Farmaceuticos en Agua

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1 <PE-AT>Multi-residue analysis of 44 pharmaceutical compounds in environmental water
2 samples by solid phase extraction coupled to liquid chromatography – tandem mass
3 spectrometry
4 Carole Miossec a, Laurent Lanceleur a and Mathilde Monperrus a*
a
5 CNRS/ UNIV PAU & PAYS ADOUR/ E2S UPPA, INSTITUT DES SCIENCES ANALYTIQUES ET DE
6 PHYSICOCHIMIE POUR L'ENVIRONNEMENT ET LES MATERIAUX – MIRA, UMR5254, 64600,
7 ANGLET, France
8 Mathilde Monperrus
9 University of Pau and Pays de l’Adour
10 1 Allée Montaury
11 64600 Anglet
12 mathilde.monperrus@univ-pau.fr
13 +33(0)5 59 57 44 16
14 Abbreviations: AEAG, agence de l'eau adour-garonne; ERDF, european regional
15 development fund; GF, glass fiber; IDL, instrumental detection limit; ME, matrix effect;
16 MeOH, methanol; MQL, method quantification limit; MRM, multiple reaction monitoring;
17 PTFE, polytetrafluoroethylene; PVDF, polyvinylidene difluoride; RT, retention time; SPM,
18 suspended particulate matter; TQ, triple quadrupole; WWTP, wastewater treatment plant
Received: 11 27, 2018; Revised: 03 06, 2019; Accepted:03 08, 2019
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jssc.201801214.
This article is protected by copyright. All rights reserved.

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19 Keywords: liquid chromatography - tandem mass spectrometry / pharmaceuticals / solid
20 phase extraction / water
21 Abstract
22 A solid phase extraction combined with a liquid chromatography - tandem mass
23 spectrometry analysis has been developed and validated for the simultaneous
24 determination of 44 pharmaceuticals belonging to different therapeutic classes (i.e.
25 antibiotics, anti-inflammatories, cardiovascular agents, hormones, neuroleptics and
26 anxiolytics) in water samples.
27 The sample preparation was optimized by studying target compounds retrieval after i) water
28 filtration, ii) solid phase extraction using Waters Oasis HLB cartridges at various pH and iii)
29 several evaporation techniques. The method was then validated by the analysis of spiked
30 estuarine waters and wastewaters before and after treatment. Analytical performances
31 were evaluated in terms of linearity, accuracy, precision, detection and quantification limits.
32 Recoveries of the pharmaceuticals were acceptable, instrumental detection limits varied
33 between 0.001 and 25 pg injected and method quantification limits ranged from 0.01 to
34 30.3 ng/L. The precision of the method, calculated as relative standard deviation, ranged
35 from 0.3 to 49.4%.
36 This procedure has been successfully applied to the determination of the target analytes in
37 estuarine waters and wastewaters. Eight of these 44 pharmaceuticals were detected in
38 estuarine water, while 26 of them were detected in wastewater effluent. As expected, the

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39 highest values of occurrence and concentration were found in wastewater influent.<PE-
40 FRONTEND>
41 1. Introduction
42 Over the past few years, there was an increasing interest to investigate the impact of
43 emerging contaminants on the environment. Among the great diversity of pollutants
44 reaching our water supplies, pharmaceutically active compounds are one of the largest
45 input [1]. As pharmaceuticals consumption is continuously increasing [2], it raises concerns
46 about their impact on the environment and undesired physiological effects they can cause
47 to aquatic organisms [3–8]. Their presence was investigated worldwide in different aquatic
48 matrices (e.g. water, sediments and biota) [9–12], and a large number of compounds were
49 detected, especially in wastewaters at concentrations ranging from nanogram to microgram
50 per liter [13–19]. Consequently, there is a growing need to develop reliable analytical
51 methods that enable rapid, robust, sensitive and selective determination of these emerging
52 pollutants at trace levels in environmental samples.
53 Simultaneously analyzing several groups of compounds with different chemical and physical
54 properties is challenging, as it generally requires a compromise in the selection of
55 experimental conditions [20]. Previous studies report the use of gas chromatography - mass
56 spectrometry (GC/MS) [21] and more popularly liquid chromatography - tandem mass
57 spectrometry (LC/MS/MS) [22–25] due to easier sample preparation requirements for polar
58 chemicals (i.e., no derivatization needed) and higher selectivity and sensitivity. This
59 technology has therefore been successfully used for the simultaneous determination of

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60 extensive lists of pharmaceuticals in complex matrices, such as human urine and sewage
61 sludge [26,27].
62 The preparation of liquid environmental samples for LC/MS/MS analysis typically involves a
63 filtration to remove suspended particles and off-line [28] or on-line [29] solid phase
64 extraction (SPE) pre-concentration step in order to ensure adequate sensitivity for MS
65 detection and therefore accurate analytes determinations at sub-ppb levels. A drawback
66 associated to LC/MS/MS methods is to manage matrix effects, which are attributed to the
67 presence of undesirable sample components that co-elute with the analytes, altering the
68 ionization process [28]. Consequences of matrix effects are suppression or enhancement of
69 the signal, which can affect both identification and quantification of analytes. Matrix effects
70 depend on each analyte/matrix combination, but also on the sample preparation, the
71 chromatographic separation, mass spectrometry instrumentation and the ionization
72 conditions [30]. The evaluation of matrix effects should be included in the validation process
73 of the method considering the different matrices studied. Several strategies were proposed
74 to solve matrix effects, including modifications of sample pre-treatment, chromatographic
75 or MS conditions and calibration techniques [30]. The use of deuterated internal standards
76 is, by far, the most used in the pharmaceutical residues analysis field [22,28].
77 The objective of the present study was to develop a multi-residue analytical methodology
78 based on a SPE protocol followed by LC/MS/MS detection for the simultaneous analysis of
79 44 commonly used human and veterinary pharmaceuticals (including antibiotics, anti-
80 inflammatories, cardiovascular agents, hormones, neuroleptics and anxiolytics) in water
81 samples, in order to provide a routine method for the evaluation of the environmental
82 quality of aquatic ecosystems. The sample preparation was optimized by studying target

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83 compounds retrieval after water filtration, SPE at various pH and several evaporation
84 techniques. Matrix effects and analytical performances of the optimal procedure were
85 evaluated. Finally, the developed method was successfully applied to investigate occurrence
86 of target pharmaceuticals in estuarine waters and wastewaters from the Basque country
87 (France).
88 2. Materials and Methods
89 2.1 Reagents and materials
90 Reference standards of acetaminophen, acetazolamide, amiodarone, ampicilline, atenolol,
91 azithromycin, caffeine, carbamazepine, ciprofloxacin, clarithromycine, cyclophosphamide,
92 diclofenac, doxycycline, erythromycin A, estrone (E1), 17β-oestradiol (E2),
93 17α-ethinylestradiol (EE2), flumequine, gemfibrozil, hydrochlorothiazide, ibuprofen,
94 josamycin, ketoprofen, lorazepam, losartan, metoprolol, metronidazole, niflumic acid,
95 nordiazepam, 19-norethindrone, norfloxacin, ofloxacin, oxazepam, oxolinic acid, phenazone,
96 piperacillin, rifampicin, roxithromycine, spiramycin, sulfadiazine, sulfamethazine,
97 sulfamethoxazole, tetracycline, trimethoprim and tylosine were supplied by Sigma Aldrich
98 (Saint-Louis, USA) and classified as analytical grade (>98%). Isotopically labeled compounds
99 used as internal standards were atenolol-d7, carbamazepine-d10, ibuprofen-d3,
100 nordiazepam-d5 and ofloxacin-d3 also purchased from Sigma Aldrich. All standard stock and
101 working solutions were prepared in methanol and stored in the dark at -20°C.
102 Methanol (MeOH) and acetonitrile (ACN) were of LC/MS grade and supplied by Fisher
103 (Hampton, USA). Acetone (laboratory reagent, 99.5%) was used for cleaning all the
104 glassware and was supplied by Fisher. Formic acid (HCOOH, content >98%), acetic acid,

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105 sodium acetate trihydrate, hydrochloric acid, ammonium hydroxide solution and
106 Ethylenediaminetetraacetic acid disodium (Na2EDTA) were purchased from Sigma Aldrich.
107 Ultrapure water was obtained with a PURELAB Classic water purification system from Veolia
108 (Paris, France). Cartridges used for solid phase extraction were Oasis HLB (60mg, 3cc) from
109 Waters (Milford, USA)
110 2.2 Cleaning procedure
111 All glassware was carefully cleaned before use by soaking into acetone and placing it for 25
112 min in an ultrasonic bath. Non-volumetric glassware was further heated at 450°C for 4 hours
113 in a B180 muffle furnace from Nabertherm (Bremen, Germany) and then stored covered
114 with aluminum sheets.
115 2.3 Samples collection and pre-treatment
116 Estuarine samples were collected from the Adour Estuary (France), while influent and
117 effluent samples were collected from a local wastewater treatment plant (WWTP) (France).
118 pH and conductivity were determined in the field with a handheld multiparameter HANNA
119 HI9829 probe (table 1). An appropriate amount of homogenized water sample was filtered
120 on the sampling day through pre-combusted GF/F membranes and suspended particulate
121 matter (SPM) concentrations were determined by weighting the particulate fraction
122 remaining on the filter after dryness.
123 Samples were filtered through 0.45 µm cellulose acetate membrane filters (Grosseron,
124 Couëron, France) and stored at -20°C prior to extraction and analysis. A suitable volume of
125 Na2EDTA solution (0.1M) was added to achieve a final concentration of 0.1% (g solute / g

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126 solution) in order to improve the extraction efficiency of target compounds as
127 recommended by previous pharmaceutical compounds studies [1,31,32].
128 2.4 Solid phase extraction procedure
129 Internal standards (atenolol-d7, carbamazepine-d10, ibuprofen-d3, nordiazepam-d5 and
130 ofloxacin-d3) were added at a concentration of 100 ng/L, 100 ng/L, 2,500 ng/L, 100 ng/L,
131 and 500 ng/L, respectively, to each sample. The solid phase extraction was carried out with
132 a SPE vacuum manifold (Agilent, France). Oasis HLB (60mg, 3mL) cartridges were
133 conditioned with 5mL of MeOH and 5mL of ultrapure water. 200 mL of samples were
134 applied to the SPE cartridges. Water was used to rinse the SPE column before the elution
135 with 8mL of MeOH. The eluate was collected in 10 mL glass tubes, evaporated to dryness
136 under a gentle air stream using a TurboVap LV Evaporator system (Zymark, Hopkinton, USA),
137 and dissolved in 1mL water/MeOH (75/25 v/v), giving a 200 times concentrated sample.
138 Finally, extracts were vortexed, transferred into vials and kept at -20°C until analysis (figure
139 1).
140 2.5 Liquid chromatography
141 Chromatographic analyses were carried out using an Acquity UPLC system (Waters)
142 equipped with a quaternary solvent manager and a sample manager. Chromatographic
143 separation was performed using an Acquity UPLC HSS T3 (1.8µm particle size, 50mm x
144 2.1mm i.d.,) column (Waters) preceded by a guard column (1.8µm particle size, 5mm x
145 2.1mm i.d.,) of the same packing material from the same manufacturer, at a flow rate of 0.4
146 mL/min. Column was kept at 40°C and sample manager was maintained at 15°C. Three
147 separate sets of solvents, and therefore three injections were used for the quantification of

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148 all compounds. The analysis in positive ionization mode was performed using ultrapure
149 water with 0.1% formic acid as eluent (A) and acetonitrile as eluent (B). For the analysis in
150 negative ionization mode, eluent (A) was ultrapure water with 0.01% formic acid and eluent
151 (B) was acetonitrile. For the analysis of hormones (estrone, 17β-oestradiol and
152 17α-ethinylestradiol) in negative ionization mode, eluent (A) was ultrapure water and eluent
153 (B) was acetonitrile. For the three methods, the initial composition was 2% B. It held for 2
154 min, increased linearly to reach 60% at 4 min and then 100% at 6 min. It held 1 min,
155 returned to the initial composition (2% B) at 7.1 min and held for 3 min, totaling a 10
156 minutes analysis run time (See example of chromatograms in Supporting Information figure
157 1). Sample injection volume was 5 µL.
158 2.6 Tandem mass spectrometry
159 Mass spectrometry was performed by a Xevo TQ MS (Triple quadrupole) with an
160 electrospray (ESI) interface (Waters). Drying gas, as well as nebulizing gas was nitrogen,
161 generated by pressurized air in a Nitrocraft nitrogen generator (Air Liquide). Cone gas and
162 desolvatation gas flows were set at 10 L/h and 600 L/h, respectively. Source temperature
163 was set to 150°C and desolvatation temperature to 600°C. Capillary voltages of 0.5 kV
164 (positive ionization mode) and 1.0 kV (negative ionization mode) were applied. Collision gas
165 was Argon with a purity greater than 99.999% (Linde). Instrument control, data acquisition
166 and data treatment were performed using Waters MassLynx software. Quantification was
167 carried out in Multiple Reaction Monitoring (MRM) mode, selecting two characteristics
168 transitions for each compound.

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169 Table 2 shows optimized MRM transitions with their respective ionization mode, retention
170 times, ion ratios and internal standards.
171 3. Results and discussion
172 3.1 Sample preparation optimization
173 3.1.1 Filtration
174 The analysis of organic pollutants in aqueous samples typically involves a filtration step to
175 remove suspended particles. To date, different filtration materials have been indifferently
176 used in pharmaceutical research: cellulose acetate [33–35], nylon [22,36],
177 polytetrafluoroethylene (PTFE) [37], polyvinylidene difluoride (PVDF) [38] and glass fiber
178 (GF/F) [22,23,36].
179 In order to study the potential loss of target compounds which may occur during the
180 filtration step (and eventually leading to an underestimation of final results), various types
181 of membrane filters were tested. Water was spiked with the target compounds at a level of
182 100 µg/L, filtered through 0.7 µm glass fiber (GF/F), 0.45 µm cellulose acetate and 0.45 µm
183 PVDF membrane filters and then analyzed following the above described LC/MS/MS
184 method.
185 Responses obtained for pharmaceutical compounds were compared with those obtained
186 from the analysis of the same spiked non-filtered water (figure 2). A large majority of
187 compounds were satisfactorily recovered (between 90 and 110%) with all types of filter.
188 Norfloxacin, ofloxacin and ciprofloxacin presented slightly low recoveries with cellulose
189 acetate filter (50-55%) and PVDF filter (66-69%), but better recoveries with the glass fiber

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190 filter (77-80%). Meanwhile, spiramycin and azithromycin were completely retained by glass
191 fiber filter but showed higher recoveries with cellulose acetate (49% and 52%) and PVDF
192 (77% and 69%) filters. Finally, amiodarone was entirely absorbed (recoveries below 5%)
193 when cellulose acetate and PVDF membranes were used, while recovery with glass fiber
194 filters was a little higher but still not satisfactory (32%).
195 In summary, cellulose acetate (0.45 µm) membrane filters seems to be the best choice for
196 the filtration of aqueous environmental samples intended for the analysis of this kind of
197 pharmaceutical compounds, showing satisfactory results for 44 out of the 45 tested
198 compounds and also cheaper than PVDF membrane filters.
199 3.1.2 Extraction pH
200 To date, different acidification rates have been used for the extraction of pharmaceuticals
201 with Oasis HLB cartridges: acidification to pH 2-3 [24,34,39], adjustment to pH 7 [23,37] and
202 pH non adjusted [22,28,36]. The aim of this method development is to be able to extract a
203 maximum of our target compounds (which includes weakly basic and weakly acid
204 molecules) in one single step.
205 In order to study effects of pH on extraction efficiencies, the pH value of ultrapure water
206 was adjusted at pH 3, 5 and 7 by adding an acetate buffer (pH 4) prepared with acetic acid
207 and sodium acetate and adjusting to pH 3 with hydrochloric acid and pH 5 with ammonium
208 hydroxide solution. 200 mL of these waters were then spiked with 500 ng/L of target
209 compounds and the extraction protocol was applied.
210 Results of extraction experiments are summarized in figure 3. Decrease in pH led to a
211 reduction in the extraction efficiency for some compounds (metronidazole, acetaminophen,

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212 acetazolamide, atenolol and erythromycin A). Extractions from samples at pH 7 provided the
213 highest retrievals, consistent with findings from Gomez et al. (2006) [40] and Gros et al.
214 (2006) [22]. As pH of environmental water samples are usually close to 7, SPE without pH
215 adjustment was selected as the optimal way to extract all target analytes in one single step.
216 3.1.3 Evaporation
217 Evaporation stage could be a critical step, especially for the most volatile compounds which
218 may be degraded. To date, different evaporation conditions have been used for the
219 evaporation of pharmaceutical compounds: evaporation to dryness under nitrogen stream
220 [22,32,36], evaporation to dryness under nitrogen stream at 40°C [23,28,37] and partial
221 evaporation under nitrogen stream. In order to investigate retrievals of compounds after
222 evaporation, 8 mL of a solution containing all compounds in methanol was evaporated
223 under three different conditions: to dryness under a gentle air stream and reconstitution
224 with 1 mL of water/MeOH (75/25 v/v), to dryness under a gentle air stream at 40°C and
225 reconstitution with 1 mL of water/MeOH (75/25 v/v), under a gentle air stream and
226 evaporation stopped when approximately 250 µL of solution remained in the tube and then
227 adjusted to 1mL with water.
228 Areas of target molecules were compared to areas obtained for a non-evaporated solution
229 (figure 4). Results showed that all compounds had the same overall behavior and resisted
230 well to evaporation (recoveries above 70% for 84% of the target compounds). A significant
231 loss was noticed for some antibiotics (tetracycline, doxycycline, josamycin, rifampicin),
232 whatever the evaporation technique was. As there were no significant differences between
233 these different techniques, evaporation to dryness under air stream at room temperature

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234 was chosen as a gentle evaporation method in order to concentrate while preserving
235 molecules as much as possible. Losses observed for some analytes were subsequently
236 bypassed by the quantification with an internal procedural calibration.
237 3.2 Method validation
238 3.2.1 Quantification and quality control
239 An internal procedural calibration with deuterated analogs of the target analytes was used
240 to quantify concentrations of target compounds in order to take in account losses during
241 SPE and evaporation steps. An eight-point calibration curve was performed in 200 mL of
242 ultrapure water spiked with increasing pollutants concentration levels ranging from 0 to
243 1,000 ng/L as well as internal standards at various concentration (described in 2.4), and the
244 optimized procedure was carried out.
245 All analyses were subjected to quality control procedures. For each group of samples,
246 solvent blanks (water/MeOH 75/25 v/v), procedural blanks and matrix spike (500 ng/L) were
247 prepared and run in the sequence to check carryover and system performances.
248 3.2.2 Matrix effects evaluation
249 Matrix effects that can cause enhancement or suppression of analytical signals are
250 frequently observed in the chemical analysis field [41,42]. This phenomenon is due to matrix
251 compounds which are eluted with the same retention time as the target compounds. Matrix
252 effects depend on the nature of the matrix and the efficiency of the sample preparation
253 step. Therefore, the sample preparation step should eliminate interfering compounds while
254 retaining target analytes. To thoroughly study this phenomenon, estuarine water, and

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255 influent and effluent wastewaters were spiked by adding 500 ng/L of the target compounds
256 and the optimized procedure was carried out.
257 Matrix effects (ME, %) were calculated according to:
( ) ( )
( ) ( )
( )
258 where A(matrix) is the area in the spiked matrix, A(blank) is the area in the non-spiked
259 matrix and A(solvent) is the area in the spiked ultrapure water. Results are shown in table 3
260 for all target analytes.
261 These results showed variability among the evaluated compounds and also among the
262 different types of waters. The majority of the compounds were negatively influenced by the
263 matrix (suppression of the signal during ionization), while only some of them were positively
264 affected (e.g. spiramycin, azithromycin, rifampicin, …). As expected, signal suppression has
265 been observed to be higher in the WWTP influent water (the most complex matrix) than in
266 the WWTP effluent water and than in the estuarine waters. The addition of deuterated
267 internal standards is therefore mandatory to correct matrix effects.
268 3.2.3 Method performances
269 Analytical performances of the optimized method are reported in table 4. Linearity (R2) was
270 calculated in the range 0-1000 ng/L. Instrumental detection limits (IDL) were determined as
271 lowest injected compound concentrations in solvent matrix that yielded a signal-to-noise
272 (S/N) ratio of 3. Method quantification limits (MQL) were determined as lowest injected
273 compound concentrations in procedural ultrapure water matrix that yielded a signal-to-

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274 noise (S/N) ratio of 10. Recoveries were also determined, and precision was expressed as
275 the relative standard deviation (RSD) of 3 replicates.
276 Coefficients of determination were higher than 0.990 for all compounds except two
277 (doxycycline and 17α-ethinylestradiol), demonstrating that the method is linear in the range
278 assayed. Recoveries achieved for a majority of target compounds in estuarine waters and
279 wastewaters were acceptable, and precision was below 50 % for all compounds.
280 3.3 Application to real water samples
281 The optimized and validated methodology was applied to estuarine samples, as well as
282 influent and effluent samples from a local wastewater treatment plant.
283 Results presented in table 5 show that only 26 to 44 molecules were detected at
284 concentrations above the MQL at least once. Concentrations ranged from the MQL to 140
285 µg/L (acetaminophen in WWTP influent). From the 26 quantified molecules, only 5
286 pharmaceutical compounds were found in all samples (atenolol, carbamazepine, caffeine,
287 oxazepam and hydrochlorothiazide). Eighteen targeted compounds were not detected
288 during this sampling campaign. Eight of these 44 pharmaceuticals were detected in
289 estuarine water, while 26 of them were detected in wastewater effluent. As expected, the
290 highest values of occurrence and concentration were found in wastewater influent with
291 values above the µg/L for acetaminophen, caffeine, ibuprofen, hydrochlorothiazide,
292 ketoprofen, diclofenac, ciprofloxacin and atenolol.
293
294

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295 4. Concluding remarks
296 A SPE-LC/MS/MS method was successfully developed for the simultaneous quantification of
297 44 pharmaceutical compounds in environmental water samples. Matrix effects were
298 calculated for estuarine waters and wastewaters and resulted in a higher signal suppression
299 in the most complex matrix. Internal calibration was used for quantification, which provided
300 acceptable recoveries. Analysis by LC/MS/MS in positive and negative ionization modes
301 provided good sensitivity and selectivity, with quantification limits ranging from 0.01 to 30.3
302 ng/L. This method was successfully applied to the determination of target compounds in
303 estuarine waters, influent and effluent wastewaters. We suggest that this analytical method
304 could be adapted to other matrices, such as biological tissues and sediments for evaluating
305 the environmental quality of aquatic ecosystems.
306 Acknowledgments
307 This work was financially supported by ERDF (European Regional Development Fund) and
308 AEAG (Agence de l'Eau Adour-Garonne) grants in the framework of the MICROPOLIT project.
309 Authors are grateful to people who helped in water samples collection.
310 Conflict of interest statement
311 Authors have declared no conflict of interest.
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386 emerging contaminants in liquid and solid environmental matrices by ultra-high-
387 performance liquid chromatography tandem mass spectrometry. J. Chromatogr. A
388 2016, 1431, 64–78.
389 [24] Grabic, R., Fick, J., Lindberg, R. H., Fedorova, G., Tysklind, M., Multi-residue method for
390 trace level determination of pharmaceuticals in environmental samples using liquid
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392 183–195.
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394 abuse and their metabolites in surface and wastewater using solid-phase extraction
395 coupled to liquid chromatography with high-resolution mass spectrometry. J. Sep. Sci.
396 2017, 40, 3621–3631.
397 [26] Klepacki, J., Davari, B., Boulet, M., Lizarraga, R., Christians, U., A High-throughput HPLC-
398 MS/MS Assay for the Detection, Quantification and Simultaneous Structural
399 Confirmation of 136 Drugs and Metabolites in Human Urine. Ther. Drug Monit. 2017,
400 39, 565–574.
401 [27] Gago-Ferrero, P., Borova, V., Dasenaki, M. E., Τhomaidis, Ν. S., Simultaneous
402 determination of 148 pharmaceuticals and illicit drugs in sewage sludge based on
403 ultrasound-assisted extraction and liquid chromatography-tandem mass spectrometry.
404 Anal. Bioanal. Chem. 2015, 407, 4287–4297.

19
405 [28] Gracia-Lor, E., Sancho, J. V., Hernández, F., Multi-class determination of around 50
406 pharmaceuticals, including 26 antibiotics, in environmental and wastewater samples by
407 ultra-high performance liquid chromatography–tandem mass spectrometry. J.
408 Chromatogr. A 2011, 1218, 2264–2275.
409 [29] Garcia-Ac, A., Segura, P. A., Viglino, L., Fürtös, A., Gagnon, C., Prévost, M., Sauvé, S.,
410 On-line solid-phase extraction of large-volume injections coupled to liquid
411 chromatography-tandem mass spectrometry for the quantitation and confirmation of
412 14 selected trace organic contaminants in drinking and surface water. J. Chromatogr. A
413 2009, 1216, 8518–8527.
414 [30] Gosetti, F., Mazzucco, E., Zampieri, D., Gennaro, M. C., Signal
415 suppression/enhancement in high-performance liquid chromatography tandem mass
417 [31] Hernández, F., Sancho, J. V., Ibáñez, M., Guerrero, C., Antibiotic residue determination
418 in environmental waters by LC-MS. TrAC Trends Anal. Chem. 2007, 26, 466–485.
419 [32] Yang, S., Cha, J., Carlson, K., Simultaneous extraction and analysis of 11 tetracycline
420 and sulfonamide antibiotics in influent and effluent domestic wastewater by solid-
421 phase extraction and liquid chromatography-electrospray ionization tandem mass
423 [33] Iparraguirre, A., Prieto, A., Vallejo, A., Moeder, M., Zuloaga, O., Etxebarria, N., Paschke,
424 A., Tetraphasic polar organic chemical integrative sampler for the determination of a
425 wide polarity range organic pollutants in water. The use of performance reference
426 compounds and in-situ calibration. Talanta 2017, 164, 314–322.

20
427 [34] Cimetiere, N., Soutrel, I., Lemasle, M., Laplanche, A., Crocq, A., Standard addition
428 method for the determination of pharmaceutical residues in drinking water by SPE-LC-
429 MS/MS. Environ. Technol. 2013, 34, 3031–3041.
430 [35] Lin, A. Y.-C., Tsai, Y.-T., Occurrence of pharmaceuticals in Taiwan’s surface waters:
431 impact of waste streams from hospitals and pharmaceutical production facilities. Sci.
432 Total Environ. 2009, 407, 3793–3802.
433 [36] Petrovic, M., Gros, M., Barcelo, D., Multi-residue analysis of pharmaceuticals in
434 wastewater by ultra-performance liquid chromatography–quadrupole–time-of-flight
435 mass spectrometry. J. Chromatogr. A 2006, 1124, 68–81.
436 [37] Archer, E., Petrie, B., Kasprzyk-Hordern, B., Wolfaardt, G. M., The fate of
437 pharmaceuticals and personal care products (PPCPs), endocrine disrupting
438 contaminants (EDCs), metabolites and illicit drugs in a WWTW and environmental
439 waters. Chemosphere 2017, 174, 437–446.
440 [38] Pillai, S. A., Chobisa, D., Urimi, D., Ravindra, N., Filters and Filtration: A Review of
441 Mechanisms That Impact Cost, Product Quality and Patient Safety. J Pharm Sci 2016, 8,
442 8.
443 [39] Gros, M., Rodríguez-Mozaz, S., Barceló, D., Rapid analysis of multiclass antibiotic
444 residues and some of their metabolites in hospital, urban wastewater and river water
445 by ultra-high-performance liquid chromatography coupled to quadrupole-linear ion
446 trap tandem mass spectrometry. J. Chromatogr. A 2013, 1292, 173–188.
447 [40] Gómez, M. J., Petrovid, M., Fernández-Alba, A. R., Barceló, D., Determination of
448 pharmaceuticals of various therapeutic classes by solid-phase extraction and liquid

21
449 chromatography-tandem mass spectrometry analysis in hospital effluent wastewaters.
450 J. Chromatogr. A 2006, 1114, 224–233.
451 [41] Rogatsky, E., Stein, D., Evaluation of matrix effect and chromatography efficiency: new
452 parameters for validation of method development. J. Am. Soc. Mass Spectrom. 2005,
453 16, 1757–1759.
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455 matrix effects in environmental analysis with LC-ESI-MS. Anal. Bioanal. Chem. 2004,
456 378, 910–916.
457 Figure captions
458 Figure 1: Schematic description of the extraction procedure.\
459

22
460 Figure 2: Recovery (n=3) of the target compounds after filtration of ultrapure water spiked
461 at 100 µg/L.
462
463 Figure 3: Influence of pH (pH=7, pH=5 and pH=3) on measured areas obtained using Oasis
464 HLB SPE cartridges (matrix: ultrapure water, spiking level 500 ng/L, n=3).
465

23
466 Figure 4: Impact of evaporation technique (evaporation to dryness, evaporation to dryness
467 at 40°C, partial evaporation) on the stability of pharmaceutical compounds (n=3).
468
469

24
Table 1: Physicochemical parameters of studied waters.
Conductivity Suspended
pH
(mS/cm) particles (mg/L)
Upstream estuary 7.73 0.40 21.8
Lower estuary 7.71 0.49 25.5
Estuarine WWTP effluent 6.80 1.70 40.3
Coastal WWTP influent 7.23 1.02 324
Coastal WWTP effluent 7.92 0.64 3.0
470
471
Table 2: MS/MS parameters for the analysis of target analytes by MRM in negative and
positive ionization modes.
Ionizatio
RT n Ion Ratios
Compounds Internal standards MRM 1 MRM 2
(min (Quant/Q
) modes ual)
274.4 >
Atenolol-d7 - 3.54 ESI + 145.1 274.4 > 190.3 3.0
365.4 >
Ofloxacin-d3 - 3.91 ESI + 261.1 365.4 > 321.2 1.3
247.4 >
Carbamazepine-d10 - 4.70 ESI + 204.2 247.4 > 186.9 10.7
276.0 >
Nordiazepam-d5 - 4.91 ESI + 140.2 276.0 > 165.1 0.8
Carbamazepine- 172.3 >
Metronidazole d10 2.49 ESI + 128.1 172.3 > 82.0 2.4
Acetaminophen d10 2.69 ESI + 110.1 152.0 > 92.8 4.8
Acetazolamide d10 3.36 ESI + 180.9 223.3 > 164.0 1.1
Sulfadiazine d10 3.51 ESI + 156.0 251.3 > 92.0 0.8
267.5 >
Atenolol Atenolol-d7 3.55 ESI + 145.1 267.5 > 190.1 1.8
Caffeine d10 3.83 ESI + 138.1 195.4 > 110.1 3.5
Ampicilline d10 3.84 ESI + 106.0 350.4 > 160.0 2.4
Trimethoprim d10 3.88 ESI + 230.1 291.4 > 123.1 0.9
320.4 >
Norfloxacin Ofloxacin-d3 3.90 ESI + 276.2 320.4 > 233.1 1.3
362.4 >
Ofloxacin Ofloxacin-d3 3.91 ESI + 261.2 362.4 > 318.2 1.3
Ciprofloxacin Ofloxacin-d3 3.93 ESI + 332.4 > 332.4 > 245.2 1.2

25
288.1
Sulfamethazine d10 4.06 ESI + 186.0 279.3 > 92.0 1.3
445.4 >
Tetracycline Ofloxacin-d3 4.07 ESI + 410.1 445.4 > 154.0 1.3
Phenazone d10 4.09 ESI + 131.0 189.4 > 55.4 26.1
Metoprolol d10 4.09 ESI + 116.1 268.4 > 121.1 2.7
843.7 >
Spiramycin Ofloxacin-d3 4.11 ESI + 174.1 843.7 > 142.1 3.4
749.7 >
Azithromycin Ofloxacin-d3 4.14 ESI + 158.1 749.7 > 591.5 1.1
Carbamazepine-
Sulfamethoxazole d10 4.31 ESI + 254.3 > 92.0 254.3 > 156.0 1.1
445.4 >
Doxycycline Ofloxacin-d3 4.32 ESI + 428.2 445.4 > 154.1 6.1
Oxolinic acid d10 4.44 ESI + 244.1 262.4 > 216.0 4.7
Erythromycin A d10 4.50 ESI + 158.2 734.7 > 576.5 5.6
Piperacillin d10 4.53 ESI + 143.1 518.4 > 114.7 5.1
Cyclophosphamide d10 4.53 ESI + 140.0 261.3 > 106.0 2.5
916.7 >
Tylosine Ofloxacin-d3 4.57 ESI + 174.1 916.7 > 100.9 5.1
Carbamazepine d10 4.72 ESI + 194.1 237.4 > 179.1 8.7
Flumequine d10 4.78 ESI + 202.0 262.4 > 244.1 0.9
287.0 >
Oxazepam Nordiazepam-d5 4.79 ESI + 241.1 287.0 > 269.1 1.2
Clarithromycine d10 4.80 ESI + 158.1 748.7 > 590.4 6.6
837.8 >
Roxithromycine Ofloxacin-d3 4.82 ESI + 158.1 837.8 > 679.4 2.6
323.1 >
Lorazepam Nordiazepam-d5 4.84 ESI + 277.0 323.1 > 305.0 2.2
Losartan d10 4.86 ESI + 207.1 423.4 > 405.2 2.1
271.0 >
Nordiazepam Nordiazepam-d5 4.92 ESI + 140.0 271.0 > 165.0 1.7
828.7 >
Josamycin Ofloxacin-d3 4.93 ESI + 109.1 828.7 > 174.1 1.3
Ketoprofen d10 5.06 ESI + 105.1 255.4 > 209.1 1.3
19-Norethindrone d10 5.15 ESI + 109.0 299.4 > 91.0 1.8
Rifampicin Ofloxacin-d3 5.16 ESI + 823.6 > 823.6 > 151.1 2.1

26
791.4
Amiodarone d10 5.45 ESI + 100.1 646.3 > 86.1 1.2
207.9 >
Ibuprofen-d3 - 5.53 ESI - 163.8 - -
Hydrochlorothiazid 296.1 >
e Ibuprofen-d3 3.68 ESI - 269.0 296.1 > 205.0 1.2
281.2 >
Niflumic acid Ibuprofen-d3 5.46 ESI - 237.0 281.2 > 177.0 5.4
294.1 >
Diclofenac Ibuprofen-d3 5.47 ESI - 250.0 294.1 > 214.1 18.5
205.2 >
Ibuprofen Ibuprofen-d3 5.53 ESI - 161.1 - -
249.2 >
Gemfibrozil Ibuprofen-d3 5.78 ESI - 121.0 249.2 > 127.1 17.9
271.1 >
17β-oestradiol Ibuprofen-d3 5.06 ESI - 145.0 271.1 > 183.0 10.4
17α- 295.1 >
ethinylestradiol Ibuprofen-d3 5.18 ESI - 145.0 295.1 > 199.0 3.1
269.1 >
Estrone Ibuprofen-d3 5.24 ESI - 145.0 269.1 > 158.9 9.6
MRM 1: transition used for quantification
MRM 2: transition used for confirmation
472
473
Table 3: Matrix effects (%) calculated for all target analytes in

different waters.
ME (%)
Upstream Lower Estuarine WWTP Coastal WWTP Coastal WWTP
estuary estuary effluent influent effluent
Atenolol-d7 118 115 62 21 85
Ofloxacin-d3 -83 -88 -96 -84 -72
Carbamazepin
100 89 -40 -50 -28
e-d10
Nordiazepam-
14 23 -26 -10 22
d5
Ibuprofen-d3 -20 -20 -68 -91 -9
Metronidazole -11 -16 -41 -22 16
Acetaminophe
-5 -17 -55 22 17
n
Acetazolamide -92 -88 -72 -87 -23
Sulfadiazine -40 -24 -46 -74 -31
Atenolol 104 100 15 -4 42
Caffeine 7 -6 -42 -55 -23

27
Ampicilline -15 2 -87 -78 -51

Trimethoprim -6 -8 -79 -71 -12
Norfloxacin -28 -57 -71 -50 -15
Ofloxacin -29 -50 -79 -57 -14
Ciprofloxacin -41 -63 -84 -64 -32
Sulfamethazin
42 39 -36 -61 -22
e
Tetracycline -74 -58 -97 -90 -72
Phenazone -2 -7 -57 -39 20
Metoprolol -6 -10 -69 -83 -58
Spiramycin 466 433 127 26 267
Azithromycin 153 165 70 -1 95
Sulfamethoxaz
20 10 -60 -62 -40
ole
Doxycycline -60 -48 -27 -83 -64
Oxolinic acid 86 30 -2 -72 23
Erythromycin
-38 -33 -85 -99 -7
A
Piperacillin -48 -9 -75 -28 11
Cyclophospha
70 46 -47 -78 -47
mide
Tylosine 46 36 24 78 127
Carbamazepin
94 84 -39 -46 -18
e
Flumequine 50 66 -12 -23 20
Oxazepam 24 27 -31 -10 28
Clarithromycin
13 21 -40 -20 27
e
Roxithromycin
8 22 -4 123 147
e
Lorazepam 34 38 -6 -19 12
Losartan 19 10 -30 20 48
Nordiazepam 17 20 -27 -6 22
Josamycin 9 13 -14 114 107
Ketoprofen 14 11 -38 -26 10
19-
-4 -15 -57 -69 -30
Norethindrone
Rifampicin 265 257 95 37 327
Hydrochlorothi
4 -4 -62 -87 -41
azide
Niflumic acid 20 12 -48 -92 30
Diclofenac -12 -16 -71 -90 -13
Ibuprofen -8 -14 -63 -91 -9
Gemfibrozil -6 -10 -59 -85 83
17β-oestradiol -25 -34 -98 N/A -30
17α-
ethinylestradio -44 -42 N/A N/A N/A
l

28
Estrone -23 -27 -98 N/A 34

N/A : Not
applicable
If there is an intense signal suppression due to the presence of the matrix, ME (%)
is close to -100.
If there is an intense signal enhancement due to the presence of the matrix, ME
(%) is close to 100.
474
475
Table 4: Analytical performances of the analytical procedure:

linearity (R2 coefficient of determination), instrumental
detection limits (IDL), method quantification limits (MQL),
recoveries (n=3) and precisions (RSD%, n=3).
Recoveries (%) Precisions (%)

Estua Coas Coas Ups Ups Lo Estua Coas
IDL
Upst Upst rine tal tal trea trea we rine tal
(pg
M rea rea Low WWT WW WW m m r WWT WW
R2 inje
QL m m er P TP TP est est est P TP
Compoun cted
(ng estu estu estu efflue influ efflu uar uar ua efflue efflu
ds )
/L) ary ary ary nt ent ent y y ry nt ent
50
100 500 0
100 500 500 500 500 500 ng/ ng/ ng 500 500
ng/L ng/L ng/L ng/L ng/L ng/L L L /L ng/L ng/L
0.99
Metronid 717 0.2 141. 134.
azole 9 0.06 6 30.8 46.1 46.1 106.8 3 9 7.9 4.3 2.4 18.0 5.0
0.99
Acetamin 606 0.9 a) 118. 20.
ophen 0 0.21 1 31.0 55.0 50.0 79.0 7 4 4.7 7.7 20.0 4.1
0.99
Acetazola 399 4.5 49. 16.
mide 7 2.03 5 N/A 5.5 8.0 47.2 25.9 93.3 N/A 4 1 24.2 6.7
0.99
Sulfadiazi 788 0.3 70. 29.
ne 1 0.27 1 15.8 30.3 40.8 91.2 49.5 89.9 6 5 4.2 9.1 1.7
0.99
728 0.1
Atenolol 4 0.06 1 91.0 95.0 94.0 85.0 69.1 89.2 4.1 0.6 1.9 14.4 1.8
0.99
679 0.6 a)
Caffeine 3 0.10 5 44.0 56.0 52.0 69.0 97.5 9.3 0.3 4.4 2.0 0.7
0.99
Ampicillin 666 1.2 64. 12.
e 0 1.25 2 41.8 46.3 58.7 23.2 43.9 72.2 0 1.1 8 46.0 7.1
Trimetho 0.99 0.2
prim 867 0.02 2 37.0 39.9 41.3 30.3 58.8 91.9 3.7 1.0 3.2 7.0 5.2

29
7
0.99
Norfloxaci 824 3.3 100. 118. 27.
n 8 0.14 6 88.7 1 91.0 179.6 7 94.9 2 9.3 6.2 19.2 13.6
0.99
997 0.00 0.0 101. 100. 105. 104.
Ofloxacin 1 3 1 5 1 7 124.7 1 99.8 3.3 2.6 1.5 14.0 1.4
0.99
Ciprofloxa 982 0.5 100. 11.
cin 9 0.03 9 96.4 0 94.2 120.0 96.0 71.7 8.5 5.8 3 11.5 10.5
0.99
Sulfamet 841 0.1
hazine 4 0.02 7 63.2 70.7 73.4 106.4 74.4 98.4 6.1 1.1 3.2 5.2 4.0
0.99
Tetracycli 500 0.3 238. 1113. 62. 29. 26.
ne 2 0.15 4 97.0 98.9 9 2 66.1 93.1 3 5 0 35.7 9.2
0.99
Phenazon 795 0.9 113. 115.
e 7 0.14 1 45.3 47.4 47.8 70.5 0 8 1.8 3.2 2.7 4.2 8.3
0.99
Metoprol 829 0.1 113.
ol 7 0.01 1 39.5 42.8 43.4 49.4 31.5 6 2.5 1.0 1.2 5.5 5.3
0.99
Spiramyci 204 8.0 125. 140.
n 5 1.25 6 2 98.7 3 178.1 20.6 37.2 4.1 4.4 2.9 21.0 8.2
0.99
Azithrom 706 0.6 110. 155. 13.
ycin 3 0.06 8 0 98.1 7 301.9 40.1 46.6 7.6 4 2.6 33.4 5.3
0.99
Sulfamet 394 0.5 23.
hoxazole 5 0.03 7 44.3 59.0 56.7 65.7 65.7 73.1 2 3.4 2.4 7.9 2.1
0.97
Doxycycli 749 1.3 111. 120. 28. 33. 26.
ne 6 1.36 2 22.1 20.4 27.6 97.6 5 4 3 0 3 27.8 9.5
0.99
Oxolinic 648 0.3 124. 17. 27.
acid 2 0.33 8 57.8 88.7 63.9 170.2 52.8 0 7.8 0 4 10.0 7.0
0.99
Erythrom 751 0.3 114. 32. 25.
ycin A 5 0.02 2 18.6 28.1 36.6 21.9 5.8 4 0 6 2.4 10.6 0.7
0.99
Piperacilli 804 1.3 119. 28. 12. 24.
n 1 0.73 2 45.5 35.3 59.5 52.2 82.5 4 6 0 9 34.9 25.3
0.99
Cyclophos 704 0.0
phamide 7 0.01 6 78.5 85.2 77.4 88.1 42.5 70.1 3.7 2.9 0.3 5.8 1.9
0.99
989 0.2 129. 180.
Tylosine 5 0.01 1 55.6 66.3 65.4 175.7 9 6 6.5 4.4 2.3 2.5 9.8
Carbamaz 0.99 0.00 0.0 97.1 97.5 97.9 119.4 103. 101. 2.0 2.6 1.4 4.7 3.6

30
epine 897 1 1 0 6
1
0.99
Flumequi 302 4.5 136. 131.
ne 0 0.19 5 68.5 76.1 90.0 152.7 2 0 7.0 4.9 2.4 5.1 4.0
0.99
Oxazepa 978 0.00 0.1 116. 117. 111. 101.
m 4 3 1 0 0 0 91.0 2 87.4 8.6 2.2 4.0 10.5 4.5
0.99
Clarithro 778 0.00 0.0 107. 110.
mycine 6 4 6 48.0 50.5 59.7 97.7 4 8 6.1 7.1 9.3 7.7 18.3
0.99
Roxithro 975 0.9 303. 145.
mycine 9 0.05 1 46.9 46.3 57.8 152.8 0 7 7.2 6.9 3.5 7.9 9.5
0.99
Lorazepa 936 3.3 113. 121. 115.
m 8 0.17 3 5 1 1 138.6 87.4 89.9 1.3 1.2 3.3 14.2 0.9
0.99
636 0.2 213. 158.
Losartan 4 0.04 2 53.5 59.4 58.5 136.0 8 1 5.4 2.9 5.5 2.1 5.3
0.99
Nordiaze 911 0.0 106. 100.
pam 5 0.02 3 99.2 6 3 104.9 98.6 98.0 5.1 0.3 3.0 12.2 2.8
0.99
019 0.2 591. 189.
Josamycin 7 0.03 3 46.9 49.4 55.3 118.8 9 1 8.7 7.0 1.8 2.1 10.7
0.99
Ketoprofe 206 1.4 123. 120.
n 8 0.10 3 44.4 59.4 61.8 68.8 0 5 2.4 7.3 3.0 0.8 4.9
19- 0.99
Norethin 768 2.2
drone 7 0.75 7 45.3 48.4 45.6 73.6 58.9 89.2 9.8 5.3 4.0 6.8 6.1
0.99
Rifampici 747 8.3 201. 134. 137. 14.
n 2 0.93 3 0 6 5 184.8 36.6 36.0 5 5.8 3.8 3.3 27.1
Hydrochl 0.99
orothiazid 824 0.2 116. 126. 116. 131. 14.
e 2 0.15 1 9 5 2 181.6 0 68.2 3 5.8 1.6 3.6 1.4
0.99
Niflumic 755 0.3 148. 146. 134. 131. 13.
acid 8 0.12 6 4 2 0 174.9 84.3 8 8.0 3 2.8 8.9 0.7
0.99
Diclofena 915 1.2 102. 107. 100. 102. 102.
c 2 0.33 5 8 1 1 104.0 4 4 6.4 5.2 6.2 7.7 1.5
0.99
401 3.2 108. 109. 101. 10.
Ibuprofen 1 0.72 1 1 7 7 108.3 81.5 96.3 5 7.6 0.9 8.3 3.5
0.99
Gemfibro 875 0.2 105. 113. 108. 122. 125.
zil 3 0.05 3 8 2 8 118.5 3 8 3.8 5.4 3.0 12.0 1.4

31
0.99
17β- 676 6.2 16.
oestradiol 8 4.41 5 71.4 74.7 61.1 31.1 N/A 65.7 0 5.1 3.7 25.5 11.6
17α- 0.98
ethinylest 271 25.0 30. 31. 11. 13.
radiol 3 0 30 74.8 60.6 61.4 N/A N/A N/A 9 9 3 N/A N/A
0.99
835 1.8 19.
Estrone 6 0.37 4 90.4 84.1 75.8 26.9 N/A 91.1 2 3.7 6.2 43.7 10.1
a) Quantification was impossible
because the sample initially
contained high amount of target
molecule
N/A : Not
applicable
476
477
Table 5: Pharmaceutical concentrations (expressed in ng/L) detected in estuarine waters, influent

and effluent wastewaters from the basque country.
Estuarine Coastal
Therapeutic
Compounds Upstrea Low WWTP WWTP WWTP
groups
m er effluent Influent Effluent
Estrone (E1) <1.8 <1.8 <1.8 <1.8 <1.8
17β-oestradiol (E2) <6.3 <6.3 <6.3 <6.3 <6.3
Steroid estrogens 17α-ethinylestradiol
(EE2) <30 <30 <30 <30 <30
19-Norethindrone <2.3 <2.3 <2.3 <2.3 <2.3
Ampicilline <1.2 <1.2 <1.2 <1.2 <1.2

Azithromycin <0.7 <0.7 146 64.6 64.5
Ciprofloxacin <0.6 <0.6 123 1,140 97.4
<0.0
Clarithromycine <0.06 6 127 34.3 13.6
Doxycycline <1.3 <1.3 <1.3 <1.3 <1.3
<0.3
Erythromycin A <0.32 2 154 <0.32 <0.32
Flumequine <4.6 <4.6 <4.6 <4.6 <4.6
Antibiotics <0.2
Josamycin <0.23 3 13.5 10.4 7.0
<0.2
Metronidazole <0.26 6 140 49.9 <0.26
Norfloxacin <3.4 <3.4 <3.4 <3.4 <3.4
<0.0
Ofloxacin <0.01 1 143 288 150
<0.3
Oxolinic acid <0.38 8 <0.38 <0.38 <0.38
Piperacillin <1.3 <1.3 <1.3 <1.3 <1.3

32
<0.9
Roxithromycine <0.91 1 58.4 <0.91 <0.91
Rifampicin <8.3 <8.3 <8.3 <8.3 <8.3
Spiramycin <8.1 <8.1 161 218 50.9
<0.3
Sulfadiazine <0.31 1 <0.31 <0.31 <0.31
<0.1
Sulfamethazine <0.17 7 <0.17 <0.17 <0.17
Sulfamethoxazole <0.57 6.5 147 250 111
<0.3
Tetracycline <0.34 4 <0.34 <0.34 <0.34
<0.2
Trimethoprim <0.22 2 88.2 55.9 4.1
<0.2
Tylosine <0.21 1 <0.21 <0.21 <0.21
Acetaminophen 34.6 60.2 1,720 140,300 <0.91

Diclofenac 1.3 <1.3 888 1,150 900
Ibuprofen <3.2 <3.2 1,020 19,500 75.9
Analgesics and Ketoprofen <1.4 <1.4 2,450 3,930 199
NSAIDs <0.3
Niflumic acid <0.36 6 130 54.2 194
<0.9
Phenazone <0.91 1 <0.91 <0.91 <0.91
Atenolol 4.5 4.1 758 1,010 17.4

<0.2
β-blockers Losartan <0.22 2 405 336 16.0
<0.1
Metoprolol <0.11 1 156 192 46.9
<0.0
Anti-cancers
Cyclophosphamide <0.06 6 <0.06 <0.06 <0.06
Anti-epileptics Carbamazepine 4.8 2.7 455 546 977
Human indicators Caffeine 18.4 54.0 10,600 82,200 86.0
Lorazepam <3.3 <3.3 36.3 21.0 22.0

<0.0
Anxiolytics
Nordiazepam <0.03 3 24.8 4.1 11.8
Oxazepam 10.6 8.3 6,190 974 1,430
Acetazolamide <4.6 <4.6 <4.6 <4.6 <4.6

<0.2
Various
Gemfibrozil <0.23 3 84.2 11.3 4.4
Hydrochlorothiazide 4.2 0.6 987 17,200 1,430
478

33

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com Page 1 Journal of Separation Science

1 <PE-AT>Multi-residue analysis of 44 pharmaceutical compounds in environmental water

2 samples by solid phase extraction coupled to liquid chromatography – tandem mass

4 Carole Miossec a, Laurent Lanceleur a and Mathilde Monperrus a*

6 PHYSICOCHIMIE POUR L'ENVIRONNEMENT ET LES MATERIAUX – MIRA, UMR5254, 64600,

9 University of Pau and Pays de l’Adour

14 Abbreviations: AEAG, agence de l'eau adour-garonne; ERDF, european regional

17 PTFE, polytetrafluoroethylene; PVDF, polyvinylidene difluoride; RT, retention time; SPM,

Received: 11 27, 2018; Revised: 03 06, 2019; Accepted:03 08, 2019

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19 Keywords: liquid chromatography - tandem mass spectrometry / pharmaceuticals / solid

20 phase extraction / water

22 A solid phase extraction combined with a liquid chromatography - tandem mass

24 determination of 44 pharmaceuticals belonging to different therapeutic classes (i.e.

25 antibiotics, anti-inflammatories, cardiovascular agents, hormones, neuroleptics and

26 anxiolytics) in water samples.

32 Recoveries of the pharmaceuticals were acceptable, instrumental detection limits varied

35 from 0.3 to 49.4%.

37 estuarine waters and wastewaters. Eight of these 44 pharmaceuticals were detected in

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39 highest values of occurrence and concentration were found in wastewater influent.<PE-

43 emerging contaminants on the environment. Among the great diversity of pollutants

45 input [1]. As pharmaceuticals consumption is continuously increasing [2], it raises concerns

49 detected, especially in wastewaters at concentrations ranging from nanogram to microgram

52 pollutants at trace levels in environmental samples.

54 properties is challenging, as it generally requires a compromise in the selection of

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64 extraction (SPE) pre-concentration step in order to ensure adequate sensitivity for MS

65 detection and therefore accurate analytes determinations at sub-ppb levels. A drawback

68 ionization process [28]. Consequences of matrix effects are suppression or enhancement of

71 chromatographic separation, mass spectrometry instrumentation and the ionization

74 to solve matrix effects, including modifications of sample pre-treatment, chromatographic

79 44 commonly used human and veterinary pharmaceuticals (including antibiotics, anti-

80 inflammatories, cardiovascular agents, hormones, neuroleptics and anxiolytics) in water

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88 2. Materials and Methods

89 2.1 Reagents and materials

90 Reference standards of acetaminophen, acetazolamide, amiodarone, ampicilline, atenolol,

91 azithromycin, caffeine, carbamazepine, ciprofloxacin, clarithromycine, cyclophosphamide,

92 diclofenac, doxycycline, erythromycin A, estrone (E1), 17β-oestradiol (E2),

93 17α-ethinylestradiol (EE2), flumequine, gemfibrozil, hydrochlorothiazide, ibuprofen,

94 josamycin, ketoprofen, lorazepam, losartan, metoprolol, metronidazole, niflumic acid,

95 nordiazepam, 19-norethindrone, norfloxacin, ofloxacin, oxazepam, oxolinic acid, phenazone,

96 piperacillin, rifampicin, roxithromycine, spiramycin, sulfadiazine, sulfamethazine,

97 sulfamethoxazole, tetracycline, trimethoprim and tylosine were supplied by Sigma Aldrich

99 used as internal standards were atenolol-d7, carbamazepine-d10, ibuprofen-d3,

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109 Waters (Milford, USA)

110 2.2 Cleaning procedure

114 with aluminum sheets.

115 2.3 Samples collection and pre-treatment

122 remaining on the filter after dryness.

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126 solution) in order to improve the extraction efficiency of target compounds as

127 recommended by previous pharmaceutical compounds studies [1,31,32].

128 2.4 Solid phase extraction procedure

129 Internal standards (atenolol-d7, carbamazepine-d10, ibuprofen-d3, nordiazepam-d5 and

140 2.5 Liquid chromatography

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157 1). Sample injection volume was 5 µL.

158 2.6 Tandem mass spectrometry

159 Mass spectrometry was performed by a Xevo TQ MS (Triple quadrupole) with an