP, high 128 SPM content). The optimized SPE and quantification method was validated in both 129 types of WWs effluent samples. 130 131 2. Experimental 132 2.1. Chemicals and materials 133 Phenolic compounds standards, 2-nitrophenol (2-NTP), 4-nitrophenol (4-NTP), 2,4-134 dimethylphenol (2,4-DMP), 2-CP, 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-135 diCP), 2,4,5-trichlorophenol (2,4,5-triCP), 2,4,6-trichlorophenol (2,4,6-triCP) and 4-136 n-NP were obtained from Fluka (Buchs, Switzerland). On the other hand, 3-137 nitrophenol (3-NTP), 4-chloro-3-methylphenol (4-C-3-MP), 4-tertOP and PCP were 138 supplied by Supelco (Bellefonte, PA, USA). Purities were always >97%. A standard 139 7 solution (100 mg L−1) of isotopically labeled PCP ([13C6]-PCP) was used as internal 140 standard (IS) and it was obtained from Dr. Erhenstofer (Augsburg, Germany). Stock 141 standard solutions of individual compounds (with concentrations ranging from 200 to 142 450 mg L-1) were prepared by exact weighing of the powder or liquid and dissolution 143 in 50 mL of acetone. These solutions were then stored under refrigeration (T <5 ºC). 144 A working standard solution of the 13 phenolic compounds (2 mg L-1 of each 145 compound) was prepared by appropriate dilution of the stock solutions with acetone, 146 and it was stored under refrigeration (T <5 ºC). A working standard solution of [13C6]-147 PCP (22 mg L-1) was prepared by appropriate dilution of the standard solution with 148 acetone and stored under the aforementioned conditions. HPLC-grade methanol 149 (MeOH), anhydride acetic acid (AAA) (99.9%), and pyridine (Py) (99.8%) were 150 purchased from Sigma-Aldrich (Madrid, Spain). Acetone and hydrochloric acid (HCl) 151 were obtained from J.T. Baker (Deventer, Netherlands). Dichloromethane (DCM) was 152 purchased from Riedel-de Haën (Seelze-Hannover, Germany). Ultrapure water was 153 obtained from a Milli-Q Gradient water system (Millipore, Bedford, MA, USA). 154 Thirty mm cellulose filters and 47-mm glass microfiber filters from Whatman 155 (Maidstone, England, UK) and 0.45-µm HNWP nylon membrane filters from 156 Millipore (Carrigtwohill, County Cork, Ireland) were also available for filtration 157 stages. For SPE, Oasis HLB (200 mg, 6 cm3) cartridges were obtained from Waters 158 (Milford, MA, USA). 159 160 2.2. Apparatus 161 A GC system Varian 3800 (Varian Instruments, Sunnyvale, CA, USA) equipped with 162 electronic flow control was interfaced to a 1200L triple quadrupole (QqQ) mass 163 8 spectrometer. Samples were injected into an SPI/1079 split/splitless programmed-164 temperature injector using a Combi Pal (CTC Analytics AG, Zwingen, Switzerland) 165 with a 100-µL syringe. A fused-silica untreated capillary column (2 m × 0.25 mm i.d.) 166 from Supelco was used as pre-column connected to a VF-5 ms Factor Four capillary 167 column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) purchased from Varian. 168 Helium was used as carrier gas (99.9999%) at a constant flow rate of 1 mL min−1, and 169 argon (99.999%) was used as collision gas. The mass spectrometer was operated in 170 electron ionization (EI) mode at 70 eV. The mass spectrometer was calibrated every 171 four days with perfluorotributylamine. Varian Workstation software was used for 172 instrument control and data analysis. 173 A Reax-2 rotary agitator from Heidolph (Schwabach, Germany) was used for 174 agitation of the derivatization mixture. An analytical balance AB204-S from Mettler 175 Toledo (Greifensee, Switzerland) and a rotary evaporator R-114 (Büchi, Flawil, 176 Switzerland) were used during extraction and standard preparation. The horizontal 177 shaker used in the distribution study was obtained from P-Selecta (Selecta, Barcelona, 178 Spain). 179 180 2.3. Sampling 181 WW urban effluents from two different treatments, namely, MBR and ANAP, with 182 low and high SPM content respectively, were collected from WW treatment plant 183 (WWTP) of the foundation Centre for New Water Technologies (“Centro de las 184 Nuevas Tecnologías del Agua”, CENTA, Seville, Spain). This WWTP has 41,000 m2 185 and it currently holds more than 20 systems with different technologies. Additional 186 physicochemical data related to the treatments evaluated in this study can be found in 187 15 Recovery and precision were evaluated using both quantification approaches. It can 329 be observed that in WW effluents with high SPM, such as ANAP, MMC did not 330 provided adequate results for the lower spiked concentrations (0.5 and 1 g L-1). 331 Recoveries and intra and inter-day precision of most of compounds were below 60% 332 and over 30%, respectively for these two concentration levels. On the contrary, for 5 333 g L-1, recovery values were in the range 60-120%, except for 4-n-NP (51%) and 334 intra and inter-day precision were <12%. These results (Table 2) suggested that MMC 335 is not a suitable option for the adequate quantification of at very low concentrations of 336 phenols in WWs effluents with high SPM. On the other hand, when SAC was used, 337 recoveries of all compounds were in range 60-125%, except for 4-tertOP (135%) at 338 the lowest fortification level (0.5 g L-1). Intra and inter-day precision values were 339 <27% and <31% for all compounds, respectively. As it is shown in Table 2, the SAC 340 approach is more appropriate for WW effluents with high SPM content. Linearity was 341 studied in the range 10-150 g L-1 (except for NTPs which was 100-300 g L-1) and 342 the obtained determination coefficients (R2) were in the range 0.9912 (3-NTP)-0.9999 343 (2-CP, 2,4,5-tTriCP, PCP and 4-n-NPvarious compounds) for ANAP (Table 4). 344 For WW effluents with low SPM, such as MBR (Table 3), the recoveries obtained 345 when MMC was used for the three levels assayed ranged from 62-119%, except for 4-346 n-NP, with recoveries lower than 56%. Despite the adequate recovery results provided 347 by MMC for all the studied fortification levels, in general, RSD values were <30% 348 only for the highest spiked level studied (5 g L-1), as it can be observed in Table 3, 349 whereas at the lowest concentration levels evaluated (0.5 and 1 g L-1), intra and 350 inter-day precision ranged from 22 to 113%. On the other hand, the application of 351 SAC on MBR WW samples yielded recovery values in the range 70-120%, except for 352 4-CP (125%) at 0.5 g L-1. Besides, RSD values were always <28% for intra-day 353 16 precision and <27% for inter-day precision in all cases, except for 2-NTP, which was 354 41% at 0.5 g L-1. In consequence, it can be concluded that for MBR treated WW 355 effluents, SAC was also the most suitable method for an adequate quantification of 356 WW effluents with low SPM content, such as MBR WW samples (Table 3). 357 Furthermore, linearity was also evaluated for MBR and R2 values ranged from 0.9943 358 (4-NTP) to 0.9999 (2-CP, 4-CP, 2,4,6-triCP and 4-n-NP). 359 Considering these results, the SAC method should be applied for a reliable 360 quantification of phenols in WW effluents samples to compensate matrix effects on 361 the signal variation during detection and this does not depend on the SPM content of 362 the WW. The SAC methodology was therefore applied for the quantification of 363 phenols in real samples. 364 365 3.4. Estimation of the lower limits of the methodology 366 Despite of the estimation of the trueness and precision carried out in the previous 367 section, other performance characteristics of the method, such as limits of detection 368 (LODs) and quantification (LOQs) were studied. LODs and LOQs were determined 369 as the lowest concentration level that yielded a signal-to-noise (S/N) ratio of 3 and 10, 370 and they are shown in Table 4. LODs and LOQs were determined in WW sample 371 blanks for each phenolic compound studied. LODs were from 0.01 to 1 g L-1 and 372 LOQs ranged from 0.03 g L-1 to 2.5 g L-1 for ANAP and MBR (Table 4). It must 373 be noticed that similar values were obtained for both types of WW effluents, except 374 for 2-NTP and 4-C-3-MP, which showed higher LOD and LOQ values in ANAP than 375 in MBR. This could be explained taking into account that the SPM content is higher 376 17 in ANAP, increasing the amount of co-extracted material and affecting the estimation 377 of the lower limits of the method. 378 379 3.5. Application to the analysis of real WW effluent samples 380 The developed methodology was applied to the analysis of 8 WW effluent samples 381 from the CENTA, obtained after the application of different WW treatments 382 employed in this WWTP. To assure the quality of the results and avoid errors, the 383 quantification of the phenolic compounds was achieved using the SAC approach. An 384 internal quality control (IQC) was performed consisting of the analysis of spiked 385 blank WW samples at 1 g L-1 (except for 3-NTP and 4-NTP at 5 g L-1), which were 386 used to assess the extraction efficiency and a SAC calibration curve to check linearity 387 and sensitivity. Several phenolic compounds were found over the LOQs established 388 by the method, showing the obtained results in Table 5. 2-CP and 2,4,6-triCP were 389 found in six and five samples, respectively, with concentrations ranging from 0.04 to 390 0.20 g L-1 for 2-CP and from 0.05 to 0.10 g L-1 for 2,4,6-triCP. 4-CP and 4-tertOP 391 were found in four samples, and the concentrations ranged from 0.04 to 0.08 g L-1 392 and 0.04 to 0.16 g L-1 respectively. 2-CP, 2,4-DiCP, 4-tertOP, PCP and 4-n-NP were 393 found simultaneously in one of the samples (Table 5). It must be highlighted that 394 phenolic compounds were not found over the MCLs and MACs established by the 395 EPA and the EU for these compounds [10,12]. Finally, Figure 4 shows a positive 396 sample of 4-tertOP detected in a WW effluent sample at 0.12 g L-1. 397 398 5. Conclusions 399 18 A single extraction method for the simultaneous extraction of CPs, APs, NTPs and 400 cresols in WW effluent samples has been developed using SPE. A distribution study 401 of the phenolic compounds between the aqueous phase and the SPM was carried out, 402 verifying that the SPM could be in fact discarded during the extraction since only 403 phenolic compounds with high log Kow were found in the SPM at a negligible 404 percentage. Due to the difficulty to find WW blank samples and to have good 405 accuracy in the quantification, a study using MMC versus SAC was performed in two 406 different treated WW effluent samples (ANAP and MBR) showing that SAC is the 407 most suitable quantification approach. The method was validated studying recovery, 408 intra and inter-day precision, lower limits (LODs and LOQs) and linearity. 409 Determination of the analytes was carried out using GC-QqQ-MS/MS operating in 410 SRM mode. The method was applied to WW effluent samples with satisfactory 411 results, observing that phenols of several families were simultaneously detected in 412 WW effluents, highlighting the potential of analytical methods that allows the 413 simultaneous determination of several classes of phenolic compounds. 414 415 Acknowledgments 416 The authors gratefully acknowledge the Andalusian Regional Government (Regional 417 Ministry of Innovation, Science, and Enterprise-FEDER) for financial support 418 (Project Ref. P08-RNM-03892). PPB acknowledges for personal funding through 419 Juan de la Cierva Program (Spanish Ministry of Science and Innovation-European 420 Social Fund). RRG is also grateful for personal funding through Ramón y Cajal 421 Program (Spanish Ministry of Science and Innovation-European Social Fund). NBB 422 is grateful for her pre-doctoral grant from the aforementioned project. CENTA is 423 gratefully acknowledged for providing WWs effluents samples. 424 19 425 426 20 References 427 [1] S. Eker, K.J. Fikret, Environ. Manage. 90 (2009) 692-698 428 [2] M. Concetta-Tomei, M. Cristina-Annesini, Environ. Sci. Technol. 39 (2005) 5059-429 5065. 430 [3] C. Li, S. Taneda, A.K. Suzuki, C. Furuta, G. Watanabe, K. Taya, Toxicol. Appl. 431 Pharm. 217 (2006) 1–6. 432 [4] D. Li, J. Park, J.R. Oh, Anal. Chem. 73 (2001) 3089-3095. 433 [5] M. Solé, M.J. López-De-Alda, M. Castillo, C. Porte, K. Ladegaard-Pedersen, D. 434 Barceló, Environ. Sci. Technol. 34 (2000) 5076-5083. 435 [6] M.A.J. Harrison, S. Barra, D. Borghesi, D. Vione, C. Arsene, R.I. Olariu, Atmos. 436 Environ. 39 (2005) 231–248. 437 [7] J. Wang, H. Pan, Z. Liu, F. Ge, J. Chromatogr. A 1216 (2009) 2499–2503. 438 [8] P. Pothitou, D. Voutsa, Chemosphere 73 (2008) 1716–1723. 439 [9] List of the 129 priority pollutants established by the United States Environmental 440 Protection Agency (U.S. EPA). Appendix A to part 423. 441 (http://www.epa.gov/waterscience/methods/pollutants.htm, last accessed February 442 2011). 443 [10] http://www.epa.gov/safewater/contaminants/index.html (last access February 444 2011). 445 [11] Decision No 2455/2001/EC of the European Parliament and of the Council of 20 446 November 2001, establishing the list of priority substances in the field of water policy 447 and amending Directive 2000/60/EC. Off. J. Eur. Communities L 331/1, 16.12.2001. 448 [12] Directive 2008/105/ECec of the European Parliament and of the Council of 16 449 December 2008 on environmental quality standards in the field of water policy, 450 amending and subsequently repealing Council Directives 82/176/EEC, 83/513/EEC, 451 21 84/156/EEC, 84/491/EEC, 86/280/EEC and amending Directive 2000/60/EC of the 452 European Parliament and of the Council. 453 [13] G. Gatidou, M.S. Tomaidis, A.S. Stasinakis, T.D. Lekkas, J. Chromatogr. A 1138 454 (2007) 32–41. 455 [14] J. Patsias, E. Papadopoulou-Morkidou, J. Chromatogr. A 904 (2000) 171-188. 456 [15] C. Mahugo-Santana, Z. Sosa-Ferrera, M.E. Torres-Padrón, J.J. Santana-457 Rodríguez, Molecules 14 (2009) 298-320. 458 [16] H. Faraji, M. Saber-Tehrani, F. Waqif-Hussain, J. Chromatogr. A 1216 (2009) 459 8569–8574. 460 [17] J. Regueiro, E. Becerril, E. García-Jares, M. Llompart, J. Chromatogr. A 1216 461 (2009) 4693–4702. 462 [18] P. Yi-Ping, T. Shigh-Wei, Anal. Chim. Acta 624 (2008) 247–252. 463 [19] J.B. Quintana, I. Rodríguez, Anal. Bioanal. Chem. 384 (2006) 1447-1461. 464 [20] M. Kawaguchi, K. Inoue, M. Yoshimura, R. Ito, N. Sakui, H. Nakazagua, Anal. 465 Chim. Acta, 505 (2004) 217–222. 466 [21] F. Sánchez-Rojas, C. Bosch-Ojeda, J.M. Cano-Pavón, Chromatographia 69 467 (2009) S79-S94. 468 [22] X. Chen, T. Zhang, P. Liang, Y. Li, Microchim. Acta 155 (2006) 415–420. 469 [23] M. Saraji, M. Marzbam, Anal. Bioanal. Chem. 396 (2010) 2685–2693. 470 [24] I. Rodriguez, M.P. Llompart, R. Cela, J. Chromatogr. A 885 (2000) 291–304. 471 [25] L. Donghao, J. Park, Anal. Chem. 73 (2001) 3089-3095. 472 [26] J.B. Baugros, C. Cren-Olivé, B. Giroud, J.Y. Gauvrit, P. Lanteri, M.F. Grenier-473 Loustalot, J. Chromatogr. A 1216 (2009) 4941–4949. 474 [27] J.L. Martínez Vidal, A. Belmonte-Vega, A. Garrido Frenich, F.J. Egea-González, 475 F.J. Arrebola-Liébanas, Anal. Bioanal. Chem. 379 (2004) 125–130. 476 22 [28] M. Petrovic, D. Barceló, A. Díaz, F.J. Ventura, Am. Soc. Mass Spectrom. 14 477 (2003) 516–527. 478 [29] M. Saitta, S. Lo Curto, F. Salvo, G. Di Bella, G. Dugo, Anal. Chim. Acta 466 479 (2002) 335–344. 480 [30] R. Loos, G. Hanke, G. Umlauf, S.J. Eisenreich, Chemosphere 66 (2007) 690–481 699. 482 [31] C. Sánchez Brunete, E. Miguel, J.L. Tadeo, J. Chromatogr. A 1216 (2009) 5497–483 5503. 484 [32] J.L. Pérez-Pavón, A.M. Casas-Ferreira, M.E. Fernández-Laespada, B. Moreno-485 Cordero, J. Chromatogr. A 1216 (2009) 1192–1199. 486 [33] J.A. Padilla-Sánchez, P. Plaza-Bolaños, R. Romero-González, A. Garrido 487 Frenich, J.L. Martínez Vidal, J. Chromatogr. A 1217 (2010) 5724-5731. 488 [34] A. Garrido Frenich, J.L. Martínez Vidal, J.L. Fernández-Moreno, R. Romero-489 González, J. Chromatogr. A 1216 (2009) 4798-4808. 490 [35] M.N. Barco-Bonilla, R. Romero-González, P. Plaza-Bolaños, A. Garrido 491 Frenich, J.L. Martínez Vidal, J.Chromatogr. A 1217 (2010) 7817–7825. 492 [36] M. Bader, W. Rosenberg, F.M. Gutzki, D. Tsikas, J. Chromatogr. B 877 (2009) 493 1402–1415. 494 [37] P. Hoffmann, M.F. Hartmann, T. Remer, K.P. Zimmer, S.A. Wudy, Steroids 75 495 (2010) 1067–1074. 496 [38] S. Terzic, I. Senta, M. Ahel, M. Gros, M. Petrovic, , J. Muller, T. 497 Knepper, I. , F. Ventura, , D. Jabucar, Sci.Total Environ. 399 (2008) 498 66-77. 499 [39] B.O. Opelou, O.S. Fatoki, J. Odendaal, Int. J. Phys. Sci. 5 (2010) 576-581. 500 23 [40] M.E. Torres-Padrón, C. Mahugo-Santana, Z. Sosa-Ferrera, J.J. Santana-501 Rodriguez, J. Chromatogr. Sci. 46 (2008) 325-331. 502 503 504 24 505 Figure Captions 506 Fig. 1. Comparison of the recovery values obtained applying different elution solvents 507 for the extraction of spiked WW samples at 0.5 g L-1. Abbreviations: DCM: 508 dichloromethane; Sequential: sequential elution. 509 Fig. 2. Total ion chromatogram (TIC) of an extracted spiked WW sample (5 g L-1) 510 obtained by GC-QqQ-MS/MS. For compound abreviations, see Table 1. 511 Fig. 3. Calibration curves in the range 10-150 g L-1 for 4-tertOP when SAC and 512 MMC were used: a) ANAP; b) MBR. Abbreviations: ANAP: anaerobic pond; MBR: 513 membrane bioreactor; MMC: matrix-matched calibration; SAC: standard addition 514 calibration; 4-tertOP: 4-tertoctylphenol 515 Fig. 4. Selected-reaction monitoring (SRM) (a) chromatogram and (b) MS/MS 516 spectrum of 4-tertOP (0.12 g L-1) found in a real WW sample and (c) SRM 517 chromatogram and (d) MS/MS spectrum of a SAC standard (50 g L-1). 518 519 520 521 3 1. Introduction 39 Phenolic compounds can be found in wastewater (WW) effluents via different 40 sources. They can be detected in this type of samples because of their use in plastics 41 [1], drug manufacturing, phytosanitary products or leather coloring [2], by 42 anthropogenic emission [2] and by the use of treatments with aerobic or anaerobic 43 microorganisms [4]. Some phenols show high toxicity, estrogenic [5] and anti44 androgenic activity [6], and they can act as endocrine disrupters [7]. 45 Phenols can be classified in a wide range of families. The most studied analytes in 46 water are chlorophenols (CPs) [1] and alkylphenols (APs) [8]. However, the United 47 States Environmental Protection Agency (US EPA) classifies CPs, nitrophenols and 48 APs as priority pollutants [9] and it has established a maximum contamination level 49 (MCL) for pentachlorophenol (PCP) of 1 µg L-1 in drinking waters [10]. On the other 50 hand, the European Union (EU) has adopted a list of priority substances in the field of 51 water policy, including 4-n-nonylphenol (4-n-NP), 4-tert-octylphenol (4-tertOP) and 52 PCP [11]. Furthermore, maximum allowable concentrations (MAC) have been 53 established for NP (2 g L-1) and PCP (1 g L-1) in inland and other surface waters 54 [12]. However, it must be pointed out that legislation for WWs is still very scarce, and 55 the values established in drinking water are usually used as guide in WWs. Bearing in 56 mind these facts, the development of sensitive analytical methodologies for the 57 simultaneous determination of phenols belonging to different groups, such as CPs, 58 APs, nitrophenols (NTPs) and cresols (also known as methyl-phenols) with different 59 polarity range (log Kow 1.77-5.01) is needed in order to provide a complete overview 60 of the occurrence of phenolic compounds in WW effluents. 61 4 Several extraction techniques have been applied for the extraction of phenols from 62 aqueous samples, such as solid-phase extraction (SPE) [8,13-15] and liquid-liquid 63 extraction (LLE) [16]. Recently, microextraction techniques, such as solid-phase 64 microextraction (SPME) [17-19], stir bar sorptive extraction (SBSE) [19-21], liquid 65 phase microextraction (LPME) [22] or dispersive liquid-liquid microextraction 66 (DLLME) [23] have been applied. However, most of them have been used for the 67 simultaneous analysis of only one or few phenols belonging to the same family such 68 as APs [18,20] and CPs [17]. It is well-known that SPE is the most used technique in 69 water analysis [24] due to the reduced exposition and contamination by organic 70 solvents, the high pre-concentration factors avoiding evaporation steps, the semi71 automation of the process, and it allows the extraction of compounds with different 72 physico-chemical properties. The application of microextraction techniques is 73 increasing but several disadvantages, such as cost and lifetime of fibers and bars, or 74 the limited scope for a wide polarity range can hinder their utilization. 75 For the determination of phenolic compounds, gas chromatography (GC) [13,25] or 76 liquid chromatography (LC) [26,27] are the predominant techniques, mainly coupled 77 to tandem mass spectrometry (MS/MS) [28-31]. When GC is used, a derivatization 78 step is required in order to improve the chromatographic performance and sensitivity 79 of the selected compounds, and several derivatizating reagents can be applied [32,33]. 80 A well-known critical point in the analysis of WW is matrix effect [34]. In order to 81 minimize it, different calibration methods such as matrix-matched calibration (MMC) 82 [33,35], standard addition calibration (SAC) [34] and the use of isotope-labeled 83 internal standards [36,37] have been employed for complex matrixes. Quantification 84 based on isotope-labeled internal standards has disadvantages due to the 85 expensiveness of these standards and their limited availability. MMC is often used 86 5 quantification method in trace analysis. However, the lack of blank matrixes and the 87 need for storing them can make this approach logistically onerous and not necessarily 88 accurate. SAC is the most adequate technique to use when it is difficult to find blank 89 samples of the studied matrix, but a calibration set is required for each sample, 90 increasing the total number of injections and the time spent in data processing. 91 Another problem related to the determination of phenols in WW is that depending 92 on the type of WW treatment, WW effluents can have different amounts of suspended 93 particulate matter (SPM). This SPM is normally discarded during the extraction 94 process by filtration in most of the analytical methods reported in literature [38]. 95 However, a recent study [35] has demonstrated that certain analytes can be retained in 96 the SPM, depending on its polarity. Therefore, it should be necessary to evaluate the 97 presence of phenols in both phases in order to determine whether the SPM must be 98 discarded or not. 99 Furthermore, it must be pointed out that many articles reporting simultaneous 100 extraction and determination of different classes of phenols (including APs, CPs and, 101 NTPs) in water [39,40] can be found. However, they have been developed for the 102 analysis of this type of compounds in surface water, and they are not valid for the 103 analysis in WW samples, due to they are more complex matrices with different 104 physico-chemical characteristics (SPM levels, organic matter, etc.). 105 Therefore, in this study, a simultaneous SPE extraction and determination of 106 different phenolic families (CPs, NTPs, cresols and APs) has been developed for WW 107 effluent samples. In addition two novel aspects of this work must be pointed out: 108 (i) a study of the presence of phenolic compounds in the SPM according to the 109 strategy recently proposed by Barco-Bonilla et al [35], and (ii) a comparison of 110 6 MMC and SAC in order to evaluate the best quantification strategy of phenolic 111 compounds in complex matrices such as WWs. For that, two different WW 112 effluents were studied individually: membrane bioreactor (MBR, low SPM content) 113 and anaerobic pond (ANAP, high SPM content). The optimized SPE and 114 quantification method was validated in both types of WWs effluent samples. 115 116 2. Experimental 117 2.1. Chemicals and materials 118 Phenolic compounds standards, 2-nitrophenol (2-NTP), 4-nitrophenol (4-NTP), 2,4119 dimethylphenol (2,4-DMP), 2-CP, 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4120 diCP), 2,4,5-trichlorophenol (2,4,5-triCP), 2,4,6-trichlorophenol (2,4,6-triCP) and 4121 n-NP were obtained from Fluka (Buchs, Switzerland). On the other hand, 3122 nitrophenol (3-NTP), 4-chloro-3-methylphenol (4-C-3-MP), 4-tertOP and PCP were 123 supplied by Supelco (Bellefonte, PA, USA). Purities were always >97%. A standard 124 solution (100 mg L−1) of isotopically labeled PCP ([13C6]-PCP) was used as internal 125 standard (IS) and it was obtained from Dr. Erhenstofer (Augsburg, Germany). Stock 126 standard solutions of individual compounds (with concentrations ranging from 200 to 127 450 mg L-1) were prepared by exact weighing of the powder or liquid and dissolution 128 in 50 mL of acetone. These solutions were then stored under refrigeration (T <5 ºC). 129 A working standard solution of the 13 phenolic compounds (2 mg L-1 of each 130 compound) was prepared by appropriate dilution of the stock solutions with acetone, 131 and it was stored under refrigeration (T <5 ºC). A working standard solution of [13C6]- 132 PCP (22 mg L-1) was prepared by appropriate dilution of the standard solution with 133 acetone and stored under the aforementioned conditions. HPLC-grade methanol 134 7 (MeOH), anhydride acetic acid (AAA) (99.9%), and pyridine (Py) (99.8%) were 135 purchased from Sigma-Aldrich (Madrid, Spain). Acetone and hydrochloric acid (HCl) 136 were obtained from J.T. Baker (Deventer, Netherlands). Dichloromethane (DCM) was 137 purchased from Riedel-de Haën (Seelze-Hannover, Germany). Ultrapure water was 138 obtained from a Milli-Q Gradient water system (Millipore, Bedford, MA, USA). 139 Thirty mm cellulose filters and 47-mm glass microfiber filters from Whatman 140 (Maidstone, England, UK) and 0.45-µm HNWP nylon membrane filters from 141 Millipore (Carrigtwohill, County Cork, Ireland) were also available for filtration 142 stages. For SPE, Oasis HLB (200 mg, 6 cm3) cartridges were obtained from Waters 143 (Milford, MA, USA). 144 145 2.2. Apparatus 146 A GC system Varian 3800 (Varian Instruments, Sunnyvale, CA, USA) equipped with 147 electronic flow control was interfaced to a 1200L triple quadrupole (QqQ) mass 148 spectrometer. Samples were injected into an SPI/1079 split/splitless programmed149 temperature injector using a Combi Pal (CTC Analytics AG, Zwingen, Switzerland) 150 with a 100-µL syringe. A fused-silica untreated capillary column (2 m × 0.25 mm i.d.) 151 from Supelco was used as pre-column connected to a VF-5 ms Factor Four capillary 152 column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) purchased from Varian. 153 Helium was used as carrier gas (99.9999%) at a constant flow rate of 1 mL min−1, and 154 argon (99.999%) was used as collision gas. The mass spectrometer was operated in 155 electron ionization (EI) mode at 70 eV. The mass spectrometer was calibrated every 156 four days with perfluorotributylamine. Varian Workstation software was used for 157 instrument control and data analysis. 158 8 A Reax-2 rotary agitator from Heidolph (Schwabach, Germany) was used for 159 agitation of the derivatization mixture. An analytical balance AB204-S from Mettler 160 Toledo (Greifensee, Switzerland) and a rotary evaporator R-114 (Büchi, Flawil, 161 Switzerland) were used during extraction and standard preparation. The horizontal 162 shaker used in the distribution study was obtained from P-Selecta (Selecta, Barcelona, 163 Spain). 164 165 2.3. Sampling 166 WW urban effluents from two different treatments, namely, MBR and ANAP, with 167 low and high SPM content respectively, were collected from WW treatment plant 168 (WWTP) of the foundation Centre for New Water Technologies (“Centro de las 169 Nuevas Tecnologías del Agua”, CENTA, Seville, Spain). This WWTP has 41000 m2 170 and it currently holds more than 20 systems with different technologies. Additional 171 physicochemical data related to the treatments evaluated in this study can be found in 172 [35]. WW effluent samples were stored at 4 ºC and processed within 5 days after 173 collection. In the MMC experiments, and due to the difficulty of finding WW effluent 174 blank samples, the corresponding signal of the blank was removed from the MMC 175 plot in those samples where analyte signal was observed. 176 177 2.4. Distribution study 178 Non-filtered WW effluent samples were spiked with 0.5 µg L-1 of the studied 179 phenolic compounds, and then, they were agitated overnight at a rate of 100 180 oscillations per min to allow a thoroughly interaction between the analytes and both 181 9 phases of WW (aqueous phase and SPM). After this, samples were filtered to separate 182 and analyze both phases. The aqueous phase was extracted by SPE, whereas for the 183 analysis of the SPM, a method developed by Padilla-Sánchez et al. [33] for the 184 extraction of phenolic compounds in agricultural soils was employed. The distribution 185 of the compounds between both phases was determined as the percentage of them 186 present in each phase. 187 188 2.5. GC-QqQ-MS/MS 189 Aliquots of 10 µL were injected into the GC system operating at a syringe injection 190 flow rate of 10 µL s−1. The injector temperature program was as follows: 70 ºC (hold 191 for 0.5 min) → 310 ºC (100 ºC min−1, hold for 10 min). The injector split ratio was 192 initially set at 10:1. Splitless mode was switched on at 0.5 min until 3.5 min. At 3.5 193 min, the split ratio was 100:1 and at 10 min, 20:1. The column oven program was as 194 follows: 70 ºC (hold for 3.5 min) → 300 ºC (20 ºC min−1) → 300 ºC (hold 4 min). 195 Cryogenic cooling with CO2 was applied when the injector temperature was 170 ºC. 196 The total running time was 19 min. 197 The QqQ mass spectrometer was mainly operated in the selected reaction 198 monitoring (SRM) mode, although selecting ion monitoring (SIM) mode was also 199 used for confirmation purposes. The electron multiplier was set +200 V above the 200 optimal value indicated by the software instrument. The temperatures of the transfer 201 line, manifold and ionization source were set at 300, 40 and 265 °C, respectively. The 202 optimal values for the scan time ranged from 0.132 to 0.240 s. Peak widths of m/z 2.0 203 and 1.5 were set in the first (Q1) and third quadrupole (Q3), respectively. The 204 optimized MS/MS parameters are indicated in Table 1. 205 10 2.6. SPE extraction and derivatization procedure 206 WW effluent samples were filtered consecutively (250 mL) using two different pore207 size filters (47-mm glass microfiber filters and 0.45-µm nylon membrane filters). The 208 filtered WW effluents showed pH values between 7.7 and 8.3. Then, pH was adjusted 209 to 2.5-2.7 with HCl (2 M) to ensure the protonated form of the phenolic compounds, 210 facilitating the absorption into the solid phase, and an adequate preservation of the 211 samples. The Oasis HLB cartridges were conditioned with 5 mL of acetone followed 212 by 5 mL of MeOH and 3 x 5 mL of ultrapure water without allowing the cartridges to 213 dry out. Then, the filtered WW sample (250 mL) was passed through the cartridges 214 under vacuum at a flow rate of 10 mL min-1. The cartridges were dried for 2 h and the 215 phenolic compounds were eluted sequentially with 3 mL of acetone and 2 mL of 216 DCM. The extracts were collected into 5-mL volumetric flasks, adjusting the total 217 volume with DCM, without any evaporation step. Then, the derivatization stage was 218 performed according to the procedure described by Padilla-Sánchez et al. [33]. 219 Briefly, 860 µL of the extract were transferred to a 2-mL vial and 20 µL of [13C6]- 220 PCP (IS), 20 µL of Py and 100 µL of AAA were added to carry out the derivatization 221 reaction. The mixture was shaking in a rotary agitator for 2 min and then injected 222 directly to the GC-QqQ-MS/MS system. 223 224 3. Results and discussion 225 WWs can be submitted to different treatments, obtaining effluents with a variety of 226 SPM contents, and thus, WW effluents can present different physico-chemical 227 properties. When an analytical method is developed for this type of samples, this 228 diversity should be taken into account. In order to cover a wide range of WW 229 11 effluents, two types of them were evaluated, MBR and ANAP, which have low and 230 high SPM content [35], respectively. The optimization of the extraction procedure as 231 well as the quantification methods, were evaluated in both types of WW effluents. For 232 that purpose, a GC-QqQ-MS/MS method recently developed [33] was applied. 233 234 3.1. Extraction method 235 For the optimization of the SPE procedure, a methodology reported by Pothitou et al. 236 [8] was first considered. This study reported the determination of only one family of 237 phenolic compounds, APs, using Oasis HLB cartridges and acetone as elution solvent. 238 Besides, certain problems regarding the evaporation stages have been previously 239 reported [33], and therefore, the extraction method was designed without any 240 evaporation step. Since the families of phenolic compounds included in this study 241 showed a wide polarity range, several elution solvents were tested to achieve a 242 simultaneous extraction [8]. Acetone (5 mL), DCM (5 mL) and a sequential elution 243 with acetone (3 mL) and DCM (2 mL) were tested. Bearing in mind that evaporation 244 steps were not included in the extraction procedure and aliquots of the extracts are 245 directly injected in the chromatographic system, the elution solvent could be partially 246 retained in the solid phase or evaporated during the elution step. This can provoke an 247 overestimation of the final concentration in relation to the theoretical value, obtaining 248 high recovery values. In order to avoid this, 5-mL volumetric flasks were used to 249 collect the extracts and the final volume was adjusted to 5 mL with the corresponding 250 solvent used during the elution step. The obtained results are shown in Figure 1 and it 251 can be observed that acetone provided adequate results for all compounds, except for 252 2,4-dMP and 4-n-NP. When DCM was used, recoveries higher than 120% were 253 12 obtained for 2-CP, 2-NTP and 4-CP, although recovery for 4-n-NP was improved. 254 Consequently, in order to obtain good recoveries for all the compounds, a sequential 255 elution with acetone and DCM was tested. In general, this elution improved the 256 recovery rates, especially for 2,4-dMP, 2-CP, 2-NTP and 4-CP. Nonetheless, 257 recoveries between 50-60% may be accepted extraordinarily in environmental 258 analysis whenever the precision values are adequate (<30%). Therefore, further 259 experiments were carried out using the sequential elution with acetone (3 mL) and 260 DCM (2 mL) as elution solvents. Finally, a total ion chromatogram (TIC) of an 261 extracted spiked WW sample at 50 g L-1 is showed in Figure 2. 262 263 3.2. Distribution study 264 Once the extraction method was optimized for the analysis of the aqueous phase of 265 WW effluent samples, a distribution study is needed to verify whether the phenolic 266 compounds are also present in the SPM. If phenolic compounds are present 267 quantitatively in the SPM, the analysis of WW effluents should not be limited to the 268 aqueous phase. The distribution study was therefore carried out, applying the 269 approach described in Section 2.4 for both type of samples. It was observed that only 270 the phenolic compounds with high log Kow were found in the SPM, but at negligible 271 percentages (<5%). On the contrary, phenolic compounds with lower log Kow where 272 not found in the SPM (data not shown). Taking into account this result, further 273 experiments were limited to the analysis of the target analytes in the aqueous phase, 274 discarding the SPM phase. These results are in accordance with a previous study [35] 275 reporting that polar compounds were not retained in the SPM. 276 277 19 References 405 [1] S. Eker, K.J. Fikret, Environ. Manage. 90 (2009) 692-698 406 [2] M. Concetta-Tomei, M. Cristina-Annesini, Environ. Sci. Technol. 39 (2005) 5059407 5065. 408 [3] C. Li, S. Taneda, A.K. Suzuki, C. Furuta, G. Watanabe, K. Taya, Toxicol. Appl. 409 Pharm. 217 (2006) 1–6. 410 [4] D. Li, J. Park, J.R. Oh, Anal. Chem. 73 (2001) 3089-3095. 411 [5] M. Solé, M.J. López-De-Alda, M. Castillo, C. Porte, K. Ladegaard-Pedersen, D. 412 Barceló, Environ. Sci. Technol. 34 (2000) 5076-5083. 413 [6] M.A.J. Harrison, S. Barra, D. Borghesi, D. Vione, C. Arsene, R.I. Olariu, Atmos. 414 Environ. 39 (2005) 231–248. 415 [7] J. Wang, H. Pan, Z. Liu, F. Ge, J. Chromatogr. A 1216 (2009) 2499–2503. 416 [8] P. Pothitou, D. Voutsa, Chemosphere 73 (2008) 1716–1723. 417 [9] List of the 129 priority pollutants established by the United States Environmental 418 Protection Agency (U.S. EPA). Appendix A to part 423. 419 (http://www.epa.gov/waterscience/methods/pollutants.htm, last accessed February 420 2011). 421 [10] http://www.epa.gov/safewater/contaminants/index.html (last access February 422 2011). 423 [11] Decision No 2455/2001/EC of the European Parliament and of the Council of 20 424 November 2001, establishing the list of priority substances in the field of water policy 425 and amending Directive 2000/60/EC. Off. J. Eur. Communities L 331/1, 16.12.2001. 426 [12] Directive 2008/105/EC of the European Parliament and of the Council of 16 427 December 2008 on environmental quality standards in the field of water policy, 428 amending and subsequently repealing Council Directives 82/176/EEC, 83/513/EEC, 429 20 84/156/EEC, 84/491/EEC, 86/280/EEC and amending Directive 2000/60/EC of the 430 European Parliament and of the Council. 431 [13] G. Gatidou, M.S. Tomaidis, A.S. Stasinakis, T.D. Lekkas, J. Chromatogr. A 1138 432 (2007) 32–41. 433 [14] J. Patsias, E. Papadopoulou-Morkidou, J. Chromatogr. A 904 (2000) 171-188. 434 [15] C. Mahugo-Santana, Z. Sosa-Ferrera, M.E. Torres-Padrón, J.J. Santana435 Rodríguez, Molecules 14 (2009) 298-320. 436 [16] H. Faraji, M. Saber-Tehrani, F. Waqif-Hussain, J. Chromatogr. A 1216 (2009) 437 8569–8574. 438 [17] J. Regueiro, E. Becerril, E. García-Jares, M. Llompart, J. Chromatogr. A 1216 439 (2009) 4693–4702. 440 [18] P. Yi-Ping, T. Shigh-Wei, Anal. Chim. Acta 624 (2008) 247–252. 441 [19] J.B. Quintana, I. Rodríguez, Anal. Bioanal. Chem. 384 (2006) 1447-1461. 442 [20] M. Kawaguchi, K. Inoue, M. Yoshimura, R. Ito, N. Sakui, H. Nakazagua, Anal. 443 Chim. Acta, 505 (2004) 217–222. 444 [21] F. Sánchez-Rojas, C. Bosch-Ojeda, J.M. Cano-Pavón, Chromatographia 69 445 (2009) S79-S94. 446 [22] X. Chen, T. Zhang, P. Liang, Y. Li, Microchim. Acta 155 (2006) 415–420. 447 [23] M. Saraji, M. Marzbam, Anal. Bioanal. Chem. 396 (2010) 2685–2693. 448 [24] I. Rodriguez, M.P. Llompart, R. Cela, J. Chromatogr. A 885 (2000) 291–304. 449 [25] L. Donghao, J. Park, Anal. Chem. 73 (2001) 3089-3095. 450 [26] J.B. Baugros, C. Cren-Olivé, B. Giroud, J.Y. Gauvrit, P. Lanteri, M.F. Grenier451 Loustalot, J. Chromatogr. A 1216 (2009) 4941–4949. 452 [27] J.L. Martínez Vidal, A. Belmonte-Vega, A. Garrido Frenich, F.J. Egea-González, 453 F.J. Arrebola-Liébanas, Anal. Bioanal. Chem. 379 (2004) 125–130. 454 21 [28] M. Petrovic, D. Barceló, A. Díaz, F.J. Ventura, Am. Soc. Mass Spectrom. 14 455 (2003) 516–527. 456 [29] M. Saitta, S. Lo Curto, F. Salvo, G. Di Bella, G. Dugo, Anal. Chim. Acta 466 457 (2002) 335–344. 458 [30] R. Loos, G. Hanke, G. Umlauf, S.J. Eisenreich, Chemosphere 66 (2007) 690– 459 699. 460 [31] C. Sánchez Brunete, E. Miguel, J.L. Tadeo, J. Chromatogr. A 1216 (2009) 5497– 461 5503. 462 [32] J.L. Pérez-Pavón, A.M. Casas-Ferreira, M.E. Fernández-Laespada, B. Moreno463 Cordero, J. Chromatogr. A 1216 (2009) 1192–1199. 464 [33] J.A. Padilla-Sánchez, P. Plaza-Bolaños, R. Romero-González, A. Garrido 465 Frenich, J.L. Martínez Vidal, J. Chromatogr. A 1217 (2010) 5724-5731. 466 [34] A. Garrido Frenich, J.L. Martínez Vidal, J.L. Fernández-Moreno, R. Romero467 González, J. Chromatogr. A 1216 (2009) 4798-4808. 468 [35] M.N. Barco-Bonilla, R. Romero-González, P. Plaza-Bolaños, A. Garrido 469 Frenich, J.L. Martínez Vidal, J.Chromatogr. A 1217 (2010) 7817–7825. 470 [36] M. Bader, W. Rosenberg, F.M. Gutzki, D. Tsikas, J. Chromatogr. B 877 (2009) 471 1402–1415. 472 [37] P. Hoffmann, M.F. Hartmann, T. Remer, K.P. Zimmer, S.A. Wudy, Steroids 75 473 (2010) 1067–1074. 474 [38] S. Terzic, I. Senta, M. Ahel, M. Gros, M. Petrovic, , J. Muller, T. 475 Knepper, I. , F. Ventura, , D. Jabucar, Sci.Total Environ. 399 (2008) 476 66-77. 477 [39] B.O. Opelou, O.S. Fatoki, J. Odendaal, Int. J. Phys. Sci. 5 (2010) 576-581. 478 22 [40] M.E. Torres-Padrón, C. Mahugo-Santana, Z. Sosa-Ferrera, J.J. Santana479 Rodriguez, J. Chromatogr. Sci. 46 (2008) 325-331. 480 481 482 23 Figure Captions 483 Fig. 1. Comparison of the recovery values obtained applying different elution solvents 484 for the extraction of spiked WW samples at 0.5 g L-1. Abbreviations: DCM: 485 dichloromethane; Sequential: sequential elution. 486 Fig. 2. Total ion chromatogram (TIC) of an extracted spiked WW sample (5 g L-1) 487 obtained by GC-QqQ-MS/MS. For compound abreviations, see Table 1. 488 Fig. 3. Calibration curves in the range 10-150 g L-1 for 4-tertOP when SAC and 489 MMC were used: a) ANAP; b) MBR. Abbreviations: ANAP: anaerobic pond; MBR: 490 membrane bioreactor; MMC: matrix-matched calibration; SAC: standard addition 491 calibration; 4-tertOP: 4-tertoctylphenol 492 Fig. 4. Selected-reaction monitoring (SRM) (a) chromatogram and (b) MS/MS 493 spectrum of 4-tertOP (0.12 g L-1) found in a real WW sample and (c) SRM 494 chromatogram and (d) MS/MS spectrum of a SAC standard (50 g L-1). 495 496 497 498 Departamento de Hidrogeología y Química Analítica C t r a . S a c r a m e n t o L a C a ñ a d a d e S a n U r b a n o 0 4 1 2 0 A l m e r í a ( E s p a ñ a ) T e l f . : 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 Almeria, 12 July 2011 Dear Editor: Please, find enclosed the revised version of the manuscript entitled “Simultaneous analysis of chlorophenols, alkylphenols, nitrophenols and cresols in wastewater effluents, using solid phase extraction and further determination by gas chromatography–tandem mass spectrometry”, Manuscript No. TAL-D-11-00529. We revised our manuscript taking into account the reviewer’s comments (changes are indicated by using “Track Changes”). Although we tried to clarify the reviewer’s comments, we have considered that it was also necessary to indicate some aspects, which can be seen in the document uploaded as “Response to Reviewers”. I hope that the reviewing process finds the revised manuscript acceptable for publication in the journal. Yours Sincerely, Prof. Antonia Garrido Frenich Cover Letter Figure(s) Click here to download high resolution image Figure(s) Click here to download high resolution image Figure(s) Click here to download high resolution image Figure(s) Click here to download high resolution image