Solid‑phase extraction and fractionation of multiclass pollutants from wastewater followed by liquid chromatography tandem‑mass spectrometry analysis

Vol.:(0123456789) 1 3 Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-022-04066-8 RESEARCH PAPER Solid‑phase extraction andfractionation ofmulticlass pollutants fromwastewater followed byliquid chromatography tandem‑mass spectrometry analysis V.Fernández‑Fernández1· M.Ramil1· R.Cela1· I.Rodríguez1 Received: 9 February 2022 / Revised: 1 April 2022 / Accepted: 5 April 2022 © The Author(s) 2022 Abstract Herein, we describe a modular solid-phase extraction (SPE) setup, combining three sorbents, for the effective extraction of neutrals, acidic, and basic micropollutants from wastewater, followed by their further elution in three independent extracts. The performance of this approach was demonstrated for a suite of 64 compounds, corresponding to different chemical families, using liquid chromatography tandem-mass spectrometry (LC–MS/MS). Target compounds were effectively extracted from wastewater samples; moreover, 62 out of 64 species were isolated in just one of the three fractions (neutrals, acids, and bases) obtained from the combination of sorbents. Globally, the efficiency and the selectivity of the SPE methodology improved the features obtained using generic SPE polymers, displaying just reversed-phase interactions. The overall recoveries of the analytical method, calculated against solvent-based calibration standards, stayed between 80 and 120% for 57 and 60 compounds, in raw and treated wastewater, respectively. Procedural limits of quantification (LOQs) varied from 1 to 20ng L−1. Analysis of urban wastewater samples identified a group of 19 pollutants showing either negligible median removal efficiencies (± 20%) during wastewater treatment, or even a noticeable enhancement (case of the biodegradation product of the drug valsartan), which might be useful as markers of wastewater discharges in the aquatic environment. Keywords Wastewater· Modular solid-phase extraction· Fractionation· Liquid chromatography tandem-mass spectrometry Introduction The number of organic compounds of environmental and toxicological concern has increased steadily for the last 20years. Many of them are introduced in the aquatic environment through urban wastewater [1, 2]. Most of the analytical procedures for the monitoring of these compounds are based on mass spectrometry (MS), combined with different chromatographic techniques, after an extraction and concentration step [3]. In this regard, the hydrophilic-lipophilic balanced (HLB) solidphase extraction (SPE) sorbents cover the effective extraction of compounds within a broad range of polarities from water samples. Thus, they are usually employed in combination with multianalyte/multiclass liquid chromatography (LC) MS-based methods [4–6]. The price of their high retention efficiency is a limited selectivity, which turns in significant variations in the efficiency of compound ionization (particularly using electrospray ionization, ESI) between sample extracts and solventbased standards [6]. The so-called matrix effects (MEs) do not only affect the accuracy of the obtained results, but also compound detectability since, in most cases, their ionization efficiency is attenuated for sample extracts when compared to solvent-based standards [5]. Mixed-mode (MM) sorbents, sharing ionic and reversedphase (RP) interactions, improve the recoveries of highly polar, ionizable compounds in comparison to RP polymers, maintaining an acceptable retention efficiency for neutrals [7, 8]. Moreover, they allow the use of fractionated elution protocols, recovering compounds retained through the RP mechanism and those establishing electrostatic interactions with sorbent in different fractions [9]. Thus, cleaner extracts are obtained for the latter group of compounds, which turns in lower MEs [10–12]. The scientific literature contains previous successful * I. Rodríguez [email protected] 1 Department ofAnalytical Chemistry, Nutrition andFood Sciences, Research Institute On Chemical andBiological Analysis (IAQBUS), Universidade de Santiago de Compostela, 15782SantiagodeCompostela, Spain Fernández-Fernández V.et al. 1 3 applications of different types of MM sorbents to the selective extraction of basic (i.e., illicit drugs [13] and pharmaceuticals [11]), or acidic compounds (such as anti-hypertension drugs [12] and perfluorinated carboxylic and sulfonic acids [14]) from water samples. Most of these studies have been compiled in a recent review [7]. However, using conventional MM sorbents, the isolation of acidic and basic compounds in two separate fractions, requires two independent SPE extractions, with different MM polymers. In both assays, neutrals are mixed, either with basic or with acidic species. In order to combine high retention efficiencies during the concentration step with fractionated elution protocols, different alternatives are under evaluation. Zwitterionic MM sorbents permitted the removal of neutrals, in a washing fraction, whilst acids and bases were recovered together [15]. Another possibility involves the combination of different types of sorbents, either packed in the same cartridge, or connected in tandem. This approach was reported to improve the extraction efficiency of low molecular size, polar, and ionizable compounds, poorly retained in RP materials; however, in these previous studies, the sequential elution of compounds in independent fractions was not investigated [8, 16]; consequently, extracts presented a high complexity. Using a multilayered cartridge setup (based on different combinations of conventional MM sorbents), Salas etal. [17] demonstrated the possibility to retain and to recover quantitatively a selection of 13 compounds in neutral, acidic, and basic fractions. In that study, the retention efficiency and the success of the fractionated elution protocol depended on the type of MM sorbents, and their relative proportions in the in-house packed cartridge [17]. The aim of this research was to develop a modular SPE approach, based on tandem combinations of commercially available cartridges, covering the effective extraction and the further fractionated elution of a suite of 64 compounds (log D values from − 1.95 to 5.5) attending to their ionizable groups: acids, bases, and neutrals, from wastewater samples. Sorbents were maintained in separate cartridges to increase the versatility of the elution protocol. Each SPE fraction was analyzed using different LC–MS/MS procedures, finely tuned to enhance compound detectability and to reduce blank contamination problems. The performance of the method was characterized in terms of extraction efficiency, MEs, and accuracy. Thereafter, it was applied to determine the levels of target compounds in raw and treated wastewater samples obtained from urban sewage treatment plants (STPs). Material andmethods Solvents, sorbents, andstandards Methanol (MeOH) and acetonitrile (ACN), both LC–MS grade; formic acid (FA, LC–MS grade); ammonia (NH3, 7M solution in MeOH); and ammonium fluoride (NH4F) were purchased from Merck (Darmstadt, Germany). Ultrapure deionized water (18.2 MΩ cm−1) was obtained from a Genie U system (Rephile, Shanghai, China). RP OASIS HLB 60-mg and 200-mg cartridges, and 150-mg MM cartridges (MCX, RP, and strong cationic exchange sorbent; and WAX, RP, and weak anionic exchange sorbent) were provided by Waters (Milford, MA, USA). Ionic exchange 500-mg cartridges containing either sulfonic functionalities (SCX), or quaternary amines (SAX), as charged groups bonded to silica particles, were obtained from Agilent (Santa Clara, CA, USA). Native standards of species involved in this research were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compounds were selected attending to their environmental and/or toxicological concern, including species compiled in the 2020 revision of the EU Watch List of contaminants to control in the aquatic environment [18]. The suite of compounds includes species without ionizable groups (neutrals, i.e., organophosphorus compounds and certain neonicotinoids), weak (phenols) and strong acids (carboxylic, tetrazolic, sulfonic, etc.), weak and strong bases (i.e., azoles and tertiary amines, respectively), and pollutants combining acidic and basic functionalities in the same molecule (e.g., certain angiotensin receptor antagonists, ARA-II, as losartan). The list of analytes, including their log D values and their categorization as acids, bases, or neutrals, is given in Table1. Individual stock solutions of each compound were prepared in MeOH, except in case of neonicotinoids (ACN). Further dilutions and mixtures were also made in MeOH. Stock solutions and diluted mixtures were maintained at − 20°C and used throughout this study. The exception was perfluorinated carboxylic acids. Their methanolic solutions were renewed every month to prevent esterification of the carboxylic moiety [19]. A selection of isotopically labeled compounds (either deuterated or 13C species) was obtained from Merck and Toronto Research Chemicals (North York, Canada), either as pure compounds, or as stocks in MeOH (usually 100–1000μg mL−1, TableS1). Mixtures of these species were also made in MeOH. They were added to water samples, as surrogate standards (SSs), before SPE extraction. Solvent-based calibration standards were prepared in MeOH, or MeOH to FA (99:1) case of acidic species, in the range of concentrations from 0.5 to 300ng mL−1. The concentration of SSs in calibration standards was 50ng mL−1. Samples andsample preparation Wastewater was obtained from four different urban STPs in Galicia (Northwest Spain). All of them apply similar wastewater treatments involving primary and biological (activated sludge) units. Grab samples were used during Solid‑phase extraction andfractionation ofmulticlass pollutants fromwastewater followed… 1 3 Table 1 Summary of target compounds, including LC–ESI–MS/MS determination conditions, instrumental LOQs, and linearity evaluation Group Compound name Precursor ion Q1 (CE) Q2 (CE) Ratio (Q2/Q1) Ret time (min) IS ESI Log D (pH 7) LOQs (ng/ mL)a Linearity (R2, 0.5–300ng mL−1) Acids 2,4-Dichlorophenoxyacetic acid 219.0 161.0 (12) 125.0 (32) 0.08 6.66 2,4-Dichlorophenoxyacetic acid-d5- − 1.14 2 0.994b 4-(2,4-Dichlorophenoxy)butyric acid 247.0 161.0 (4) 35.0 (52) 0.11 9.54 2,4-Dichlorophenoxyacetic acid-d50.97 2 0.990b Candesartan 441.1 235.1 (20) 192.1 (32) 0.90 8.17 Irbesartan-d4 + 0.59 0.3 0.993 Eprosartan 425.1 135.1 (36) 97.1 (28) 0.30 8.10 Irbesartan-d4 + − 0.57 0.3 0.997 Fenoprop 267.0 195.0 (12) 159.0 (32) 0.20 8.57 Irbesartan-d4- − 0.13 0.5 0.993 Irbesartan 429.3 207.1 (24) 195.2 (24) 0.17 9.71 Irbesartan-d4 + 3.31 0.1 0.998 Losartan 423.2 207.1 (28) 405.2 (8) 0.37 9.22 Irbesartan-d4 + 1.51 0.2 0.997 2-Methyl-4-chlorophenoxyacetic acid 199.0 140.8 (12) 35.1 (48) 0.15 6.85 2,4-Dichlorophenoxyacetic acid-d5- − 1.3 1 0.996 Mecoprop 213.0 140.8 (12) 35.1 (48) 0.13 7.68 2,4-Dichlorophenoxyacetic acid-d5- − 0.92 1 0.996 Olmesartan 447.2 207.1 (24) 429.2 (8) 0.34 7.60 Irbesartan-d4 + − 0.78 0.2 0.997 Pentafluoropropanoic acid 163.0 118.8 (8) 68.9 (40) 0.02 1.18 Perfluorooctanoic acid 13C2- − 1.33 0.2 0.999 Perfluorobutanoic acid 213.0 169.0 (5) n.a - 2.27 2,4-Dichlorophenoxyacetic acid-d5- − 0.36 0.1 0.999 Perfluorobutano sulfonic acid 299.0 80.0 (41) 99.0 (33) 0.37 6.99 2,4-Dichlorophenoxyacetic acid-d5- − 1.81 0.1 0.997 Perfluorooctanoic acid 413.0 369.0 (17) 169.0 (5) 0.29 9.90 Perfluorooctanoic acid 13C22.69 0.1 0.996 Perfluorooctano sulfonic acid 499.0 80.0 (49) 99.0 (60) 0.20 10.45 Perfluorooctano sulfonic acid 13C81.01 0.4 0.998 Telmisartan 515.1 497.2 (40) 276.1 (40) 0.45 9.72 Irbesartan-d4 + 3.65 0.2 0.996 Valsartan 436.2 207.1 (32) 235.1 (20) 0.95 9.00 Irbesartan-d4 + − 0.68 0.2 0.996 Valsartan acid 267.1 206.1 (20) 178.1 (36) 0.55 6.62 Valsartan acid-d4 + − 1.95 0.1 0.998 Fernández-Fernández V.et al. 1 3 Table 1 (continued) Group Compound name Precursor ion Q1 (CE) Q2 (CE) Ratio (Q2/Q1) Ret time (min) IS ESI Log D (pH 7) LOQs (ng/ mL)a Linearity (R2, 0.5–300ng mL−1) Bases Acetamipridc223.1 126.0 (27) 56.1 (12) 0.41 5.69 Acetamiprid-d3 + 1.55 0.1 0.999 Amitriptyline 278.2 233.1 (16) 91.1 (36) 1.04 7.76 Imazalil-d5 + 2.28 0.1 0.998 Citalopram 325.2 109.1 (28) 262.1 (16) 0.28 6.53 Flecainide-d4 + 1.02 0.1 0.997 Climbazole 293.1 197.1 (16) 141.0 (24) 0.21 7.54 Climbazole-d4 + 3.47 0.1 0.999 Clomipramine 315.2 86.1 (20) 58.1 (56) 0.75 8.34 Clotrimazole-d5 + 2.6 0.1 0.999 Cloperastine 330.2 201.1 (16) 166.1 (40) 0.62 8.01 Clotrimazole-d5 + 2.9 0.1 0.998 Clotrimazole 277.1 239.1 (60) 165.0 (28) 0.42 8.07 Clotrimazole-d5 + 4.87 0.1 0.998 Fenticonazole 454.9/456.9 198.9 (36) 198.9 (36) 0.63 10.10 Miconazole-d5 + 4.56 0.1 0.998 Flecainide 415.1 398.1 (24) 301.0 (40) 0.59 6.63 Flecainide-d4 + 0.72 0.1 0.999 Fluconazole 307.1 219.9 (20) 70.0 (44) 0.55 5.23 Tramadol 13C d3 + 0.45 0.2 0.998 Haloperidol 376.2 123.0 (44) 165.1 (24) 0.96 7.03 Flecainide-d4 + 2.58 0.1 0.999 Imazalil 297.1 255.0 (12) 158.9 (20) 4.00 7.38 Imazalil-d5 + 3.37 0.5 0.996 Imidaclopridc256.1 175.1 (12) 209.0 (12) 1.00 4.62 Imidacloprid-d4 + 0.07 0.4 0.999 Lamotrigine 256.0 43.1 (40) 210.8 (32) 0.20 4.88 Lamotrigine 13C3 + 1.23 0.5 0.999 Metconazole 320.1 70.0 (28) 124.9 (52) 0.09 10.31 Tebuconazole-d9 + 3.72 0.3 0.999 Miconazole 417.0 158.8 (40) 160.8 (36) 0.94 9.51 Miconazole-d5 + 4.81 0.2 0.999 Myclobutanil 289.1 70.1 (16) 125.1 (32) 0.26 9.27 Myclobutanil-d4 + 3.07 0.1 0.999 N-Desethyl amiodarone 617.9 72.1 (28) 546.9 (24) 0.47 10.15 Miconazole-d5 + 5.51 0.1 0.999 N-Desmethyl citalopram 311.2 108.9 (28) 262.1 (16) 0.41 6.58 Venlafaxine-d6 + 1.02 0.2 0.998 Norsertraline 275.0 158.8 (20) 129.1 (30) 0.10 8.38 Norsertraline 13C6 + 2.8 0.5 0.999 O-Desmethyl venlafaxine 264.2 58.1 (17) 246.2 (13) 0.25 4.59 Venlafaxine-d6 + − 0.37 0.1 0.997 Penconazole 284.1 70.1 (15) 159.0 (30) 0.47 9.98 Tebuconazole-d9 + 4.64 0.1 0.998 Prochloraz 376.0 308.0 (4) 70.1 (24) 0.82 9.78 Myclobutanil-d4 + 4.59 0.2 0.998 Propiconazole 342.1 159.0 (32) 69.1 (16) 0.79 10.17 Myclobutanil-d4 + 3.65 0.2 0.999 Propranolol 260.2 116.1 (20) 183.1 (20) 0.50 6.33 Tramadol 13C-d3 + 0.45 0.2 0.998 Sertaconazole 437.0/439.0 180.9 (40) 180.9 (36) 0.64 9.50 Miconazole-d5 + 5.6 0.1 0.999 Sertraline 306.1 158.9 (36) 275.0 (12) 0.70 8.25 Norsertraline 13C6 + 2.7 0.2 0.999 Tebuconazole 308.1 70.0 (40) 124.9 (47) 0.09 10.07 Tebuconazole-d9 + 3.77 0.2 0.999 Terbutryn 242.1 185.9 (20) 68.0 (60) 0.35 8.07 Imazalil-d5 + 3.38 0.1 0.999 Tetraconazole 372.0 158.9 (32) 70.0 (24) 0.93 9.59 Myclobutanil-d4 + 3.56 0.2 0.997 Thiabendazole 202.0 175.0 (28) 131.1 (40) 0.75 3.97 Tramadol 13C d3 + 2.47 0.1 0.998 Tioconazole 386.9 130.9 (32) 68.9 (24) 0.06 8.76 Miconazole d5 + 4.11 0.5 0.999 Tramadol 264.2 58.1 (20) n.a - 4.79 Tramadol 13C d3 + − 0.06 0.1 0.996 Trazodone 372.2 176.1 (24) 147.9 (40) 0.77 5.73 Tramadol 13C d3 + 2.41 0.3 0.997 Venlafaxine 278.2 58.1 (25) 260.2 (9) 0.25 6.08 Venlafaxine-d6 + 0.39 0.1 0.999 Solid‑phase extraction andfractionation ofmulticlass pollutants fromwastewater followed… 1 3 method development. Integrated (24-h time proportional) samples were employed to measure the concentrations of target compounds, and to evaluate their removal efficiencies during wastewater treatment. Samples were received in glass bottles, sequentially passed through quartz (0.7-μm cutoff) and cellulose acetate filters (0.45-μm pore-size), and stored at 4°C, for a maximum of 24h, before extraction. During method development, different combinations of sorbents were tested. Under final working conditions, a modular SPE setup consisting of a MM 150mg WAX cartridge (top) on-line connected to a RP 60mg HLB one (bottom) was employed. Samples (100mL volume aliquots), spiked with SSs and adjusted at neutral pH (6.5–7.5) when required, were passed through both cartridges at a flowrate of c.a. 5mL min−1. After washing sample containers and connections with SPE sorbents, using 10mL of ultrapure water, cartridges were dried using a gentle stream of nitrogen and connected to an ionic exchange (SCX) one, previously conditioned with MeOH. Neutrals and weak acids were recovered with MeOH flowing through the three sorbents (extract volume 5mL). After disconnecting the three cartridges, compounds with a strong acidic functionality (carboxylic, sulfonic, or tetrazolic groups) were recovered from the WAX cartridge with 2mL of MeOH to NH3 (98:2). Basic species were eluted from the SCX one using 5mL of MeOH to NH3 (95:5) (Fig.1). Every extract was evaporated and adjusted to a final volume of 1mL; moreover, that from the WAX sorbent was acidified with 0.020mL of FA. Reference SPE extractions were carried out using RP HLB cartridges (200mg sorbent), for the concentration of 100mL samples. In this case, all compounds were recovered in the same fraction of methanol (5mL), which was further concentrated to 1mL. Extracts were filtered (0.22-μm pore-size syringe filter) before LC–MS/MS analysis. LC–MS/MS determination conditions Compounds were determined using an ultra-performance liquid chromatography (UPLC) triple quadrupole-type MS system provided by Agilent. The UPLC was 1290 Infinity II connected through a jet-stream ESI source to an i-funnel Agilent 6495 QqQ instrument. Different analytical (LC or UPLC) and delay columns were employed for the separation of target compounds, and to discriminate responses for contaminants existing in the mobile phase from those corresponding to injected compounds. Detailed UPLC conditions for each group of compounds, including type of columns, mobile phase composition, flowrate, and column temperature are compiled in TableS2. The injection volume was set at 2 μL in all methods. Voltages of the ESI source were 3000V and 2000V for positive and negative ionization modes, respectively. The fragmentor voltage was 166V and the MRM parameters for each compound, including Table 1 (continued) Group Compound name Precursor ion Q1 (CE) Q2 (CE) Ratio (Q2/Q1) Ret time (min) IS ESI Log D (pH 7) LOQs (ng/ mL)a Linearity (R2, 0.5–300ng mL−1) Neutrals Clothianidin 250.0 169.1 (8) 131.9 (8) 0.60 5.32 Chlothianidin-d3 + − 1.26 0.2 0.999 Cresyl diphenyl phosphate 341.1 90.9 (44) 151.9 (48) 0.60 9.20 Tributyl phosphate-d27 + 4.76 0.3 0.999 Dimoxystrobin 327.2 205.1 (8) 116.05 (24) 1.05 8.47 Tributyl phosphate-d27 + 5.09 0.1 0.998 Octyl isothiazolinone 214.1 101.9 (16) 43.1 (28) 0.50 7.91 Triclosan 13C6 + 3.69 0.1 0.995 Tris(2-Chloroethyl) phosphate 284.9 98.9 (20) 124.9 (16) 0.85 5.81 Tris(1-Chloro-2-propyl) phosphate-d18 + 1.47 1 0.998 Tris(1-Chloro-2-propyl) phosphate 327.0 98.9 (28) 174.9 (12) 0.33 7.85 Tris(1-Chloro-2-propyl) phosphate-d18 + 2.53 0.1 0.992 Triclosan 286.8/288.9 35.1 (5) 35.1 (5) 0.65 9.05 Triclosan 13C65.28 0.3 0.999 Thiamethoxam 292.0 211.1 (8) 132.0 (24) 0.40 5.00 Thiamethoxam-d4 + 0.16 0.1 0.999 Tributoxyethyl phosphate 399.3 299.2 (13) 199.1 (13) 0.97 9.33 Tributyl phosphate-d27 + 3.28 0.1 0.994 Tributyl phosphate 267.1 98.8 (20) 80.9 (60) 0.22 8.91 Tributyl phosphate-d27 + 3.83 0.1 0.992 Triphenyl phosphate 327.1 77.0 (28) 152.1 (48) 0.40 8.86 Tributyl phosphate-d27 + 4.59 0.2 0.993 a Instrument al LOQs b R2 values for standards in the range of concentrations from 2 to 300ng mL−1 c Transitions of these compounds were included also in the group of neutrals Fernández-Fernández V.et al. 1 3 ionization mode and ratio between qualification (Q2) and quantification (Q1) transitions, are compiled in Table1. In a few cases (e.g., perfluorobutanoic acid and tramadol, TRA), only one transition was available. MRM parameters for compounds employed as SSs are given in TableS1. In addition to the QqQ system, a time-of-flight (TOF) instrument (Agilent 6550) was employed to investigate the distribution of additional compounds in the SPE fractions obtained from non-spiked wastewater samples. In this case, the pseudo-molecular ions ([M + H]+ or [M − H]−) of each species were extracted using a mass window of 20ppm. Compound identities were further confirmed against authentic standards. In this case, the employed LC conditions were those reported in TableS2 for basic species. Extraction efficiency, matrix effects, andaccuracy evaluation The extraction efficiency (EEs, %) of the modular SPE protocol described in “Samples and sample preparation” (accounting for yields of extraction, fractionated elution, and extract concentration to 1mL) was assessed as the ratio of responses (peak area for the Q1 transition without SSs correction) obtained for spiked wastewater aliquots and spiked SPE extracts multiplied by 100. Matrix effects (MEs, %) during ESI were evaluated comparing the difference of responses for spiked and non-spiked extracts of each sample (raw and treated wastewater) with those observed for a solvent-based standard of the same concentration. Values close to 100% correspond to similar ionization efficiencies for sample extracts versus solvent-based standards. On the other hand, normalized response ratios below and above 100% mean suppression and enhancement of compound ionization in sample extracts versus solvent-based standards [20]. The above parameters (EEs and MEs) were evaluated using an additional level of 1ng mL−1 referred to the water sample (equivalent to 100ng mL−1 in the corresponding SPE extract). The accuracy of the final procedure was investigated using samples spiked at three levels: 50, 200, and 1000ng L−1. For each type of wastewater, non-spiked (n = 3 replicates) and spiked fractions (n = 3, for each addition level) were fortified with SSs (500ng L−1) and processed as reported in “Samples and sample preparation.” Responses obtained for each compound were corrected with that measured to the assigned SS (Table1), and compared to those obtained for solvent-based standards (concentration range from 0.5 to 300ng mL−1). Two different kinds of blanks were considered during method development and application. Instrumental blanks corresponded to simulated (false) injections. That is, the injection valve changes from the by-pass to the main-pass Fig. 1 Scheme of sample concentration and elution steps in the modular solid-phase extraction procedure WAX HLB SCXWAX WAX HLB SCX Extraction Elution Sample Waste Neutrals AcidsBases MeOH MeOH:NH3 (98:2) MeOH:NH 3 (95:5) Solid‑phase extraction andfractionation ofmulticlass pollutants fromwastewater followed… 1 3 position, with the mobile phase flowing through the injector loop and the injection needle to the LC column; however, the autosampler does not select any vial (sample, procedural blank, standard or solvent). These experiments permitted identifying contamination problems related to the UPLC system and/or the mobile phase (mainly the aqueous phase). Procedural blanks were prepared using ultrapure water samples, spiked only with the selection of SSs, and submitted to the adopted modular SPE protocol. This type of blanks is useful to detect contamination problems related to the sample preparation process. Instrumental limits of quantification (LOQs) were calculated as the concentration of each compound producing a response with a signal to noise ratio (S/N) of 10 for the less intense of the selected transitions (usually Q2) in solventbased standards. Procedural LOQs were estimated from instrumental LOQs, considering a 100-fold concentration factor, corrected with EEs and MEs when they were outside the range of values between 80 and 120%. Moreover, for compounds found in the procedural blanks, the LOQs of the method were calculated as the average concentration measured in blank extracts plus10 times its standard deviation. Results anddiscussion LC–ESI–MS/MS conditions Three LC-QqQ-MS methods were employed to enhance the detectability of each group of considered compounds (acids, bases, and neutrals). In case of IMI and ACE, their transitions were included in methods developed for neutral and basic species. Except for TCS, the rest of neutrals and bases were determined in ESI ( +); thus, FA was used as mobile phase modifier (0.1%) to promote their ionization. ACN, instead of MeOH, was preferred as organic mobile phase to reduce the retention of some highly lipophilic organophosphate flame retardants included in the group of neutrals, and to decrease the pressure in the UPLC system considering that two identical columns (delay and analytical columns) were required to cope with instrumental blanks noticed for some compounds within this group. As regards acidic compounds, FA (0.1%) and NH4F (1mM) were tested as mobile phase additives. The ARAII drugs showed a higher ionization efficiency under ESI ( +). On the other hand, herbicides and perfluorinated compounds led only to their [M − H]− ions. Thus, ESI ( +) and ESI ( −) modes were combined in this method. Depending on the type of modifier, differences between 20 and 40% in responses obtained for herbicides and perfluorinated compounds were noticed. However, most ARA-II drugs rendered one order of magnitude higher responses using NH4F as modifier (Fig.S1). The instrumental LOQs of compounds considered in this research were not only conditioned by their ionization efficiencies, but also by the existence of instrumental contamination sources. These problems were noticed in LC–MS/ MS records obtained for simulated injections. Particularly, mobile phases contributed significantly to the presence of several perfluorinated and organophosphate compounds in instrumental blanks. To discriminate the response due to instrumental contamination from that of the standard, delay columns were connected between the mobile phase mixer and the injector of the UPLC system. These columns are described in TableS2. Fig.S2 illustrates the separation of the chromatographic peak (earlier signal) for a low concentration standard of selected compounds: tris(2-chloro-isopropyl) phosphate (TCPP), tributoxyethyl phosphate (TBEP), and perfluorooctanoic acid (PFOA), from that corresponding to mobile phase contamination (latter peak), after installing the corresponding delay column. Under final working conditions, the LC–ESI–MS/MS methods achieved instrumental LOQs in the range from 0.1 to 0.5ng mL−1 for most of the target compounds. In all cases, linear responses were attained for concentrations up to 300ng mL−1 (Table1). Solid‑phase extraction andfractionated compound elution Preliminary SPE experiments were carried out using spiked aliquots of ultrapure water, considering different combinations of sorbents. The first tested setup involved retention of the suite of compounds (neutrals, acids, and bases) in a MM MCX sorbent [9]. In this case, samples were adjusted at pH 3 to improve the retention of highly polar, acidic species, in the MCX sorbent, through RP interactions. Neutrals are expected to be retained by the same mechanism and bases through electrostatic interactions with negatively charged sites of the polymer. Obviously, the latter interactions are favored in acidified samples. During the elution step, the MCX cartridge was connected to an anionic exchange (SAX) one. Distribution of compounds was investigated in the following solvent fractions: MeOH (5mL) flowing through both cartridges connected in series, MeOH to NH3 (95:5) recovered from the upper MCX sorbent (after removing the SAX one), and MeOH to FA (95:5) collected from the SAX cartridge. Under these conditions, neither the retention nor the fractionation of neutrals and acidic compounds was satisfactory. As example, the short-chain perfluorinated compounds (C3 carboxylic acid and C4 carboxylic and sulfonic acids) were not retained by the MCX sorbent; so, their EEs remained below 20%. Compounds with acidic and basic moieties in their structures (i.e., most of the ARA-II pharmaceuticals) were found in the fraction of basic drugs, whilst acidic drugs (e.g., valsartan, VAL, and valsartan acid, Fernández-Fernández V.et al. 1 3 VALA), herbicides (phenoxy acids), and C8 perfluorinated compounds eluted together with neutrals in the methanolic fraction. That is, they were not fractionated from neutrals by the SAX sorbent. To sum up, this setup did not show any advantage compared to the single use of the MM MCX sorbent, enabling the fractionated elution of bases from neutral and acids. The second SPE setup considered concentration of water samples, at neutral pH, using a weak anionic exchange MM sorbent (WAX) [12]. In the elution step, this sorbent was connected to a pure cationic exchanger (SCX) cartridge. As in the former case, three different fractions were collected. MeOH was passed through both cartridges connected in series to recover neutrals. Thereafter, they were disconnected and eluted with MeOH to NH3 (98:2). Above 95% of the responses (peak areas) observed for the suite of selected compounds was noticed in the expected SPE fraction accordingly to their preliminary classification given in Table1. The only exceptions were the neonicotinoid insecticides imidacloprid (IMI) and acetamiprid (ACE), distributed between the neutral and the basic fractions in similar percentages. On view of these preliminary results, the second setup was adopted, and retention and elution conditions were reevaluated using spiked wastewater samples. Some highly polar and basic species, such as TRA, venlafaxine (VEN) and O-desmethyl venlafaxine (O-DVEN), citalopram (CIT), and N-desmethyl citalopram (N-DCIT) (their log D values ranged from − 0.4 to 1.0 at neutral pH, Table1), were not retained quantitatively by the WAX sorbent. For 100mL volume wastewater samples, between 5 and 18% of the responses measured for these compounds were noticed in the extract from a second WAX cartridge on-line connected to the first one. In order to improve their retention, the mixedmode WAX cartridge was combined (placed on top) with 60mg HLB one to reinforce the RP retention mechanism during sample concentration. As regards the volume and the type of solvents employed in the fractionated elution protocol, 5mL of MeOH was passed through the ternary combination of sorbents (MM, RP, and strong cationic exchange) to recover neutrals (Fig.1). Triclosan (TCS), selected as representative of weak acidic phenolic species (predicted pKa 7.8), was also quantitatively eluted in this fraction. Again, IMI and ACE were partially retained by the SCX sorbent, being detected in neutral and, mostly, basic fractions. The other two neonicotinoids included in the study (thiamethoxam, THM, and clothianidin, CLO) were found only in the neutral fraction (methanolic extract). Likely, the chloronicotinic ring existing in the structures of IMI and ACE leads to a weak interaction of both compounds with the strong anionic exchange sorbent. None of the tested acidic compounds was released from the WAX cartridge during elution of neutrals. So, the HLB cartridge was discarded after this step (Fig.1). Acids were recovered using just 2mL of MeOH with a 2% of NH3, which is in agreement with the data published by G. Castro and co-workers [12] for SPE of ARA-II species using WAX cartridges. Finally, basic compounds showed a strong interaction with the SCX sorbent. Their quantitative elution (particularly in case of those containing tertiary amine groups) was required to increase the percentage of NH3 added to MeOH from 2 to 5%, using 5mL of this mixture. Performance ofthemethod The EEs of the sample preparation process, calculated as defined in “Extraction efficiency, matrix effects, and accuracy evaluation,” are summarized in Table2. For most compounds, EEs ranged from 80 to 120%. In a few cases, values between 70 and 130% were noticed. On the other hand, six compounds showed non-quantitative extraction yields. Within the group of bases, EEs around 50% were observed for the pharmaceuticals: fenticonazole, miconazole, and sertaconazole, and the drug metabolite N-desethyl amiodarone. The four are relatively lipophilic compounds, with log D values above 4.5 (Table1). Very likely, non-quantitative EEs are the result of sorption losses on glassware and connections with SPE cartridges. Although it was attempted to improve their recoveries by addition of MeOH to the water samples (10–20mL of methanol per 100mL of sample), this approach led to retention problems for polar basic species, positively charged at neutral pH values. Since the latter ones have a higher potential to be present in the water phase than more lipophilic drugs, no organic solvent was added to samples before SPE. The 2nd group of compounds displaying non-satisfactory recoveries was the neonicotinoids IMI and ACE. As commented in “LC–ESI–MS/MS conditions,” both species were distributed between neutral and basic fractions. In raw wastewater, the overall EEs for each group of pollutants were 94% (acids), 91% (bases) and 86% (neutrals). For treated wastewater, average SPE EEs were 96%, 76%, and 94% for acids, bases, and neutrals, respectively. The selectivity of the modular SPE methodology was assessed comparing the responses obtained for the three groups of compounds in spiked extracts from raw and treated wastewater, with those corresponding to solvent-based standards prepared in MeOH [20]. Moreover, the normalized response ratios were compared to those obtained using a HLB sorbent, applied to 100mL aliquots of the same water samples. In case of acids, most species showed normalized responses in the range from 80 to 120% (Fig.2A). The only exception was VALA affected by moderate (68%) and strong signal suppression (44%) effects in the modular SPE and HLB extracts, respectively (Fig.2A). It is worth noting that, for acidic compounds, the RP methodology (based on the use of an HLB 200-mg cartridge for concentration of 100-mL samples) failed to Solid‑phase extraction andfractionation ofmulticlass pollutants fromwastewater followed… 1 3 Table 2 Extraction efficiencies for spiked samples of raw and treated wastewater using the modular SPE procedure, n = 3 replicates Group Compound Raw wastewater Treated wastewater Group Compound Raw wastewater Treated wastewater Mean RSDs (%) Mean RSDs (%) Mean RSDs (%) Mean RSDs (%) Acids 2,4-Dichlorophenoxyacetic acid 97% 3% 102% 1% Bases Metconazole 97% 4% 84% 4% 4-(2,4-Dichlorophenoxy) butyric acid 97% 7% 99% 5% Miconazole 52% 19% 67% 1% Candesartan 88% 2% 99% 2% Myclobutanil 102% 4% 88% 5% Eprosartan 97% 3% 96% 2% N-Desethyl amiodarone 42% 22% 64% 4% Fenoprop 101% 5% 100% 2% N-Desmethyl citalopram 93% 3% 81% 5% Irbesartan 92% 2% 97% 2% Norsertraline 75% 8% 75% 4% Losartan 88% 3% 95% 4% O-Desmethyl venlafaxine 118% 3% 94% 4% 2-Methyl-4-chlorophenoxyacetic acid 98% 6% 97% 3% Penconazole 103% 3% 85% 5% Mecoprop 98% 8% 101% 3% Prochloraz 90% 5% 79% 4% Olmesartan 98% 3% 99% 2% Propiconazole 97% 4% 85% 5% Pentafluoropropanoic acid 83% 3% 82% 4% Propranolol 101% 3% 82% 4% Perfluorobutanoic acid 88% 3% 100% 2% Sertaconazole 42% 29% 65% 2% Perfluorobutano sulfonic acid 92% 4% 102% 3% Sertraline 90% 3% 77% 2% Perfluorooctanoic acid 86% 1% 116% 5% Tebuconazole 98% 2% 84% 4% Perfluorooctano sulfonic acid 88% 4% 75% 2% Terbutryn 100% 3% 79% 3% Telmisartan 92% 3% 97% 2% Tetraconazole 100% 4% 86% 5% Valsartan acid 104% 3% 84% 4% Thiabendazole 128% 15% 85% 5% Valsartan 106% 2% 94% 3% Tioconazole 71% 9% 70% 2% Bases Acetamiprid 61% 19% 57% 17% Tramadol 113% 3% 87% 5% Amitriptyline 102% 4% 79% 3% Trazodone 94% 4% 75% 5% Citalopram 105% 5% 82% 4% Venlafaxine 112% 3% 86% 5% Climbazole 96% 3% 82% 4% Neutrals Clothianidin 94% 4% 95% 2% Clomipramine 94% 3% 73% 3% Cresyl diphenyl phosphate 80% 8% 84% 5% Cloperastine 91% 4% 20% 31% Dimoxystrobin 89% 5% 99% 4% Clotrimazole 80% 6% 71% 2% Octyl isothiazolinone 81% 7% 85% 7% Fenticonazole 57% 26% 53% 5% Tris(2-Chloroethyl) phosphate 87% 7% 101% 2% Flecainide 110% 5% 87% 4% Tris(1-Chloro-2-propyl) phosphate 85% 13% 106% 4% Fluconazole 105% 4% 87% 5% Triclosan 87% 12% 79% 5% Haloperidol 109% 7% 79% 4% Thiamethoxam 86% 3% 96% 2% Imazalil 102% 6% 80% 3% Tributoxyethyl phosphate 93% 7% 105% 5% Imidacloprid 45% 15% 56% 21% Tributyl phosphate 77% 10% 96% 3% Lamotrigine 118% 5% 88% 4% Triphenyl phosphate 90% 19% 92% 4% Fernández-Fernández V.et al. 1 3 Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s0021602204066-8. Acknowledgements We acknowledge Agilent for providing access to the LC-ESI-MS/MS instrumentation employed in this study. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was supported by Spanish Government and Xunta de Galicia through grants PGC2018-094613-BI00 and ED431C 2021/06, both co-funded by the EU FEDER program. Declarations Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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