Development of On-Line Solid-Phase Extraction-Liquid Chromatography Coupled with Tandem Mass Spectrometry Method to Quantify Pharmaceutical, Glucuronide Conjugates and Metabolites in Water Lea Bazus, Nicolas Cimetiere, Dominique Wolbert, Guy Randon
To cite this version: Lea Bazus, Nicolas Cimetiere, Dominique Wolbert, Guy Randon. Development of On-Line Solid-Phase Extraction-Liquid Chromatography Coupled with Tandem Mass Spectrometry Method to Quantify Pharmaceutical, Glucuronide Conjugates and Metabolites in Water. Journal of Chromatography & Separation Techniques, OMICS International, 2016, 7 (5), pp.1000337. �10.4172/2157-7064.1000337�. �hal-01438201�
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Development of on-line solid-phase extraction-liquid chromatography
2
coupled with tandem mass spectrometry method to quantify pharmaceutical,
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glucuronide conjugates, and metabolites in water.
4 5
Léa Bazusa, Nicolas Cimetière*a, Dominique Wolberta and Guy Randonb
6
a
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35708 Rennes Cedex 7, France
8
c
9
Cedex 9, France
Ecole National de Chimie de Rennes, CNRS, UMR 6226, 11 Allée de Beaulieu, CS 50837,
Veolia Eau, Direction Technique Région Ouest, 8 allée Rodolphe Bopierre, 35020 Rennes
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11
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*Corresponding author
phone : +33 2 23 23 80 14 e-mail address :
[email protected]
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Abstract:
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The present work describes the development of an analytical method, based on automated
16
on-line solid phase extraction followed by ultra-high-performance liquid chromatography
17
coupled with tandem mass spectrometry (SPE-LC-MS/MS) for the quantification of 37
18
pharmaceutical residues, covering various therapeutic classes, and some of their main
19
metabolites, in surface and drinking water. A special attention was given to some
20
glucuronide conjuguates and metabolites of active subtances. Multiple Reaction Monitoring
21
(MRM) was chosen and two transitions per compound are monitored (quantification and
22
confirmation transitions). Quantification is performed by standard addition approach to
23
correct matrix effect. The method provides limit of quantification inferior to 20 ng.L-1 for all
24
compounds. The methodology was successfully applied to the analysis of surface water and
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drinking water of 8 drinking water treatment plant in west of France. The highest drug
26
concentrations in surface water and drinking water were reported for ketoprofen,
27
hydroxyibuprofen, acetaminophen, caffeine and danofloxacin.
28 29
Key
words:
pharmaceuticals,
automated
on-line
solid
30
chromatography, tandem mass spectrometry, water analysis
phase
extraction,
liquid
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1. Introduction
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Pharmaceuticals are an important group of emerging contaminants in the environment [1].
33
In recent years many reports have been made on the occurrence of the large, differentiated
34
group of pharmaceuticals in wastewater, surface water, ground water and drinking water in
35
many countries [2-9]. After administration, most pharmaceuticals are not completely
36
metabolized. The unmetabolized parent pharmaceutical and some metabolites are
37
subsequently excreted from the body via urine and faeces [10]. Reports have shown that
38
many pharmaceuticals do not totally degrade during conventional wastewater treatment
39
[11,12]. The concentrations of individual compounds in wastewater, surface water, ground
40
water and drinking water are typically in the range of ng/L to µg/L. The effect on long-term
41
pharmaceutical residues in aquatic environments remains largely unknown. In addition, the
42
risks to the environment are evaluated for a particular drug, while we find a mixture of all
43
these compounds in aquatic environments. Studies have shown that combinations of drugs
44
may be more powerful than the simple addition of two drugs individually toxic effects [13-
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14].
46
Wastewater effluent is a major source for the input of pharmaceuticals to the environment
47
[11;12], which can then migrate through water systems and into source water intended for
48
drinking water supplies. Advanced wastewater treatment processes have been shown to
49
significantly reduce the concentrations of emerging contaminants. However, some
50
compounds are not completely removed even if treatment techniques are used [15].
51
Moreover, most of the WWTP do not include these specifically designed treatment units.
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In this context, sensitive analytical methods allowing the quantification of many pollutants at
53
trace concentration is essential. Solid Phase Extraction (SPE) is the most commonly used
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technique to prepare sample before analysis. SPE allows the concomitance of analyte
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concentration and interferences removal [16;17]. To date, most of the published multi-
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residue methods for the determination of ultra traces of pharmaceuticals compounds in
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surface and drinking water use off-line SPE followed by gas chromatography mass
58
spectrometry (GC–MS) or by liquid chromatography-tandem mass spectrometry (LC–
59
MS/MS) [2-5;7;9;12]. However, On-line Solid Phase Extraction is an emerging method for
60
analysis of the trace compounds of organic micropollutants (reactive drugs, pesticides…).
61
This technique has many advantages: saving time, automated method, reproducibility, very
62
low solvent consumption, small sample handling, SPE cartridges reuse… [17]. The cartridges
63
used to concentrate pharmaceuticals residues are usually OasisTM HLB or hydrophobic resins.
64
[18;19]. This technique is generally coupled to liquid chromatography with UV, MS or MS/MS
65
detector with reversed phase column [20-24].
66
The objectives of this work has been to develop a fully automated method to analyze a
67
number of target compounds belonging to different therapeutical classes and some by
68
product using on-line SPE directly coupled to liquid chromatography tandem mass
69
spectrometry (LC–MS/MS). This analytical technique limits matrix effect impact. However
70
remaining, interfering species can affect the analytical train, especially natural organic
71
matter may coeluate with targeted compounds which leads to a signal disturbance causing
72
over/underestimation or false positive results, or some compounds may react with targeted
73
molecules during sampling and storage [25].
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This method was evaluated in different water matrices: UltraPure Water (UPW) to develop
75
the analytical method, surface water and drinking water for validation.
76 77 78 79 80
2. Material and methods 2.1. Compound selection
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32 pharmaceuticals and 3 metabolites and 2 glucuronide conjugates were selected for this
82
study (Table 1 and Table S1). These molecules were chosen based on the following criteria: i)
83
selected compounds should exhibit a variety of physical properties, such as functional
84
groups and polarity, ii) they should represent of a diversity of pharmaceutical classes, iii)
85
high frequencies of environmental occurrence, iv) low removal efficiencies by drinking water
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and wastewater treatment techniques in France or others countries [2-9]. Table 1 lists the 37
87
molecules selected for our study and their optimized parameters for quantification, chemical
88
structure is provided in the figure S1 in Supporting Information. Thereafter, the molecules
89
will be called by the short identifiers which are given in the table 1. The pharmaceutical
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classes represented are cardiovascular drugs, anticancer agents, human or veterinary
91
antibiotics, neuroleptics, non-steroidal anti-inflammatory drugs and hormones.
92 93
2.2. Pharmaceutical standards and reagents
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All pharmaceutical compounds have minimum 90% purity, used as received in solid form and
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were obtained from Sigma Aldrich (FRANCE). Ultra pure water (UPW) was delivered by a
96
ElgaPureLab System (resistivity 18.2 M.cm, COT< 50µg C/L ). Chromatographic and SPE
97
solvents, acetonitrile (ACN) with or without 0.1 % formic acid (FA) and methanol (MeOH)
98
were purchased from JT Baker (LC-MS grade) and were used in association with UPW in also
99
or not with 0.1 % formic acid.
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All concentrated stock solution of individual pharmaceuticals were prepared in methanol
101
with a concentration of 500 mg.L-1 and stored at −20 ◦C. The mixed spiking solutions were
102
prepared in methanol at 500 µg.L-1 and stored at 4◦C during 15 days maximum. This mixed
103
spiking solution is daily diluted in water to obtained 500 ng.L-1 before use for standard
Page 6 sur 27 104
addition. Concentrations prepared for analytical development and to quantify the target
105
compounds in the different matrices are: 5, 10, 20, 50, 100, 250 and 500 ng.L-1.
106 107
2.3. On-line solid phase extraction and liquid chromatography
108
The analytical system consists of an automated SPE sampler coupled with an LC-MS/MS. The
109
online extraction was carried out using a 2777 autosampler equipped with two parallel
110
OasisTM HLB cartridge (Direct Connect HP 20µm, 2.1x30mm) working sequentially. The
111
switching from the loading flow pattern, to elution, then conditioning and back to loading is
112
performed using two six positions EverflowTM valves. Loading eluent (UPW) and conditioning
113
eluent (methanol) were provided by a quaternary pump (AcquityTM QSM). Elution of the
114
analytes from the SPE cartridge to LC system was achieved by connected the cartridge to the
115
inlet of the separation column and using the initial chromatographic elution solution.
116
Separation was carried out using a reversed phase column (AcquityTM BEH C18, 100 mm x 2.1
117
mm ID, 17μm) placed in an oven (45°C). The elution gradient was produced by a binary
118
pump (AcquityTM BSM) and was optimized and will be described later in the manuscript.
119 120
2.4. Mass spectrometry
121
The mass spectrometer (Quattro Premier, MicromassTM) operates with the following
122
conditions: cone gas (N2, 50 L.h-1, 120 °C), desolvation gas (N2, 750 L.h-1, 350 °C), collision
123
gas (Ar, 0.1 mL.min-1), capillary voltage (3000V). The ionization source of the mass
124
spectrometer is an electrospray (ESI) used either in the positive or the negative mode
125
according to pharmaceutical compounds structure (table 1). All the analysis, are made in
126
"multiple reaction monitoring" (MRM) mode, the parent ion from the ESI source is selected
127
in the first quadrupole (pseudomolecular ion in most cases) and fragmented in the collision
Page 7 sur 27 128
cell. One or more fragments (quantification ion and, when available, confirmation ions) are
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then selected by the third quadrupole before being detected by a photomultiplier. This
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mode allows high sensitivity and selectivity.
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Table 1: List of the 35 pharmaceuticals with pharmaceutical class Molecule (short identifier), N°CAS, MW (g/mol), formula, mass parameter and retention time Pharmaceutical class
Cardiovascular drugs
anticancer agent
Human Antibiotic
Veterinarian Antibiotic
Neuroleptic
Non-steroidal antiinflammatory drugs (NSAID)
Miscellaneous
Hormone
Molecule (short identifier)
N°CAS
Amlodipin (AML) Atenolol (ATE) Losartan (LOS) Naftidrofuryl (NAF) Pravastatin (PRA) Propanolol (PRO) Gemfibrozil (GEM) Trimetazidin (TRI) Tamoxifen (TAM) Hydroxytamoxifen (OH-TAM) Ifosfamide (IFO) Doxycycline (DOX) Erythromyicin (ERY) Ofloxacin (OFX) Sulfaméthoxazole (SUL) Trimetoprime (TRP) Danofloxacin (DANO) Lincomycin (LINCO) Sulfadimerazine (SFZ) Tylosin (TYL) Carbamazepine (CBZ) Epoxycarbamazepine (Ep-CBZ) Oxazepam (OZP) Oxazepam (Glu-OZP) Diclofenac (DICLO) Ibuprofen (IBU) Hydroxyibuprofen (OH-IBU) Ketoprofen (KETO) Salicylic acid (SCA) Acetaminophen (PARA) Acetaminophen Glucuronide (Glu-PARA)
111470-99-6 29122-68-7 124750-99-8 03200-6-4 81131-70-6 525-66-6 25812-30-0 13171-25-0 10540-29-1 68047-06-3 3778-73-2 24390-14-5 114-07-8 82419-36-1 723-46-6 738-70-5 112398-08-0 859-18-7 57-68-1 74610-55-2 298-46-4 36507-30-9 604-75-1 6801-81-6 15307-79-6 15687-27-1 51146-55-5 22071-15-4 69-72-7 103-90-2 16110-10-4 58-08-2 58-93-5 57-63-6 50-28-2 53-16-7 57-83-0
Caffeine (CAF) Hydrochlorothiazide (HCTZ) Ethyinylestradiol (EE) 17β-Estradiol (βE) Estrone (EO) Progesterone (PGT)
MW (g/mol) 567.05 266.34 461 473.56 446.51 259.4 250.33 339.26 371.5 387.2 261 512.94 769.96 361.37 253.278 290.3 357.38 461.37 278.33 1066.19 236.27 252.27 286.71 462.84 294.14 206.28 222.28 254.28 138.12 151.16 327.29 194.19 297.74 296.4 272.38 270.37 314.46
formula of the active substance C20H25ClN2O5 C14H22N2O3 C22H23ClN6O C24H33NO3 C23H36O7 C16H21NO2 C15H22O3 C14H24Cl2N2O3 C26H29NO C26H29NO2 C7H15Cl2N2O2P C22H24N2O8 C37H67NO13 C18H20FN3O4 C10H11N 3O3S C14H18N4O3 C19H20FN3O3 C18H34N2O6S C11H12N4O2S C46H77NO17 C15H12N2O C15H12N2O2 C15H11ClN2O2 C21H19ClN2O8 C14H11Cl2NO2 C13H18O2 C13H18O3 C16H14O3 C7H6O3 C8H9NO2 C14H17NO8 C8H10N4O2 C7H8ClN3O4S2 C20H24O2 C18H24O2 C18H22O2 C21H30O2
ESI + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Parents ion 409.6 267 423.6 384.6 423.2 260.2 249 267.4 372.5 388.2 261.02 445.5 734.2 362 254 291.2 358.5 407.6 279.4 917 237.1 253.3 287.4 463.2 296.1 205 221.2 255 137 152 350 195.1 296.2 295.2 271.1 269.1 315.2
Daughter ion(Q) 238.1 145 405.2 99.7 100.6 116 121 180.9 72 72 153.95 428.2 158 318 92 230 314 125.9 185.9 174 194 179.9 241 287.1 250 161 177 209 92.6 110 173.8 137.7 77.6 144.9 145 145 97
Cones (V) 18 34 30 40 34 34 34 21 45 45 25 30 28 34 26 24 35 40 29 60 28 28 34 26 22 17 19 29 30 25 33 37 42 54 50 53 32
Collisions (V) 11 26 12 21 23 18 23 16 14 14 22 18 30 19 28 24 19 28 16 37 19 28 20 15 10 7 9 12 14 15 15 18 28 40 38 35 24
13 22 28 10
Dwell time (ms) 50 50 50 50 50 50 50 50 50 50 75 50 50 80 50 50 50 50 50 50 50 50 50 15 100 50 50 100 70 50
Tr (min) 4.03 1.18 4.25 4.29 2.63 3.33 4.95 1.18 5.42 4.58 3 2.95 3.68 1.35 2.74 1.18 1.53 1.23 1.91 3.84 3.85 3.2 4.08 3.34 5.5 4.06 1.2 4.14 1.16 1.24 1.64
22 22 35 41 36 26
50 50 50 70 70 50
1.35 1.5 4.07 3.89 4.14 5.77
Confirmation ion 409.6 74 207 84.7 321.1 183
Collisions (V) 13 23 22 25 16 18
165.8
26
92.04 153.8 576.2 261 156 261.1 283 359.3 91.7 773 179 236 269.1 269 214.1
25 28 19 28 16 26 25 18 26 29 39 12 14 26 25
158.7 105 64.7 90 109.7 204.8 183 183 183 109
Page 9 sur 27 132
133
3. Results and discussion
134
3.1. Mass spectrometry optimization
135
The selection of optimum detection parameters (collision energy, cone voltage, ionization
136
mode) for each targeted compound was carried out by introducing a standard diluted single
137
solute solution at 5 mg.L-1 directly in the mass spectrometer (without separation). The
138
pseudo-molecular ion ([M+H]+ or [M-H]-) was selected as the parent ion. Acetaminophen-
139
glucuronide was ionized as sodium adducts ([M+Na]+) and the daughter ion correspond to
140
the sodium adduct of paracetamol obtained by the loss of glucuronic acid. Similar
141
fragmentation pattern with loss of carbohydrate group was observed with Glu-OZP ([M+H]+
142
[M-Glu+H]+). In some cases, the standard molecules were purchased as sodium or
143
chloride salt so molecular weight of the commercial product indicated in the table 1 does
144
not correspond to the formula of active compounds. So the molecular weights indicated in
145
the table 1 do not correspond to the mass of the pseudo molecular ion (AML, LOS, NAF, PRA,
146
TRI, DOX, ERY, LINCO and TYL). Positive mode was selected for most of the molecules and 8
147
analytes were ionized under negative mode because of their tendancy to loose a proton.
148
Two transitions are chosen for quantification and confirmation. If possible transition
149
corresponding to the loss of simples fragments (ie. –H2O or –CO2) has been prefered for
150
quantification or confirmation transition. Only one transition could be found to 4 molecules:
151
Ibuprofen, Gemfibrozil, Tamoxifen and Hydroxy-Tamoxifen. The results are presented in
152
table 1.
153
3.2. On-line SPE method development
Page 10 sur 27 154
The efficiency of the SPE step was studied using two different types of SPE cartridge phases :
155
Oasis HLB (Direct Connect HP 20µm, 2.1x30mm) and XBridge C18 (Direct Connect HP 10µm,
156
2.1x30mm). The low energie interactions are predominant with the C18 phases, unlike for
157
HLB phases where the dipole-dipole interactions are brought into play. Table 2 presents
158
characteristics (log(Kow), pka, coefficient of dissociation, dipolar moment) of molecules. The
159
extraction yield was then calculated according to the following equation:
160
For each compounds, the area obtained with the injection of 5mL of solution at 100 ng.L-1 in
161
SPE mode was compared to the area obtain in conventional mode (Vinj=5µL; C=100 µg.L-1).
162
The results are presented in figure 1. In a global overview the extraction yields are better
163
with the Oasis HLB phase in comparison to the C18 phase. 11 molecules have slightly better
164
extraction yields with the XBridge C18 media. Given these results, Oasis HLB phase was
165
chosen for the SPE cartridges. The extraction yields are between 24% and 96%. Six
166
molecules, among them three hormones (ATE, TRI, DOX, EE, βE and EO) have extraction
167
yields inferior or equal to 50% but the signal is sufficient for our analysis given the
168
reproducibility of the extraction step. The loading time and flow rate influence the analyte
169
retention onto the preconcentration cartridge. If the loading time is too short, a part of the
170
molecules of interest will not be collected in the cartridge. MeOH is used for the cartridge
171
conditioning during 3 minutes and UPW for the loading sample during 5.5 minutes at
172
2mL/min. 5mL of sample are injected onto the cartridge. Elution of our compounds is made
173
using the initial chromatographic conditions. The preconcentration method takes 8.5
174
minutes. The pH of samples and eluents was also optimized to try to improve the extraction
175
yields. The figure 2 shows the effect of pH (3, 7 and 9) on molecule’s recovery yields. Most of
Page 11 sur 27 176
the targeted compounds were efficiently extracted at neutral pH values. The recovery yields
177
of thirteen molecules (LOS, GEM, TAM, OH-TAM, IFO, TYL, DICLO, PARA, CAF, CBZ, OZP, PGT
178
and ERY) do not show significant pH dependence. ATE, NAF and LINCO were comparatively
179
more recovered under neutral condition due to the amine/ammonium repartition for the
180
low pH values. DANO and OFX are amphoteric molecules and exhibit higher recovery yields
181
under acid extraction than under neutral conditions. AML and OFX have extraction yields
182
superior to 100%, the differences may be included within the experimental errors. Three
183
hormones have a better extraction yields at basic pH while below 23% for an acid pH. The
184
SPE appears globally controlled by the carboxylic functions. The best compromise to our
185
analytical method is the neutral pH.
186
Tableau 2: log(Kow), pka, coefficient of dissociation and dipolar moment of molecules
Molecule
Log(Kow)
pka
coefficient of dissociation
AML
3 0.16 1.19 4.56 1.35 3.48 4.77 1.04 3.24 4.74 0.86 2,37 3,02 0.65 0.79 0.91 0,44 0,56 0.19 1.63 2,77
8.6 9.6 5,5 8.7 4.5 9.5 4.7 4.3/8.9 8.76 3.2/6.4 13.2 3.5/7.7 8.8 6.1 5.7 7.1 6.0 7.6 7 7.7 7
5.00 10-5 1.50 10-5 8.80 10-3 4.70 10-5 5.60 10-3 1.70 10-5 4.40 10-3 7.00 10-3 4.20 10-5 6.30 10-4 2.50 10-7 1.40 10-4 3.90 10-5 9.40 10-4 1.40 10-3 2.80 10-4 9.90 10-4 1.60 10-4 3.20 10-4 1.40 10-4 1.00 10-7
ATE LOS NAF PRA PRO GEM TRI TAM OH-TAM IFO DOX ERY OFX SUL TRP DANO LINCO SFZ TYL CBZ
dipolar moment
5.71 2.83
7.2
7.34 3.66
Page 12 sur 27 Ep-CBZ OZP DICLO IBU OH-IBU KETO SCA PARA CAF HCTZ EE βE EO PGT
1.58 2,24 4,51 3,79 3,97 3.12 1,19 0,49 -0.091 -0,07 3,67 3.57 3.69 4
15.9 1.7/11.6 4 4.5 4.8 4.45 3 9.5 14 7.9 10.3 10.71 10.4 18.9
1.00 10-8 1.30 10-1 8.00 10-3 5.30 10-3 3.90 10-3 6.00 10-3 3.10 10-2 1.80 10-5 2.10 10-1 1.00 10-4 7.00 10-6 4.40 10-6 6.00 10-6 3.50 10-10
4.55 4.95
4.55 3.71
1.56 3.45
187
188 189
Figure 1: Extraction yields calculated for the two cartridges (Oasis HLB and Xbridge C18) tested for all
190
molecules in neutral pH
191
Page 13 sur 27
192 193
Figure 2: Extraction yields calculated for the 3 pH (3, 7 and 9) for all analytes
194
3.3. Chromatographic conditions
195
Three chromatographic columns packed with different stationary phases were studied, two
196
using the reversed phase mode: Acquity BEH C18 (100 mm x 2.1 mm ID, 1.7 μm) and Acquity
197
HSST3 (100 mm x 2.1 mm ID, 1.7 μm). These two columns have the same stationary phase
198
but Acquity HSST3 should allow for better separation of polar molecules due to the greater
199
proportion of residual silanol groups. The third column has a polar stationary phase: BEH
200
amide (100 mm x 2.1 mm ID, 1.7 μm) in order to separate the analyte using hydrophilic
201
interaction liquid chromatography (HILIC). Comparing the chromatograms obtained for the
202
C18 and HSST3 column, the results are quite similar. Seven minutes are required to obtain
203
sufficient separation. It should be underlined that the resolution between two consecutive
204
peaks was quite low. However, because the quantification was done using different MRM
205
channels this poor resolution does not affects the analytical performances.
206
Figure 3 summarizes the results by plotting the polarity (log Kow) as function of the capacity
207
factor of the molecule, molecules with k’<1 form the unretained groups with no log(kow)
208
dependances. For the others, correlation between k’ and log(kow) shows two adverse
Page 14 sur 27 209
behaviours in relation with the different stationary phase, BEH and HSST3 on the one part
210
and HILIC on the second part. Reversed phase HPLC columns (BEH C18 and HSST3) provide a
211
satisfactory separation with k’ ranging from 0.93 to 9.91 according to the polarity of the
212
considered compounds. However numerous analytes exhibit a high polarity and were poorly
213
retained using reversed-phase HPLC. Normal phase HPLC column (BEH Amide) provides
214
separation with k’ ranging from 0.1 to 9.6. Molecules retained by the reversed phase HPLC
215
column are not retained in normal phase HPLC with k’<1. Moreover, peak tailing are
216
observed for some molecules with HSST3 (SUL, GEM, DOX) and with HILIC column (PARA,
217
DANO, HCTZ, TRI). The best compromise for our analyses is to use the BEH C18 column.
218 219 220 221
Figure 3: Polarity (log Kow) as function of the capacity factor for all molecules and for 3 chromatographic columns
222 223
The mobile phase flow rate was 0.4mL.min-1, corresponding to the optimum zone of the Van
224
Deemter curve with this column [26]. The elution conditions were optimized. Two
225
chromatographic separation methods were needed to quantify all the target analytes.
Page 15 sur 27 226
Indeed, analytes with ESI+ detection have better sensitivity with acidified eluents (with 0.1%
227
of formic acid) unlike molecules with ESI- detection which have better sensitivity with
228
neutral eluents. Moreover, the combination of both positive and negative ionization mode
229
during the same run does lead to a decrease of the sensibility.
230
The elution conditions start with 20% ACN/80% UPW during 1 minute followed by a gradient
231
90% ACN within 6 minutes and remain constant for 1 min before returning to initial
232
conditions, details of the method are presented in Supporting information (Section B –
233
Figures S1-S3)
234
Examples of chromatograms obtained with a solution of 50 ng.L-1 in UPW and the eluent
235
program are presented in Figure 4. 12 molecules elute within two minutes for the ESI+/acid
236
eluent method. As mentioned above, the detection mode (MRM) allows an accurate
237
quantification even if the resolution is low. a.
238
Page 16 sur 27
b.
239 240
Figure 4: Chromatogram obtained at 50ng/L in UPW. a. first method with ESI+. b. Second method with ESI-
241 242
3.4. Quantification limit and matrix effect
243
Standard addition method was selected for calibration method in order to minimize or
244
eliminated matrix effects. Figures 5 present examples of calibration curve for CBZ in UPW,
245
Groundwater (GW), Drinking water (DW) and Surface water (SW). Limit of quantification
246
(LOQ) were determined for all targeted compounds in UPW and GW with the equation given
247
in figure 5a, in accordance with the AFNOR NF-T-90-210 norm for all analytes. GW could be
248
considered free of pharmaceuticals residues because GW is drawn from a well recovering
249
the waters on a small watershed without collective or on-site sanitation water release, and
250
UPW can be considered as a matrix blank. Negatively ionized molecules (EO, BE, EE, HCTZ,
251
SCA, IBU, OH-IBU, GEM, PRA) have higher limits of quantification because the background
252
noise is more important than for ESI+. The values of the quantification limit of targeted
253
compounds are presented in figure 6a. LOQ values obtained range from 5 to 17ng/L. These
254
limits of quantification are sufficient for our purpose.
Page 17 sur 27 255
Measurement errors were incorporated by defining the 90% confidence intervals (figure 5b).
256
Figures 5c and d show standard addition calibration lines of CBZ in GW and DW.
257
Comparisons of the slopes obtained with real waters to the slope obtain in the blank
258
(aGW/aUPW and aDW/aUPW) allow a comprehensive approach of the matrix effects. These slope
259
ratios are presented in figure 6b for all analytes. The matrix effect is a classical phenomenon
260
which can be very important in liquid chromatography coupled with mass spectrometry
261
because of the ionization process may be drastically influenced by the presence of
262
interfering species. Many studies have already described this phenomenon especially with
263
wastewaters. The presence of organic or inorganic substance can cause inhibition (<1) or
264
enhancement (>1) of a compound’s signal [27-29]. In our case, natural organic matter may
265
disturb the SPE step or mass ionization so the rationalization of the slopes provides a global
266
overview of matrix effect but do not allow to identify the critical step.
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In figure 6b, matrix effects are not significant when the ratio is close to 1. In drinking water
268
this ratio was close to 1 for most of the analytes, only AML has a ratio superior to 5.
269
Page 18 sur 27 270
271 272 273 274
Figure 5: a. equation of LOQ determanation. b. Exemple of standard addition for CBZ with 90% confidence interval. c. and d. Exemple of standard addition in GW and DW for CBZ
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275 276
Figure 6: a. LOQ in UPW and GW for all molecules b. Matrix effects of all analytes
277 278
3.6. Analysis of surface water and drinking water
279
The developed method was used to determine the concentration of 37 pharmaceuticals
280
substances in inflow and outflow waters of 8 drinking water treatment plants (DWTP) in
281
west of France. The samples were collected once a month between october 2013 and april
282
2015, resulting in an average of 100 inflow and 100 outflow concentration values for each
283
molecule. Nine pharmaceuticals have not been detected or with concentrations below the
284
LOQ (AML, TAM, OH-TAM, IFO, ERY, LINCO, EE, βE and PGT). Figure 7 shows the
Page 20 sur 27 285
concentrations of 27 pharmaceuticals or metabolites in surface water as a box plot; this
286
statistical representation summarizes the data, for each compound, by the mean values,
287
median value, first and third quartiles and observed extrema. 7 molecules (PARA-GLU, KETO,
288
OH-IBU, DANO, PARA, SCA, CAF) have a mean concentration greater than 50 ng.L-1. 10
289
molecules were quantifieded with mean concentrations higher than 10 ng.L-1 (GEM, CBZ,
290
DICLO, OZP, OFX, IBU, HCTZ, ATE, PRO and DOX). The last detected 10 molecules exhibit
291
mean concentration lower than 10 ng.L-1 (SFZ, SUL, TRI, PRA, Ep-CBZ, TRP, EO, NAF, TYL,
292
LOS). For some molecules, large differences between the extrema are observed (PARA-Glu,
293
KETO, OH-IBU, SCA). These differences depend on the sampling date essentially. It should be
294
underlined that median values are close to mean values indicating that extrema values do
295
not play an important role. The maximum observed concentration in surface water was 650
296
ng.L-1 for KETO. Detection frequencies depend on compounds and range from 100%
297
occurrence for CAF and PARA and 9% for TYL. 13 molecules (PARA-Glu, KETO, OH-IBU,
298
DANO, PARA, CAF, SCA, DICLO, GEM, CBZ, OZP, OFX and ATE) were quantified in more than
299
50% of surface water samples. In drinking water (figure 8), six molecules (KETO, PARA-Glu,
300
OH-IBU, DANO, PARA and CAF) were quantified in 90% or more of the drinking water
301
samples. These 6 molecules were also the most quantified molecules in surface water. The
302
overall mean concentration values are between 4 (OZP) and 327 ng/L. The maximum
303
concentration found was 650 ng/L for KETO. For drinking water, the same remark than for
304
surface water may be made concerning the gap between minimum and maximum
305
concentrations: the eight drinking water treatment plants operate different treatment
306
chains with different type of water resources.
Page 21 sur 27
600 100 500
Cmax = 650 ng/L
Concentration (ng/L)
60 300
40
200
20
100
0
307 308 309 310
0 PARA-GLUKETO OH-IBU DANO PARA
SCA
CAF
GEM
CBZ
DICLO
OZP
OFX
IBU
HCTZ
ATE
PRO
DOX
SFZ
SUL
TRI
PRA Ep-CBZ
TRP
EO
NAF
TYL
LOS
Figure 7: overall mean concentrations (), median value, first and third quartiles and extrema of 27 molecules detected on average above LOQ in surface waters and detection frequencies (%, broken line).
311 300
100
90 250 80
70
60
150
50
40 100
30
20 50
10
312 313 314 315
0
KETO
PARA-GLU
OH-IBU
DANO
PARA
SCA
CAF
GEM
IBU
ATE
OFX
DICLO
CBZ
OZP
Figure 8: overall mean concentrations(), median value, first and third quartiles and extrema for 14 molecules detected in tap waters and detection frequencies (%, broken line).
0
Detection Frequencies (%)
Concentration (ng/L)
200
Detection Frequencies (%)
80 400
Page 22 sur 27 316
4. Conclusion
317
A multiresidue analysis was developed using on-line solid phase extraction connected to
318
liquid chromatography coupled with tandem mass spectrometry in order to quantify residue
319
trace levels 35 pharmaceuticals compounds in surface and drinking water. The short
320
implementation time needed to achieve the preconcentration and the analysis, 17 minutes
321
for the positive mode method and 15 minutes for the negative mode method is among the
322
most significant advantages of this method compared to off-line solid phase extraction. The
323
developed method with a preconcentration factor of one thousand showed detection limits
324
compatible with the study of environmental matrices with very low analyte concentrations.
325
The limits of detection and quantification are between 1.5 and 4 ng/L and 4 and 17ng /L,
326
respectively. Standard addition was chosen for the quantification of molecules in water
327
samples to overcome the matrix effects and provide an accurate determination of targeted
328
compounds. Among all studied substances, doxicycline appeared to be the most affected by
329
a matrix effect. The developed methods were applied to eight surfaces and drinking water.
330
In surface water, 12 molecules could be quantified in almost all analyzed samples with a
331
maximum concentration value of 650ng/L for Ketoprofen. In drinking water, 5 molecules
332
could be regularly detected, with overall mean concentration values between 20 à 120ng/L.
333
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