Quadrupole-Linear Ion Trap

Wastewater samples used in this study were collected from five municipal STPs located in the north (Cantabria and Barcelona), center (Madrid), and sou...
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Anal. Chem. 2007, 79, 9372-9384

Application of Liquid Chromatography/ Quadrupole-Linear Ion Trap Mass Spectrometry and Time-of-Flight Mass Spectrometry to the Determination of Pharmaceuticals and Related Contaminants in Wastewater Marı´a Jesu´s Martı´nez Bueno, Ana Agu 1 era,* Marı´a Jose´ Go´mez, Marı´a Dolores Hernando, Juan Francisco Garcı´a-Reyes, and Amadeo R. Ferna´ndez-Alba

Pesticide Residues Research Group, Department of Hydrogeology and Analytical Chemistry, University of Almerı´a, 04120 La Can˜ada de San Urbano, Almerı´a, Spain

This paper describes an enhanced liquid chromatography-mass spectrometry (LC-MS) strategy for the analysis of a selected group of 56 organic pollutants in wastewater. This group comprises 38 pharmaceuticals and 10 of their most frequent metabolites, 6 pesticides, and 2 disinfectants. The LC-MS methodology applied is based in the use of a hybrid triple-quadrupole linear ion trap mass spectrometer (QTRAP) in combination with time-of-flight mass spectrometry (TOF-MS). The join application of both techniques provided very good results in terms of accurate quantification and unequivocal confirmation. Quantification was performed by LC-QTRAP-MS operating under selected reaction monitoring (SRM) mode in both positive and negative electrospray ionization. Unequivocal identification was provided by the acquisition of three SRM transitions per compound in most of the cases and by LCTOF-MS analysis, which allows obtaining accurate mass measurements of the identified compounds with errors lower than 2 ppm. Additionally, the use of TOF-MS permits retrospective analysis, since the full spectrum is recorded at all times with a high sensitivity. Thus, review of recorded chromatograms looking for new compounds or transformation products suspected to be present in the samples is feasible allowing one to increase the scope of the method along the monitoring program. The analytical performance of the quantitative LC-QTRAP-MS method was evaluated in effluent wastewater samples. Linearity of response over 3 orders of magnitude was demonstrated for most compounds (R2 > 0.99). Method limits of detection were between 0.04 and 50 ng L-1. Finally, the methodology was successfully applied to a monitoring study intended to characterize wastewater effluents of six sewage treatment plants in Spain. The presence of most of compounds was detected at concentrations ranging from 9 ng L-1 (atrazine) to 15 µg L-1 (paraxanthine). It is clear that nowadays and in the near future, our society has to face serious problems, such as overexploitation of resources, degradation of the environment, and deterioration of ecological goods and services, for sustainable development. * Corresponding author. Tel.: (+34)950015531. Fax: (+34)950015483. Email: [email protected].

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Among them, availability of water of good quality is a critical issue, since it represents an essential component for sustainable socioeconomic development. Water quality damage, mainly a consequence of anthropogenic activities, and water deficit are causes of social concern and demand prompt actions. Urban wastewaters, which include domestic and some industrial waters, among others, typically have a strongly contaminating effect on the natural aquatic systems.1-3 Even when they are submitted to treatment, it has been demonstrated by many studies that multiple organic compounds, such as pharmaceuticals, personal care products, hormones, and other disrupting compounds, escape conventional wastewater treatments and some of them are becoming ubiquitous in the environment.4 This impact can be reduced accordingly with the treatment applied. In Spain, although more than 50% of urban wastewaters are currently being treated, only half of them are subjected to biological treatments and only 3% undergo advanced treatment technologies. In a similar manner, despite the potential of water reuse and the actual legislation that promotes the reuse of treated water, only a low water reuse (∼5%) takes place. Consequently, the application of more exhaustive wastewater treatment protocols, including the use of new and improved technologies, the application of wider and integrated quality control strategies, comprising chemical, microbiological, and toxicological analysis, and the study and development of wastewater reuse strategies are tasks that are necessary undertake.5 With this aim, an ambitious program (TRAGUA) financed by the Spanish Government, which involves the participation of a multidisciplinary team formed by 24 research groups, has been initiated. The program attempts to tackle the different aspects (1) Go´mez, M. J.; Martı´nez Bueno, M. J.; Lacorte, S.; Ferna´ndez-Alba, A. R.; Agu ¨ era, A. Chemosphere 2007, 66, 993-1002. (2) Hernando, M. D.; Mezcua, M.; Ferna´ndez-Alba, A. R.; Barcelo´, D. Talanta 2006, 69, 334-342. (3) Zuccato, E.; Castiglioni, S.; Fanelli, R. E. J. Hazard. Mater. 2005, 122, 205209. (4) Carballa, M.; Omil, F.; Lema, J. M.; Llompart, M.; Garcı´a-Jares, C.; Rodrı´guez, I.; Go´mez, M.; Ternes, T. A. Water Res. 2004, 38, 2918-2926. (5) Hernando, M. D.; Ferrer, I.; Agu ¨ era, A.; Ferna´ndez-Alba, A. R. In Emerging organic pollutants in waste waters and sludge; Barcelo, D., Ed.; SpringerVerlag: Berlin, 2004; pp 53-77. 10.1021/ac0715672 CCC: $37.00

© 2007 American Chemical Society Published on Web 11/15/2007

involved in the reuse of wastewater coming from the sewage treatment plants (STPs). As a part of this program, the present work has been focused on the development of a suitable analytical methodology, which allows an exhaustive characterization of wastewater effluents. LC-MS have arisen in the last years as the technique of choice for this purpose because of the ability to analyze polar analytes; such as the many organic pollutants or their metabolites or degradation products.6,7 Target analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides excellent performance for quantitative analysis because of its inherent selectivity and sensitivity working in selected reaction monitoring (SRM) mode.8,9 However, it fails in the determination of nontarget compounds, not initially included in the multiresidue methods, which remain ignored. Identification of these compounds allows their inclusion in the monitoring programs and further evaluation of their occurrence and possible impact. In order to increase the scope of the methods, the combination of two LCMS techniques with complementary features or the use of hybrid systems, which combine different analyzer designs to increase instruments versatility, are the latest trends. In this work, an analytical methodology combining the use of two LC-MS techniques has been investigated to achieve both accurate and reliable target compound monitoring and identification of nontarget compounds. The instrumental techniques applied have been a hybrid triple-quadrupole/linear ion trap (QTRAP) and a time-of flight (TOF) instrument. QTRAP systems combine a triple-quadrupole (QqQ) scanning functionality with sensitive linear ion trap (LIT) scans.10 This is possible because of their singular configuration, which is based on a QqQ where the third quadrupole (Q3) can be closed by repulsive dc voltages and eventually used as a LIT with axial ion ejection. Working in LIT mode, the QTRAP provides improved performance and enhanced sensitivity in full scan MS (EMS) and product ion scan (enhanced product ion (EPI)) modes. Additionally, the instrument can also operate under all the triple-quadrupole scans including SRM mode as well as precursor and constant neutral loss scanning. An interesting choice of this hybrid system suggests the possibility of combining in the same run SRM and EPI scans by the built-in information-dependent acquisition (IDA) software, thus obtaining at the same time accurate quantification and additional structural information. This alternative has been explored in the present work. TOF-MS instruments represent at present a valuable tool for screening of target and nontarget compounds in wastewater.11,12 Accurate mass measurements, along with specific retention times, usually provide highly reliable identification of target species, avoiding isobaric interferences in complex samples. Additionally, accurate mass measurements allow obtaining the elemental (6) Hernando, M. D.; Go´mez, M. J.; Agu ¨ era, A.; Ferna´ndez-Alba, A. R. Trends Anal. Chem. 2007, 26, 581-594. (7) Perez, S.; Barcelo, D. Trends Anal. Chem. 2007, 26, 494-514. (8) Herna´ndez, F.; Sancho, J. V.; Iba´n ˜ez, M.; Guerrero, C. Trends Anal. Chem. 2007, 26, 466-485. (9) Go´mez, M. J.; Petrovic´, M.; Ferna´ndez-Alba, A. R.; Barcelo´, D. J. Chromatogr., A 2006, 1114, 224-233. (10) Hager, J. W.; Le Blanc, J. C. Y. Rapid Commun. Mass Spectrom. 2003, 17, 1056-1064. (11) Lacorte, S.; Ferna´ndez-Alba, A. R. Mass Spectrom. Rev. 2006, 25, 866-880. (12) Go´mez, M. J.; Malato, O.; Ferrer, I.; Agu ¨ era, A.; Ferna´ndez-Alba, A. R. J. Environ. Monit, 2007, 9, 718-729.

composition of parent and fragment ions, useful for identification of nontarget compounds, metabolites, or degradation products. Furthermore, having full scan information of monitoring samples allows further data treatment and facilitates that compounds of interest can be detected “a posteriori”, thus avoiding additional cost and time. This paper discusses the advantages and limitations of these two instrumental approaches and the developed methods, showing their application to wastewater samples. Preliminary results of the monitoring program are also presented. EXPERIMENTAL SECTION Chemicals and Reagents. All the chemicals included in this study were purchased from Sigma-Aldrich (Steinheim, Germany) at analytical grade (purity >90%), except codeine and diazepan, which were obtained by dissolving a Codeisan tablet (30 mg of codeine) from Lab. Belmac (Madrid, Spain) and a valium tablet (10 mg of diazepan) from Lab. Andreu (Barcelona, Spain), respectively. The reference compound, used as surrogate standard, 13C-phenacetin, was purchased from Lab. Dr. Ehrenstorfer (Augsburg, Germany). Individual stock standard solutions of the target compounds were prepared in methanol at a concentration between 1 and 2 mg/mL and stored at -20 °C. Working solutions, at different concentrations, were prepared by appropriate dilution of the stock solutions in MeOH/water, 10:90 (v/v). Methanol and acetonitrile HPLC grade were supplied from Merck (Darmstadt, Germany). Water used for LC-MS analysis was generated from a Direct-Q 5 Ultrapure Water System from Millipore (Bedford, MA) with a specific resistance of 18.2 MΩ· cm. Formic acid (purity, 98%) and ammonium formate (purity, 98%) were obtained from Fluka (Buchs, Germany). Commercial cartridges packed with OasisTM HLB (divinylbenzene/N-vinylpyrrolidone copolymer, 200 mg, 6 cm3) were purchased from Waters (Milford, MA). Analytes Selected. Analytes included in this study were selected on the basis of previous experience and published literature.1,13,14 They comprise a group of 56 organic pollutants belonging to different compound categories: pharmaceuticals, pesticides, disinfectants, and some of their more relevant metabolites. Among the pharmaceuticals, there are representatives of different therapeutical groups, such as analgesics/anti-inflammatories (acetaminophen, indomethacine, codeine, mefenamic acid, ketorolac, naproxen, ibuprofen, diclofenac, fenoprofen, ketoprofen), antibiotics (metronidazole, sulfamethoxazole, trimethoprim, ciprofloxacin, cefotaxime, ofloxacin, erythromycin), lipid regulators (fenofibrate, bezafibrate, gemfibrozil), β-blockers (atenolol, propranolol, sotalol, metoprolol), antidepressants (fluoxetine, paroxetine), antiepileptic/psychiatrics (carbamazepine, diazepam), ulcer healings (ranitidine, omeprazole), corticosteroides (methylprednisolone), diuretics (furosemide, hydrochlorothiazide), and bronchodilatadors (salbutamol, terbutaline). Occurrence of many of these compounds has been already reported in environmental waters.15,16 Because of their relevance and, in some cases, (13) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Environ. Sci. Technol. 2002, 36, 1202-1211. (14) Ternes, T. A. Water Res.. 1998, 32, 3245-3260. (15) Bendz, D.; Paxeus, N. A.; Ginn, T. R.; Loge, F. J. J. Hazard. Mater. 2005, 122, 195-204. (16) Bound, J. P., Voulvoulis, N. Chemosphere 2004, 56, 1143-1155.

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significant presence, a group of metabolites such as carbamazepine 10,11-epoxide, 1,7-dimethylxanthine (paraxanthine), clofibric acid, and fenofibric acid were also included. Of special interest was the case of the metabolites of the antipyretic drug dypirone and its active product 4-methylaminoantipyrine (4-MAA), such as N-acetyl4-aminoantipiryne (4-AAA), N-formyl-4-aminoantipiryne (4-FAA), 4-dimethylaminoantipiryne (4-DAA), 4-amino-antipiryne (4-AA), and antipyrine, which were included a posteriori because of the evidence of a high level of 4-MAA in the samples and further identification by LC-TOF-MS. Because of the location of one of the sampled STPs in an agricultural area, a group of six pesticides, atrazine, chlorpyriphos methyl, chlorfenvinphos, diuron, isoproturon and simazine, was considered for their study. Finally, two well-known disinfectants, biphenylol and chlorophene, completed the group of target compounds. Sampling and Sample Preparation. Wastewater samples used in this study were collected from five municipal STPs located in the north (Cantabria and Barcelona), center (Madrid), and southeast of Spain (Almerı´a). They are representative of different activities (urban, agricultural, industrial). All plants apply a pretreatment for solid removal, a primary treatment to eliminate suspended material, an activated sludge biological treatment, and a final clarification. Integrated samples representative of 1-days work in the STP were taken. They were taken at 1-h intervals, during the period comprised from January 2007 to May 2007. Sampling was carried out by an automatic device (0.5 l/3 h). Effluent samples were collected by using prerinsed amber glass bottles and sent to the laboratory in Almerı´a for analysis. All samples were filtered through a 0.7-µm glass fiber filter (Teknokroma, Barcelona, Spain) and extracted within 48 h in all the cases. A solid-phase extraction (SPE) procedure was applied to the wastewater samples using commercial Oasis HLB (divinylbenzene/N-vinylpyrrolidone copolymer) cartridges (200 mg, 6 cm3) from Waters. To optimize the extraction method, three SPE conditions were tested: pH 5, 7, and 8. In the light of the results of these preliminary trials, HLB cartridges at pH 8 were selected for further experiments. An automated sample processor ASPEC XL fitted with an 817 switching valve and an external 306 LC pump from Gilson (Villiers-le-Bel, France) was used for this purpose. The Oasis HLB cartridges were preconditioned with 6 mL of MeOH and 5 mL of deionized water HPLC-grade (pH adjusted to 5 and 7, with HCl 2 N, and to 8, if it is necessary, with 20% NH4OH) at a flow rate of 1 mL/min. After the conditioning step, aliquots of 200 mL of sample (pH adjusted to 5, 7, or 8.5) were loaded into the cartridge. Samples were previously spiked with 10 µL of 10 mg/L solution of the surrogate standard 13Cphenacetin. Samples were passed through the cartridges at a flow rate of 10 mL/min and then rinsed with 5 mL of deionized water prior to the elution. After that, the cartridges were dried by a nitrogen stream for ∼5 min to remove excess of water, and finally, the analytes retained were eluted with 2 × 4 mL of MeOH at 1 mL/min. The extracts were evaporated until almost dry using a Turbo-Vap from Zymark (Hopkinton, MA), with a water temperature at 35 °C. The samples were then reconstituted with 1 mL of MeOH/water, 10:90 (v/v) and were then filtered directly into an analysis vial using a 0.45-µm PTEF syringe filter (Millipore). Before analysis, a dilution 1:1 with MeOH/water (10:90) was applied. 9374

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Liquid Chromatography-QTRAP-Mass Spectrometry. The method for the analysis of target compounds was developed for the 3200 QTRAP MS/MS system (Applied Biosystems, Concord, ON, Canada). The QTRAP analyzer combines a fully functional triple-quadrupole and ion trap mass spectrometer within the same platform. Separation of the analytes were performed using an HPLC (series 1100, Agilent Technologies, Palo Alto, CA) equipped with a reversed-phase C-18 analytical column (Zorbax SB, Agilent Technologies) of 5-µm particle size, 250-mm length, and 3.0-mm i.d. For the analysis in positive mode, the compounds were separated using acetonitrile (mobile phase A) and HPLC-grade water with 0.1% formic acid (mobile phase B) at a flow rate of 0.2 mL/min. A linear gradient progressed from 10% A (initial conditions) to 100% A in 40 min, after which the mobile-phase composition was maintained at 100% A for 10 min. The reequilibration time was 15 min. Compounds analyzed in negative mode were separated using acetonitrile (mobile phase A) and HPLC-grade water (mobile phase B) at a flow rate of 0.3 mL/ min. LC gradient started with 30% A and linearly was increased to 100% A, in 7 min, after which the mobile-phase composition was maintained at 100% A for 8 min. The re-equilibration time was 10 min. The volume of injection was of 20 µL in both modes. The analyses were performed using a turbo ion spray source in positive and negative modes. The operation conditions for the analysis in positive ionization mode were the following: ion spray voltage, 5000 V; curtain gas, 10 (arbitrary units); GS1 and GS2, 50 and 40 psi, respectively; probe temperature, 500 °C. The parameters used for the analysis in negative ionization mode were as follows: ion spray voltage, -35000 V; curtain gas, 10 (arbitrary units); GS1 and GS2, 50 psi; probe temperature, 500 °C. Nitrogen served as nebulizer gas and collision gas in both modes. Mass calibration and resolution adjustments on the resolving quadrupoles were performed automatically by using a 10-5 mol/L solution of poly(propylene glycol) introduced via a syringe pump and connected to the interface. SRM experiments were carried out to obtain the maximum sensitivity for the detection of the target molecules. The optimization of MS parameters (declustering potential (DP), entrance potential (EP), for precursor ions and collision energy (CE), collision cell exit potential (CXP) for product ions) was performed by flow injection analysis for each compound. Table 1 shows the values of the parameters optimized and the SRM transitions selected. The MS was operated in SRM mode with a resolution set to low and unit for Q1 and Q3, respectively. An additional experiment was developed for the ibuprofen, where further structural information was necessary for confirmatory purposes. For this case, the QTRAP system operated using EPI mode and MS3 mode. The additional compound-dependent parameters optimized were as follows: collision energy spread at 0 (arbitrary units) and scan rates at 4000 amu/s. Confirmation of each compound was performed by means of two SRM transitions and the monitoring of the SRM ratio. The most intense SRM transition was selected for quantitation purposes. Applied Biosystems/MDS Sciex Analyst software was used for data acquisition and processing. Liquid Chromatography-Time-of-Flight-Mass Spectrometry. A liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF MS) system, in positive and negative ionization modes, was used to confirm the target

Table 1. Optimized Parameters for the QTRAP-MS/MS Analysis of the Selected Compoundsa compound

precursor ion (m/z)

DP

nicotine salbutamol atenolol terbutaline ranitidine sotalol 4-MAA 4-DAA 4-AA paraxanthine acetaminophen metronidazole codeine caffeine 4-AAA 4-FAA trimethoprim cefotaxime ofloxacin ciprofloxacin mepivacaine metoprolol antipyrine omeprazole propanolol sulfamethoxazole carbamaz.10,11-epoxide erythromycin paroxetine carbamazepine simazine fluoxetine ketorolac methylprednisolone isoproturon atrazine ketoprofen naproxen diazepan biphenylol indomethacine fenofibric acid mefenamic acid chlorfenvinphos chlorpyriphos methyl fenofibrate

163.1 240.3 267.3 226.3 315.3 273.3 218.2 232.2 204.2 181.2 152.1 172.1 300.2 195.1 246.2 232.2 291.3 456.1 362.3 332.3 247.4 268.2 189.2 346.3 260.0 254.2 253.2 734.6 330.3 237.2 202.2 310.3 256.2 475.3 207.3 216.1 255.2 231.2 285.2 171.2 358.2 319.1 242.2 359.1 322.1 361.2

40 44 30 47 38 45 35 48 45 50 40 35 30 40 46 45 45 40 47 50 28 30 48 35 35 47 75 58 70 50 45 30 70 40 40 45 47 35 50 40 50 65 36 55 43 60

Positive Ionization 117.2 34 148.2 26 145.2 35 152.2 20 176.2 21 133.2 37 56.1 30 113.2 17 56.2 30 124.2 25 110.1 20 128.1 20 165.2 50 138.2 25 228.1 18 214.2 18 230.2 28 396.1 10 318.3 25 231.2 48 98.1 23 116.2 25 77.1 51 198.2 15 116.2 23 108.2 30 180.2 40 158.3 40 192.2 25 194.3 25 132.0 26 44.2 25 105.1 25 321.1 17 72.1 35 174.1 24 209.2 16 185.3 20 193.2 40 153.1 23 139.1 25 233.1 22 180.2 53 155.2 15 125.1 21 233.1 25

furosemide hydrochlorothiazide clofibric acid bezafibrate diclofenac diuron fenoprofen ibuprofen chlorophene gemfibrozil

329.1 296.0 213.0 360.1 294.0 231.1 241.1 205.1 217.0 249.2

35 56 30 30 25 50 17 30 61 35

Negative Ionization 205.1 30 205.0 29 127.1 25 274.2 30 250.0 15 186.1 23 197.1 13 161.2 10 181.2 27 121.0 25

a

SRM 1

CE 1

SRM 2

CE 2

SRM 3

CE 3

84.1 166.2 190.2 107.1 130.1 213.2 97.2 98.2 159.2 69.2 64.8 82.1 215.3 110.2 83.1 104.1 123.2 324.1 261.3 314.3 70.1 133.2 104.1 136.2 183.2 156.1 236.2 576.5 123.2 192.1 124.2 148.2 178.1 339.3 165.1 104.1 105.1 170.1 154.2 152.0 174.3 139.1 224.2 127.1 290.1 139.1

27 16 20 40 30 22 16 25 16 43 45 30 30 30 40 28 30 15 33 25 53 30 32 35 23 21 11 28 30 28 23 10 34 16 19 40 33 32 35 37 15 42 34 21 19 35

130.1 222.2 116.1 125.2 224.2 255.2 159.2 111.2 94.2 96.1 93.0 111.1 199.2 123.1 104.2 83.1 261.2 241.3 245.1

27 14 25 32 20 14 17 21 28 32 35 30 35 40 28 28 30 20 32

159.2 106.2 151.2 155.2 147.2 210.2 316.1 151.2 104.1 457.2 134.2 132.1 177.2 222.3 127.2

28 40 25 30 22 20 25 30 34 9 29 31 22 35 45

121.0 209.2 205.1 109.1 121.1

40 20 24 30 40

285.1 269.1 85.0 154.1 214.1 149.8 93.1

20 25 17 40 28 33 48

126.2 126.0

45 41

85.0

35

122.1

46

153.0 127.1

30 15

101.0

43

DP, declustering potential (V); CE, collision energy (eV); EP, entrance potential, 5 V; CXP, collision cell exit potential, 2.5 V.

pharmaceuticals in the samples. The analytes were separated using a HPLC system (consisting of vacuum degasser, autosampler, and binary pump) (Agilent Series 1100, Agilent Technologies) equipped with a reversed-phase C18 analytical column of 3 × 250 mm, 5-µm particle size (Zorbax SB-C18, Agilent Technologies). Gradient LC elution was performed with 0.1% formic acid and 5% MilliQ water in acetonitrile as mobile phase A and 0.1% formic

acid in water (pH 3.5) as mobile phase B. For the analysis in positive mode, the optimized chromatographic method held the initial mobile-phase composition (10% A) constant for 2 min, followed by a linear gradient to 100% A in 40 min, after which the mobile-phase composition was maintained at 100% A for 10 min. A 15-min postrun time back to the initial mobile-phase composition was used after each analysis. The flow rate used was 0.2 mL/ Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

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min. Compounds analyzed in negative mode were separated using acetonitrile as mobile phase A and 0.05% ammonium formate in HPLC-grade water as mobile phase B (pH 6.2) at a flow rate of 0.3 mL/min. LC gradient started with 30% A and linearly was increased to 100% A, in 7 min, after which the mobile-phase composition was maintained at 100% A for 8 min. The reequilibration time was 10 min. The volume of injection was of 20 µL in both modes. The HPLC system was connected to a time-of-flight mass spectrometer (MSD-TOF, Agilent Technologies, Santa Clara, CA) equipped with an electrospray interface operating under the following conditions: capillary, 4000 V; nebulizer, 40 psi; drying gas, 9 L/min; gas temperature, 300°; skimmer voltage, 60 V; octapole dc1, 37.5 V; octapole rf, 250 V; fragmentor 190 V. The mass axis was calibrated using the mixture provided by the manufacturer over the m/z 50-3200 range. A second orthogonal sprayer with a reference solution was used as a continuous calibration in positive ion using the following reference masses: 121.0509 and 922.0098 m/z (resolution: 9700 ( 500 at 922.0098 m/z). With the electrospray source in negative (ESI-), reference masses were 119.036 320 and 966.000 725 m/z (resolution: 10000 ( 500 at 966.000 725 m/z). Spectra were acquired over the m/z 50-1000 range at a scan rate of 1 s/spectrum. The full mass spectra data recorded were processed with Applied Biosystem/ MDS-Sciex Analyst QS software (Frankfurt, Germany) with accurate mass application-specific additions from Agilent MSD TOF software. Based on the accurate mass obtained, all possible elemental compositions for ion fragments with a maximum deviation of 5 ppm from the measured mass were calculated. Validation Study. All the validation studies were performed by using sewage extracts taken from the STP effluent of Almerı´a. Because of the impossibility to obtain blanks, the samples were previously analyzed and the presence of the target compounds considered. To minimize matrix effects, the consequence of the presence of sample matrix components, matrix-matched calibration curves were used for quantitative determinations. The linearity in the response was studied by using matrix-matched calibration solutions prepared by spiking sewage extracts at five concentration levels, ranging from the determination limit of each analyte to 1 mg/L in the final extract. Each point was obtained as the average of three injections. Integrated peak area data of the selected quantification SRM transitions (SRM1; see Table 1) were used to construct the curves. The recovery studies (n ) 3) were carried out by spiking sewage samples at the concentration level of 0.5 µg/L. Precision of the chromatographic method, determined as relative standard deviation (RSD), was obtained from repeated injection (n ) 5) of a spiked extract during the same day (repeatability) and on different days (reproducibility). The method detection limit (MDL) and method quantification limit (MQL) were determined experimentally from the injection of spiked wastewater samples and calculated using the minimum concentration of analyte providing signal-to-noise ratios of 3 and 10, respectively. They were estimated from the spiked extracted ion chromatograms at the lowest analyte concentration assayed. Confirmation criteria applied to the target compounds in the sewage samples were as follows: presence of two characteristic SRM transitions at the correct retention time, with the correct relative ion intensity and accurate mass. 9376

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For assessment of ion suppression/enhancement effects, chromatograms of standard solutions and spiked effluent wastewater extracts (1 mg L-1) have been compared. The influence of dwell time on peak area and signal-to-noise ratio (S/N) was assayed using extracts of effluent samples spiked at a concentration of 100 µg L-1, which were analyzed using different dwell time values per SRM transition. RESULTS AND DISCUSSION Sample Preparation. Optimization of the extraction procedure was made with the aim of reaching good recoveries for the widest group of compounds in a single extraction step. Oasis HLB was the sorbent selected because of its proved versatility and efficiency in the extraction of analytes of a wide range of polarities, as is the case.12,17 Experiments were performed adjusting the pH of the samples at three different values: 5, 7, and 8. As was expected, better recoveries were obtained at pH 5 for some selected compounds (cefotaxime), but as a general approach, neutral and basic pH yielded better results for the majority of analytes, with slight differences in favor of pH 8, which was finally selected. Recoveries obtained with the proposed method are shown in Table 2. Lower recoveries (